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1
Davis, Mary Beth, Jon Dietz, David M. Standiford, and Charles P. Emerson. "Transposable Element Insertions Respecify Alternative Exon Splicing in Three Drosophila Myosin Heavy Chain Mutants." Genetics 150, no. 3 (November 1, 1998): 1105–14. http://dx.doi.org/10.1093/genetics/150.3.1105.
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Abstract Insertions of transposable elements into the myosin heavy chain (Mhc) locus disrupt the regulation of alternative pre-mRNA splicing for multi-alternative exons in the Mhc2, Mhc3, and Mhc4 mutants in Drosophila. Sequence and expression analyses show that each inserted element introduces a strong polyadenylation signal that defines novel terminal exons, which are then differentially recognized by the alternative splicing apparatus. Mhc2 and Mhc4 have insertion elements located within intron 7c and exon 9a, respectively, and each expresses a single truncated transcript that contains an aberrant terminal exon defined by the poly(A) signal of the inserted element and the 3′ acceptor of the upstream common exon. In Mhc3, a poly(A) signal inserted into Mhc intron 7d defines terminal exons using either the upstream 3′ acceptor of common exon 6 or the 7d acceptor, leading to the expression of 4.1- and 1.7-kb transcripts, respectively. Acceptor selection is regulated in Mhc3 transcripts, where the 3′ acceptor of common Mhc exon 6 is preferentially selected in larvae, whereas the alternative exon 7d acceptor is favored in adults. These results reflect the adult-specific use of exon 7d and suggest that the normal exon 7 alternative splicing mechanism continues to influence the selection of exon 7d in Mhc3 transcripts. Overall, transposable element-induced disruptions in alternative processing demonstrate a role for the nonconsensus 3′ acceptors in Mhc exons 7 and 9 alternative splicing regulation.
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Lou, Hua, Karla M. Neugebauer, Robert F. Gagel, and Susan M. Berget. "Regulation of Alternative Polyadenylation by U1 snRNPs and SRp20." Molecular and Cellular Biology 18, no. 9 (September 1, 1998): 4977–85. http://dx.doi.org/10.1128/mcb.18.9.4977.
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ABSTRACT Although considerable information is currently available about the factors involved in constitutive vertebrate polyadenylation, the factors and mechanisms involved in facilitating communication between polyadenylation and splicing are largely unknown. Even less is known about the regulation of polyadenylation in genes in which 3′-terminal exons are alternatively recognized. Here we demonstrate that an SR protein, SRp20, affects recognition of an alternative 3′-terminal exon via an effect on the efficiency of binding of a polyadenylation factor to an alternative polyadenylation site. The gene under study codes for the peptides calcitonin and calcitonin gene-related peptide. Its pre-mRNA is alternatively processed by the tissue-specific inclusion or exclusion of an embedded 3′-terminal exon, exon 4, via factors binding to an intronic enhancer element that contains both 3′ and 5′ splice site consensus sequence elements. In cell types that preferentially exclude exon 4, addition of wild-type SRp20 enhances exon 4 inclusion via recognition of the intronic enhancer. In contrast, in cell types that preferentially include exon 4, addition of a mutant form of SRp20 containing the RNA-binding domain but missing the SR domain inhibits exon 4 inclusion. Inhibition is likely at the level of polyadenylation, because the mutant SRp20 inhibits binding of CstF to the exon 4 poly(A) site. This is the first demonstration that an SR protein can influence alternative polyadenylation and suggests that this family of proteins may play a role in recognition of 3′-terminal exons and perhaps in the communication between polyadenylation and splicing.
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Lou, Hua, David M. Helfman, Robert F. Gagel, and Susan M. Berget. "Polypyrimidine Tract-Binding Protein Positively Regulates Inclusion of an Alternative 3′-Terminal Exon." Molecular and Cellular Biology 19, no. 1 (January 1, 1999): 78–85. http://dx.doi.org/10.1128/mcb.19.1.78.
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ABSTRACT Polypyrimidine tract-binding protein (PTB) is an abundant vertebrate hnRNP protein. PTB binding sites have been found within introns both upstream and downstream of alternative exons in a number of genes that are negatively controlled by the binding of PTB. We have previously reported that PTB binds to a pyrimidine tract within an RNA processing enhancer located adjacent to an alternative 3′-terminal exon within the gene coding for calcitonin and calcitonin gene-related peptide. The enhancer consists of a pyrimidine tract and CAG directly abutting on a 5′ splice site sequence to form a pseudoexon. Here we show that the binding of PTB to the enhancer pyrimidine tract is functional in that exon inclusion increases when in vivo levels of PTB increase. This is the first example of positive regulation of exon inclusion by PTB. The binding of PTB was antagonistic to the binding of U2AF to the enhancer-located pyrimidine tract. Altering the enhancer pyrimidine tract to a consensus sequence for the binding of U2AF eliminated enhancement of exon inclusion in vivo and exon polyadenylation in vitro. An additional PTB binding site was identified close to the AAUAAA hexanucleotide sequence of the exon 4 poly(A) site. These observations suggest a dual role for PTB in facilitating recognition of exon 4: binding to the enhancer pyrimidine tract to interrupt productive recognition of the enhancer pseudoexon by splicing factors and interacting with the poly(A) site to positively affect polyadenylation.
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Le Sommer, Caroline, Michelle Lesimple, Agnès Mereau, Severine Menoret, Marie-Rose Allo, and Serge Hardy. "PTB Regulates the Processing of a 3′-Terminal Exon by Repressing both Splicing and Polyadenylation." Molecular and Cellular Biology 25, no. 21 (November 1, 2005): 9595–607. http://dx.doi.org/10.1128/mcb.25.21.9595-9607.2005.
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ABSTRACT The polypyrimidine tract binding protein (PTB) has been described as a global repressor of regulated exons. To investigate PTB functions in a physiological context, we used a combination of morpholino-mediated knockdown and transgenic overexpression strategies in Xenopus laevis embryos. We show that embryonic endoderm and skin deficient in PTB displayed a switch of the α-tropomyosin pre-mRNA 3′ end processing to the somite-specific pattern that results from the utilization of an upstream 3′-terminal exon designed exon 9A9′. Conversely, somitic targeted overexpression of PTB resulted in the repression of the somite-specific exon 9A9′ and a switch towards the nonmuscle pattern. These results validate PTB as a key physiological regulator of the 3′ end processing of the α-tropomyosin pre-mRNA. Moreover, using a minigene strategy in the Xenopus oocyte, we show that in addition to repressing the splicing of exon 9A9′, PTB regulates the cleavage/polyadenylation of this 3′-terminal exon.
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Kim, J., J. J. Yim, S. Wang, and D. Dorsett. "Alternate use of divergent forms of an ancient exon in the fructose-1,6-bisphosphate aldolase gene of Drosophila melanogaster." Molecular and Cellular Biology 12, no. 2 (February 1992): 773–83. http://dx.doi.org/10.1128/mcb.12.2.773-783.1992.
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The fructose-1,6-bisphosphate aldolase gene of Drosophila melanogaster contains three divergent copies of an evolutionarily conserved 3' exon. Two mRNAs encoding aldolase contain three exons and differ only in the poly(A) site. The first exon is small and noncoding. The second encodes the first 332 amino acids, which form the catalytic domain, and is homologous to exons 2 through 8 of vertebrates. The third exon encodes the last 29 amino acids, thought to control substrate specificity, and is homologous to vertebrate exon 9. A third mRNA substitutes a different 3' exon (4a) for exon 3 and encodes a protein very similar to aldolase. A fourth mRNA begins at a different promoter and shares the second exon with the aldolase messages. However, two exons, 3a and 4a, together substitute for exon 3. Like exon 4a, exon 3a is homologous to terminal aldolase exons. The exon 3a-4a junction is such that exon 4a would be translated in a frame different from that which would produce a protein with similarity to aldolase. The putative proteins encoded by the third and fourth mRNAs are likely to be aldolases with altered substrate specificities, illustrating alternate use of duplicated and diverged exons as an evolutionary mechanism for adaptation of enzymatic activities.
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Kim, J., J. J. Yim, S. Wang, and D. Dorsett. "Alternate use of divergent forms of an ancient exon in the fructose-1,6-bisphosphate aldolase gene of Drosophila melanogaster." Molecular and Cellular Biology 12, no. 2 (February 1992): 773–83. http://dx.doi.org/10.1128/mcb.12.2.773.
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The fructose-1,6-bisphosphate aldolase gene of Drosophila melanogaster contains three divergent copies of an evolutionarily conserved 3' exon. Two mRNAs encoding aldolase contain three exons and differ only in the poly(A) site. The first exon is small and noncoding. The second encodes the first 332 amino acids, which form the catalytic domain, and is homologous to exons 2 through 8 of vertebrates. The third exon encodes the last 29 amino acids, thought to control substrate specificity, and is homologous to vertebrate exon 9. A third mRNA substitutes a different 3' exon (4a) for exon 3 and encodes a protein very similar to aldolase. A fourth mRNA begins at a different promoter and shares the second exon with the aldolase messages. However, two exons, 3a and 4a, together substitute for exon 3. Like exon 4a, exon 3a is homologous to terminal aldolase exons. The exon 3a-4a junction is such that exon 4a would be translated in a frame different from that which would produce a protein with similarity to aldolase. The putative proteins encoded by the third and fourth mRNAs are likely to be aldolases with altered substrate specificities, illustrating alternate use of duplicated and diverged exons as an evolutionary mechanism for adaptation of enzymatic activities.
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Parra, Marilyn K., Sherry L. Gee, Mark J. Koury, Narla Mohandas, and John G. Conboy. "Alternative 5′ exons and differential splicing regulate expression of protein 4.1R isoforms with distinct N-termini." Blood 101, no. 10 (May 15, 2003): 4164–71. http://dx.doi.org/10.1182/blood-2002-06-1796.
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Abstract Among the alternative pre-mRNA splicing events that characterize protein 4.1R gene expression, one involving exon 2′ plays a critical role in regulating translation initiation and N-terminal protein structure. Exon 2′ encompasses translation initiation site AUG1 and is located between alternative splice acceptor sites at the 5′ end of exon 2; its inclusion or exclusion from mature 4.1R mRNA regulates expression of longer or shorter isoforms of 4.1R protein, respectively. The current study reports unexpected complexity in the 5′ region of the 4.1R gene that directly affects alternative splicing of exon 2′. Identified far upstream of exon 2 in both mouse and human genomes were 3 mutually exclusive alternative 5′ exons, designated 1A, 1B, and 1C; all 3 are associated with strong transcriptional promoters in the flanking genomic sequence. Importantly, exons 1A and 1B splice differentially with respect to exon 2′, generating transcripts with different 5′ ends and distinct N-terminal protein coding capacity. Exon 1A-type transcripts splice so as to exclude exon 2′ and therefore utilize the downstream AUG2 for translation of 80-kDa 4.1R protein, whereas exon 1B transcripts include exon 2′ and initiate at AUG1 to synthesize 135-kDa isoforms. RNA blot analyses revealed that 1A transcripts increase in abundance in late erythroblasts, consistent with the previously demonstrated up-regulation of 80-kDa 4.1R during terminal erythroid differentiation. Together, these results suggest that synthesis of structurally distinct 4.1R protein isoforms in various cell types is regulated by a novel mechanism requiring coordination between upstream transcription initiation events and downstream alternative splicing events.
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Lou, H., Y. Yang, G. J. Cote, S. M. Berget, and R. F. Gagel. "An intron enhancer containing a 5' splice site sequence in the human calcitonin/calcitonin gene-related peptide gene." Molecular and Cellular Biology 15, no. 12 (December 1995): 7135–42. http://dx.doi.org/10.1128/mcb.15.12.7135.
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Regulation of calcitonin (CT)/calcitonin gene-related peptide (CGRP) RNA processing involves the use of alternative 3' terminal exons. In most tissues and cell lines, the CT terminal exon is recognized. In an attempt to define regulatory sequences involved in the utilization of the CT-specific terminal exon, we performed deletion and mutation analyses of a mini-gene construct that contains the CT terminal exon and mimics the CT processing choice in vivo. These studies identified a 127-nucleotide intron enhancer located approximately 150 nucleotides downstream of the CT exon poly(A) cleavage site that is required for recognition of the exon. The enhancer contains an essential and conserved 5' splice site sequence. Mutation of the splice site resulted in diminished utilization of the CT-specific terminal exon and increased skipping of the CT exon in both the mini-gene and in the natural CT/CGRP gene. Other components of the intron enhancer modified utilization of the CT-specific terminal exon and were necessary to prevent utilization of the 5' splice site within the intron enhancer as an actual splice site directing cryptic splicing. Conservation of the intron enhancer in three mammalian species suggests an important role for this intron element in the regulation of CT/CGRP processing and an expanded role for intronic 5' splice site sequences in the regulation of RNA processing.
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Datson, Nicole A., Geoffrey M. Duyk, Gert-Jan B. van Ommen, and Johan T. Den Dunnen. "Specific isolation of 3′-terminal exons of human genes by exon trapping." Nucleic Acids Research 22, no. 20 (1994): 4148–53. http://dx.doi.org/10.1093/nar/22.20.4148.
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Tabaska, J. E., R. V. Davuluri, and M. Q. Zhang. "Identifying the 3'-terminal exon in human DNA." Bioinformatics 17, no. 7 (July 1, 2001): 602–7. http://dx.doi.org/10.1093/bioinformatics/17.7.602.
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Fedchenko, V. I., and A. A. Kaloshin. "A Simplified Method for Obtaining cDNA of Low-Copy and Silent Eukaryotic Genes Using Human Renalase as an the Example." Biomedical Chemistry: Research and Methods 2, no. 2 (2019): e00101. http://dx.doi.org/10.18097/bmcrm00101.
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A simplified «exon» method was developed for producing cDNA of low-copy and silent eukaryotic genes. It is based on assembly of the target gene from genomic DNA by direct synthesis of its exons, followed by their PCR-based joining without further purification of the amplicons. During the synthesis of exons, direct primers were used; these included about ~ 20 nucleotides of the 3`-terminal sequence previous (from the amplified) exon and ~ 20 nucleotides of the 5`-initial sequence of the amplified exon. Reverse primers included ~ 20 nucleotides complementary to the terminal sequence of the amplified exon. Forward and reverse primers flanking the gene to be assembled included the restriction sites necessary for insertion into the expression vector. Using this approach it is possible to assemble almost any eukaryotic gene with a known nucleotide sequence of genomic DNA available in the database.
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Rani, Abdul Qawee Mahyoob, Tetsushi Yamamoto, Tatsuya Kawaguchi, Kazuhiro Maeta, Hiroyuki Awano, Hisahide Nishio, and Masafumi Matsuo. "Intronic Alternative Polyadenylation in the Middle of the DMD Gene Produces Half-Size N-Terminal Dystrophin with a Potential Implication of ECG Abnormalities of DMD Patients." International Journal of Molecular Sciences 21, no. 10 (May 18, 2020): 3555. http://dx.doi.org/10.3390/ijms21103555.
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The DMD gene is one of the largest human genes, being composed of 79 exons, and encodes dystrophin Dp427m which is deficient in Duchenne muscular dystrophy (DMD). In some DMD patient, however, small size dystrophin reacting with antibody to N-terminal but not to C-terminal has been identified. The mechanism to produce N-terminal small size dystrophin remains unknown. Intronic polyadenylation is a mechanism that produces a transcript with a new 3′ terminal exon and a C-terminal truncated protein. In this study, intronic alternative polyadenylation was disclosed to occur in the middle of the DMD gene and produce the half-size N-terminal dystrophin Dp427m, Dpm234. The 3′-rapid amplification of cDNA ends revealed 421 bp sequence in the downstream of DMD exon 41 in U-251 glioblastoma cells. The cloned sequence composing of the 5′ end sequence of intron 41 was decided as the terminal exon, since it encoded poly (A) signal followed by poly (A) stretch. Subsequently, a fragment from DMD exon M1 to intron 41 was obtained by PCR amplification. This product was named Dpm234 after its molecular weight. However, Dpm234 was not PCR amplified in human skeletal and cardiac muscles. Remarkably, Dpm234 was PCR amplified in iPS-derived cardiomyocytes. Accordingly, Western blotting of cardiomyocyte proteins showed a band of 234 kDa reacting with dystrophin antibody to N-terminal, but not C-terminal. Clinically, DMD patients with mutations in the Dpm234 coding region were found to have a significantly higher likelihood of two ECG abnormal findings. Intronic alternative splicing was first revealed in Dp427m to produce small size dystrophin.
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Seidman, S., M. Sternfeld, R. Ben Aziz-Aloya, R. Timberg, D. Kaufer-Nachum, and H. Soreq. "Synaptic and epidermal accumulations of human acetylcholinesterase are encoded by alternative 3'-terminal exons." Molecular and Cellular Biology 15, no. 6 (June 1995): 2993–3002. http://dx.doi.org/10.1128/mcb.15.6.2993.
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Tissue-specific heterogeneity among mammalian acetylcholinesterases (AChE) has been associated with 3' alternative splicing of the primary AChE gene transcript. We have previously demonstrated that human AChE DNA encoding the brain and muscle AChE form and bearing the 3' exon E6 (ACHE-E6) induces accumulation of catalytically active AChE in myotomes and neuromuscular junctions (NMJs) of 2- and 3-day-old Xenopus embryos. Here, we explore the possibility that the 3'-terminal exons of two alternative human AChE cDNA constructs include evolutionarily conserved tissue-recognizable elements. To this end, DNAs encoding alternative human AChE mRNAs were microinjected into cleaving embryos of Xenopus laevis. In contrast to the myotomal expression demonstrated by ACHE-E6, DNA carrying intron 14 and alternative exon E5 (ACHE-I4/E5) promoted punctuated staining of epidermal cells and secretion of AChE into the external medium. Moreover, ACHE-E6-injected embryos displayed enhanced NMJ development, whereas ACHE-I4/E5-derived enzyme was conspicuously absent from muscles and NMJs and its expression in embryos had no apparent effect on NMJ development. In addition, cell-associated AChE from embryos injected with ACHE-I4/E5 DNA was biochemically distinct from that encoded by the muscle-expressible ACHE-E6, displaying higher electrophoretic mobility and greater solubility in low-salt buffer. These findings suggest that alternative 3'-terminal exons dictate tissue-specific accumulation and a particular biological role(s) of AChE, associate the 3' exon E6 with NMJ development, and indicate the existence of a putative secretory AChE form derived from the alternative I4/E5 AChE mRNA.
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ISHIKAWA, Takahiro, Kazuya YOSHIMURA, Masahiro TAMOI, Toru TAKEDA, and Shigeru SHIGEOKA. "Alternative mRNA splicing of 3′-terminal exons generates ascorbate peroxidase isoenzymes in spinach (Spinacia oleracea) chloroplasts." Biochemical Journal 328, no. 3 (December 15, 1997): 795–800. http://dx.doi.org/10.1042/bj3280795.
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We have isolated two cDNA clones encoding spinach (Spinacia oleracea) stromal and thylakoid-bound ascorbate peroxidase isoenzymes [Ishikawa, Sakai, Yoshimura, Takeda and Shigeoka (1996) FEBS Lett. 384, 289-293]. The gene (ApxII) encoding both chloroplastic ascorbate peroxidase isoenzymes was isolated and the organization of the gene was determined. Alignment between the cDNAs and the gene for chloroplastic ascorbate peroxidase isoenzymes indicates that both enzymes arise from a common pre-mRNA by alternative splicing of two 3ʹ-terminal exons. Genomic Southern-blot analysis supported this finding. The gene spanned nearly 8.5 kbp and contained 13 exons split by 12 introns. The penultimate exon 12 (residues 7376-7530) for the stromal ascorbate peroxidase mRNA consisted of one codon for Asp365 before the TAA termination codon, and the entire 3ʹ-untranslated region, including a potential polyadenylation signal (AATAAA). The final exon 13 (residues 7545-7756) for the thylakoid-bound ascorbate peroxidase mRNA consisted of the corresponding coding sequence of the hydrophobic C-terminal region, the TGA termination codon and the entire 3ʹ-untranslated region, including a potential polyadenylation signal (AATATA). Both exons were interrupted by a 14 bp non-coding sequence. Northern-blot and reverse transcription-PCR analysis showed that the transcripts for stromal and thylakoid-bound ascorbate peroxidase are present in spinach leaves.
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Joiner, Clinton H., Scott Crable, and Patrick G. Gallagher. "Alternative Splicing within Exon 1 of the KCl Cotransporter-3 (KCC3) Gene Results in Novel Transcripts in Erythroid Cells." Blood 108, no. 11 (November 16, 2006): 1564. http://dx.doi.org/10.1182/blood.v108.11.1564.1564.
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Abstract The KCl Cotransporter (KCC) is a key component of the volume regulation system of human reticulocytes, and its excessive activity in sickle cells contributes to cellular dehydration and therefore to sickling pathology. Three of the four KCC genes, including KCC3, are expressed in erythroid cells (Exp.Hematol.2005;33:624), but their relative contribution to KCC fluxes and volume regulation in red cells remains unknown. Heterogenity of the 5′ ends of the KCC3 mRNA transcripts has been described by Mount and colleagues (Mercado et al. Am J Physiol289:F1246, 2005), including two untranslated exons (ex1D and 1C) 1.2 kb upstream from ex1A of KCC3a, which contains the originally described translation initiation site. We used RT-PCR, 5′ RACE (rapid amplification of cDNA ends), and primer extension analyses to study the 5′ ends of KCC3 cDNA transcripts in erythroid cells. The large exon 1 of KCC3 was identified in fetal liver and bone marrow RNA as a 1646 bp region (containing exons 1A, 1C, and 1D described by Mercado et al) that undergoes complex patterns of alternate splicing to generate 5 different transcripts. Three major splicing isoforms are expressed in hematopoietic cell RNA. One isoform incorporates the first 103bp of the exon (exon 1D) as 5′ untranslated sequence which splices to the last 208bp of the exon, including additional 5′ untranslated sequence and an alternative in-frame initiator methionine. The translated protein exhibits a 59 amino acid N-terminal truncation of KCC3a lacking several potential phosphorylation sites (KCC3a-Short of Mercado et al). This transcript was the most abundant isoform in hematopoietic cell RNA. A second major isoform contains the first 668bp of the exon (including both exons 1D and 1C) as 5′ untranslated sequence, then splices out the next 735bp to join the last 243bp of the exon, including additional 5′ untranslated sequence and the alternate initiator methionine of KCC3a-Short. A third novel transcript includes the entire exon, utilizing the first initiator methionine of ‘full-length’ KCC3a. Two other novel transcripts were found, both of which code for KCC3a-Short. These transcripts were also identified in EST databases. We examined the genomic region around exon 1 for promoter activity using luciferase promoter constructs expressed in erythroid K562 cells. Promoter activity was minimal with constructs which spanned from exon 1D to the beginning of exon 1A, but increased substantially in constructs that included this region plus 900 bp 5′ sequence. Reporter activity increased 3 fold upon removal of the 590 bp 3′ segment of such constructs, suggesting the presence of negative regulatory elements within the exon immediately upstream of exon 1A. Thus, KCC3 exhibits a complex pattern of alternative splicing in erythroid cells, producing several novel transcripts, some of which encode an N-terminal truncation of KCC3a. Identifying the factors modulating transcriptional control of KCC3 expression and the functional behavior of this truncated protein in erythroid cells is important to understanding volume regulation in reticulocytes and its abnormalities in sickle cells.
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del Arco, Araceli. "Novel variants of human SCaMC-3, an isoform of the ATP-Mg/Pi mitochondrial carrier, generated by alternative splicing from 3′-flanking transposable elements." Biochemical Journal 389, no. 3 (July 26, 2005): 647–55. http://dx.doi.org/10.1042/bj20050283.
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CaMCs (calcium-dependent mitochondrial carriers) represent a novel subfamily of metabolite carriers of mitochondria. The ATP-Mg/Pi co-transporter, functionally characterized more than 20 years ago, has been identified to be a CaMC member. There are three isoforms of the ATP-Mg/Pi carrier in mammals, SCaMC-1 (short CaMC-1), -2 and -3 (or APC-1, -3 and -2 respectively), corresponding to the genes SLC25A24, SLC25A25 and SLC25A23 respectively, as well as six N-terminal variants generated by alternative splicing for SCaMC-1 and -2 isoforms. In the present study, we describe four new variants of human SCaMC-3 generated by alternative splicing. The new mRNAs use the exon 9 3′-donor site and distinct 5′-acceptor sites from repetitive elements, in regions downstream of exon 10, the last exon in all SCaMCs. Transcripts lacking exon 10 (SCaMC-3b, -3b′, -3c and -3d) code for shortened proteins lacking the last transmembrane domain of 422, 456 and 435 amino acids, and were found in human tissues and HEK-293T cells. Mitochondrial targeting of overexpressed SCaMC-3 variants is incomplete. Surprisingly, the import impairment is overcome by removing the N-terminal extension of these proteins, suggesting that the hydrophilic N-terminal domain also participates in the mitochondrial import process, as shown for the CaMC members aralar and citrin [Roesch, Hynds, Varga, Tranebjaerg and Koehler (2004) Hum. Mol. Genet. 13, 2101–2111].
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Rigo, Frank, and Harold G. Martinson. "Functional Coupling of Last-Intron Splicing and 3′-End Processing to Transcription In Vitro: the Poly(A) Signal Couples to Splicing before Committing to Cleavage." Molecular and Cellular Biology 28, no. 2 (October 29, 2007): 849–62. http://dx.doi.org/10.1128/mcb.01410-07.
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ABSTRACT We have developed an in vitro transcription system, using HeLa nuclear extract, that supports not only efficient splicing of a multiexon transcript but also efficient cleavage and polyadenylation. In this system, both last-intron splicing and cleavage/polyadenylation are functionally coupled to transcription via the tether of nascent RNA that extends from the terminal exon to the transcribing polymerase downstream. Communication between the 3′ splice site and the poly(A) site across the terminal exon is established within minutes of their transcription, and multiple steps leading up to 3′-end processing of this exon can be distinguished. First, the 3′ splice site establishes connections to enhance 3′-end processing, while the nascent 3′-end processing apparatus makes reciprocal functional connections to enhance splicing. Then, commitment to poly(A) site cleavage itself occurs and the connections of the 3′-end processing apparatus to the transcribing polymerase are strengthened. Finally, the chemical steps in the processing of the terminal exon take place, beginning with poly(A) site cleavage, continuing with polyadenylation of the 3′ end, and then finishing with splicing of the last intron.
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Thomas, J. T., C. J. Cresswell, B. Rash, H. Nicolai, T. Jones, E. Solomon, M. E. Grant, and R. P. Boot-Handford. "The human collagen X gene. Complete primary translated sequence and chromosomal localization." Biochemical Journal 280, no. 3 (December 15, 1991): 617–23. http://dx.doi.org/10.1042/bj2800617.
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We report on the complete primary translated sequence of human alpha 1(X) collagen, deduced from a genomic clone, and the chromosomal localization of the human collagen X gene. The primary translated product of human collagen X is encoded by two exons of 169 bp and approx. 2940 bp. The 169 bp exon encodes 15 bp of 5′-end untranslated sequence, 18 amino acid residues (54 bp) of signal peptide and 33 1/3 amino acid residues (100 bp) of the N-terminal non-collagenous domain. The 2940 bp exon encodes 4 2/3 amino acid residues (14 bp) of the N-terminal non-collagenous domain, the complete triple-helical domain of 463 amino acid residues (1389 bp), the complete C-terminal non-collagenous domain of 161 amino acid residues (483 bp) and 1054 bp of 3′-end untranslated sequence up to and including a potential cleavage/polyadenylation signal. The size of the intron separating the two exons, as estimated by partial sequencing and Southern-blot analyses, is approx. 3200 bp. By a combination of somatic cell hybrid screening and hybridization in situ the human collagen X gene (COL10A1) has been assigned to the distal end of the long arm of chromosome 6 at the locus 6q21-6q22.3.
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Duff, J., P. Davies, K. Watt, and I. J. McEwan. "Structural dynamics of the human androgen receptor: implications for prostate cancer and neurodegenerative disease." Biochemical Society Transactions 34, no. 6 (October 25, 2006): 1098–102. http://dx.doi.org/10.1042/bst0341098.
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The AR (androgen receptor) is a ligand-activated transcription factor that mediates the action of the steroids testosterone and dihydrotestosterone. Alterations in the AR gene result in a number of clinical disorders, including: androgen-insensitivity, which leads to disruption of male development; prostate cancer; and a neuromuscular degenerative condition termed spinal bulbar muscular atrophy or Kennedy's disease. The AR gene is X-linked and the protein is coded for by eight exons, giving rise to a C-terminal LBD (ligand-binding domain; exons 4–8), linked by a hinge region (exon 4) to a Zn-finger DBD (DNA-binding domain; exons 2 and 3) and a large structurally distinct NTD (N-terminal domain; exon 1). Identification and characterization of mutations found in prostate cancer and Kennedy's disease patients have revealed the importance of structural dynamics in the mechanisms of action of receptors. Recent results from our laboratory studying genetic changes in the LBD and the structurally flexible NTD will be discussed.
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Haag, F. A., G. Kuhlenbäumer, F. Koch-Nolte, E. Wingender, and H. G. Thiele. "Structure of the gene encoding the rat T cell ecto-ADP-ribosyltransferase RT6." Journal of Immunology 157, no. 5 (September 1, 1996): 2022–30. http://dx.doi.org/10.4049/jimmunol.157.5.2022.
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Abstract Cellular functions, such as the cytolytic potential of CTLs, can be regulated by mono-ADP-ribosylation of target proteins. Recently, the T cell differentiation marker RT6 has been shown to possess mono-ADP-ribosyltransferase activity. Defects in RT6 expression coincide with increased susceptibility in animal models for insulin-dependent diabetes mellitus and other autoimmune diseases. We present an analysis of the rat RT6 gene, providing a basis for studying the regulation of this gene in T cells of normal and diabetes-prone rats. It is the first structural analysis of a mammalian mono-ADP-ribosyltransferase gene. The RT6 gene consists of eight exons spanning approximately 20 kb. The proximal four exons encode 5' untranslated region sequences and are found in multiple alternatively spliced variants. Exon 5 encodes the N-terminal signal sequence. An unusually large exon 7 encodes the entire native polypeptide. The final exon 8 encodes the C-terminal signal sequence for glycosylphosphatidylinositol anchor attachment and the 3' untranslated region. Two independent TATA box-containing promoters associated with exons 1 and 2 were identified, and their activity was verified in transient transfection assays. The distal promoter displays elements contained in the regulatory regions of T cell-specific genes, such as ets and ikaros. Analysis of RT6 transcripts showed that this promoter is the major one in adult rat spleen cells. The 3' end of the gene does not display alternative splicing. However, two polyadenylation signals are found in the 3' untranslated region.
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Enayat, M. Said, Andrea M. Guilliatt, Gurcharan K. Surdhar, P. Vincent Jenkins, K. John Pasi, Cheng Hock Toh, Michael D. Williams, and Frank G. H. Hill. "Aberrant dimerization of von Willebrand factor as the result of mutations in the carboxy-terminal region: identification of 3 mutations in members of 3 different families with type 2A (phenotype IID) von Willebrand disease." Blood 98, no. 3 (August 1, 2001): 674–80. http://dx.doi.org/10.1182/blood.v98.3.674.
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Abstract The 3′ end of the VWF gene was screened in the affected members of 3 different families with type 2A (phenotype IID) von Willebrand disease (vWD). Exons 49 to 52 of the VWF gene were amplified and screened for mutations by chemical cleavage mismatch detection. Mismatched bands were detected in exon 52 of 2 patients and in exon 51 of a third patient. Using direct DNA sequencing, a heterozygous G8562A transition leading to a Cys2008Tyr substitution was found in all the patients in family 1, and a T8561A transversion leading to a Cys2008Ser substitution was found in both patients from family 2. In a patient from a third family, an 8-base deletion from nucleotide 8437 to 8444 was identified in exon 51. The 2 mutations in exon 52 were reproduced by in vitro site-directed mutagenesis of full-length von Willebrand factor (vWF) cDNA and transiently expressed in COS-7 cells. The corresponding recombinant VWFs for these 2 mutations exhibited the typical aberrant vWF:Ag multimer pattern seen in the plasma of the patients. These 3 mutations demonstrate the importance of other carboxy-terminal cysteines in addition to the reported Cys2010 residue, in the normal dimerization of vWF, and their essential role in the assembly of normal multimeric vWF.
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Winardi, R., M. Reid, J. Conboy, and N. Mohandas. "Molecular analysis of glycophorin C deficiency in human erythrocytes." Blood 81, no. 10 (May 15, 1993): 2799–803. http://dx.doi.org/10.1182/blood.v81.10.2799.2799.
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Abstract Human erythrocyte glycophorin C plays a functionally important role in maintaining erythrocyte shape and regulating membrane mechanical stability. We report here the characterization of the glycophorins C and D deficiency in erythrocytes of the Leach phenotype. Glycophorin C gene is encoded by 4 exons. Amplification of reticulocyte cDNA from Leach phenotype and normal individuals generated a 140-bp fragment when using primers spanning exons 1 and 2. However, no polymerase chain reaction (PCR) products were detected in the Leach phenotype using primers flanking either exons 1 and 3 or exons 1 and 4, suggesting that the 3' end of the mRNA was missing or altered. Exon 4 also appeared to be missing from Leach genomic DNA, based on both Southern hybridization and PCR. These results indicate that an absence of glycophorin C and glycophorin D in erythrocytes from these Leach phenotype individuals is a consequence of a deletion or marked alteration of exon 3 and exon 4 of their glycophorin C gene. Surprisingly, the mutant gene encodes an mRNA stable enough to be detected in circulating reticulocytes. Although this mRNA could encode an N-terminal fragment of glycophorin C, these protein isoform(s) would not be expressed in the membrane because they lack the transmembrane and cytoplasmic domains.
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Winardi, R., M. Reid, J. Conboy, and N. Mohandas. "Molecular analysis of glycophorin C deficiency in human erythrocytes." Blood 81, no. 10 (May 15, 1993): 2799–803. http://dx.doi.org/10.1182/blood.v81.10.2799.bloodjournal81102799.
Full textAbstract:
Human erythrocyte glycophorin C plays a functionally important role in maintaining erythrocyte shape and regulating membrane mechanical stability. We report here the characterization of the glycophorins C and D deficiency in erythrocytes of the Leach phenotype. Glycophorin C gene is encoded by 4 exons. Amplification of reticulocyte cDNA from Leach phenotype and normal individuals generated a 140-bp fragment when using primers spanning exons 1 and 2. However, no polymerase chain reaction (PCR) products were detected in the Leach phenotype using primers flanking either exons 1 and 3 or exons 1 and 4, suggesting that the 3' end of the mRNA was missing or altered. Exon 4 also appeared to be missing from Leach genomic DNA, based on both Southern hybridization and PCR. These results indicate that an absence of glycophorin C and glycophorin D in erythrocytes from these Leach phenotype individuals is a consequence of a deletion or marked alteration of exon 3 and exon 4 of their glycophorin C gene. Surprisingly, the mutant gene encodes an mRNA stable enough to be detected in circulating reticulocytes. Although this mRNA could encode an N-terminal fragment of glycophorin C, these protein isoform(s) would not be expressed in the membrane because they lack the transmembrane and cytoplasmic domains.
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Awasthi, Sita, Jennifer A. Isler, and James C. Alwine. "Analysis of Splice Variants of the Immediate-Early 1 Region of Human Cytomegalovirus." Journal of Virology 78, no. 15 (August 1, 2004): 8191–200. http://dx.doi.org/10.1128/jvi.78.15.8191-8200.2004.
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ABSTRACT The major immediate-early (MIE) gene of human cytomegalovirus (HCMV) produces multiple mRNAs through differential splicing and polyadenylation. Reverse transcriptase PCR was used to characterize transcripts from exons 1, 2, 3, and 4 (immediate-early 1 [IE1]). The expected IE72 and IE19 mRNAs were detected, as well as two heretofore-uncharacterized transcripts designated IE17.5 and IE9. The IE72, IE19, and IE17.5 transcripts utilized the same 5′-splice site in exon 3. IE9 utilized a cryptic 5′-splice site within exon 3. The IE19, IE17.5, and IE9 transcripts all used different 3′-splice sites within exon 4. These spliced species occur in infected human foreskin fibroblast (HFF) cells, with accumulation kinetics similar to those of IE72 mRNA. IE19 and IE9 RNAs were much more abundant than IE17.5 RNA. Transfection of CV-1 cells with cDNAs resulted in IE19 and IE17.5 proteins detectable by antibodies to either N-terminal or C-terminal epitopes. No IE9 protein product has been detected. We have not been able to detect IE19, IE17.5, or IE9 proteins during infection of HFF, HEL, or U373MG cells. Failure to detect IE19 protein contrasts with a previous report (M. Shirakata, M. Terauchi, M. Ablikin, K. Imadome, K. Hirai, T. Aso, and Y. Yamanashi, J. Virol. 76:3158-3167, 2002) of IE19 protein expression in HCMV-infected HEL cells. Our analysis suggests that an N-terminal breakdown product of IE72 may be mistaken for IE19. Expression of IE19 or IE17.5 from its respective cDNA results in repression of viral gene expression in infected cells. We speculate that expression of these proteins during infection may be restricted to specific conditions or cell types.
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Amendt, B. A., Z. H. Si, and C. M. Stoltzfus. "Presence of exon splicing silencers within human immunodeficiency virus type 1 tat exon 2 and tat-rev exon 3: evidence for inhibition mediated by cellular factors." Molecular and Cellular Biology 15, no. 8 (August 1995): 4606–15. http://dx.doi.org/10.1128/mcb.15.8.4606.
Full textAbstract:
Human immunodeficiency virus type 1 (HIV-1) pre-mRNA splicing is regulated in order to maintain pools of unspliced and partially spliced viral RNAs as well as the appropriate levels of multiply spliced mRNAs during virus infection. We have previously described an element in tat exon 2 that negatively regulates splicing at the upstream tat 3' splice site 3 (B. A. Amendt, D. Hesslein, L.-J. Chang, and C. M. Stoltzfus, Mol. Cell. Biol. 14:3960-3970, 1994). In this study, we further defined the element to a 20-nucleotide (nt) region which spans the C-terminal vpr and N-terminal tat coding sequences. By analogy with exon splicing enhancer (ESE) elements, we have termed this element an exon splicing silencer (ESS). We show evidence for another negative cis-acting region within tat-rev exon 3 of HIV-1 RNA that has sequence motifs in common with a 20-nt ESS element in tat exon 2. This sequence is juxtaposed to a purine-rich ESE element to form a bipartite element regulating splicing at the upstream tat-rev 3' splice site. Inhibition of the splicing of substrates containing the ESS element in tat exon 2 occurs at an early stage of spliceosome assembly. The inhibition of splicing mediated by the ESS can be specifically abrogated by the addition of competitor RNA. Our results suggest that HIV-1 RNA splicing is regulated by cellular factors that bind to positive and negative cis elements in tat exon 2 and tat-rev exon 3.
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van den Heuvel, R. M., G. J. van Eys, F. C. Ramaekers, W. J. Quax, W. T. Vree Egberts, G. Schaart, H. T. Cuypers, and H. Bloemendal. "Intermediate filament formation after transfection with modified hamster vimentin and desmin genes." Journal of Cell Science 88, no. 4 (November 1, 1987): 475–82. http://dx.doi.org/10.1242/jcs.88.4.475.
Full textAbstract:
Previously we cloned and characterized the hamster intermediate filament genes coding for vimentin and desmin. It was demonstrated that the cloned desmin gene was expressed after gene transfer and that the newly synthesized protein assembles into intermediate filaments. Here we present data on the transfection of modified vimentin and desmin genes onto simian virus 40-transformed hamster lens cells and HeLa cells. Modifications included: (1) removal of exons encoding the desmin COOH-terminal domain; (2) exchange of exons encoding the COOH-terminal domain of vimentin and desmin; and (3) deletion of part of exon I of desmin, coding for the NH2-terminal amino acids 4–148. In transient transfection assays it was shown that the modifications in the COOH region had no detectable effects on the filament forming potential of the encoded proteins as demonstrated with desmin antibodies in the indirect immunofluorescence test. On the other hand, deletion of a considerable part of the first exon of the desmin gene results in a lack of bona fide intermediate filament formation. Immunoblotting with desmin antibodies of cell populations enriched for the transfected modified genes showed that the presence of the modified genes results in the synthesis of the corresponding proteins with the expected molecular weights. From our results we conclude that in vivo: (1) the presence of the COOH terminus is not essential for filament formation; (2) that an exchange of COOH-terminal parts of vimentin and desmin does not prevent assembly into intermediate filaments; and (3) that removal of the NH2 terminus of desmin affects intermediate filament formation.
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Kudrycki, K. E., and G. E. Shull. "Rat kidney band 3 Cl-/HCO3- exchanger mRNA is transcribed from an alternative promoter." American Journal of Physiology-Renal Physiology 264, no. 3 (March 1, 1993): F540—F547. http://dx.doi.org/10.1152/ajprenal.1993.264.3.f540.
Full textAbstract:
We have previously shown that the rat kidney band 3 Cl-/HCO3- exchanger mRNA encodes an NH2-terminal truncated form of band 3 and that its 5' end differs from that of the erythrocyte band 3 mRNA (K. E. Kudrycki and G. E. Shull. J. Biol. Chem. 264: 8185-8192, 1989). To determine the genetic basis for the alternative kidney and erythroid mRNAs, we 1) isolated and characterized a rat erythroid band 3 cDNA, 2) isolated the rat band 3 gene and determined the exon/intron organization of sequences corresponding to the alternative 5' ends of the rat kidney and erythroid mRNAs, and 3) identified the transcription initiation sites of the two transcripts. The unique sequences at the 5' end of the rat erythroid mRNA are derived from exons 1-3 and are followed directly by sequences from exon 4 that are common to both mRNAs. In the kidney mRNA, sequences upstream of exon 4 are derived entirely from intron 3. Primer extension and S1 nuclease protection analyses demonstrate the presence of multiple transcription initiation sites for the rat erythroid band 3 mRNA at the beginning of exon 1, whereas the transcription initiation site for the kidney mRNA is located within intron 3. Thus two distinct promoters, separated by almost 5 kb of genomic sequence, are responsible for the highly tissue-specific transcription of the alternative rat erythroid and kidney band 3 mRNAs.
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Markova, T. V., E. L. Dadali, S. S. Nikitin, A. F. Murtazina, O. L. Mironovich, and I. V. Kanivets. "Clinical and genetic characteristics of distal arthrogryposis caused by mutations in the PIEZO2 gene." Neuromuscular Diseases 11, no. 2 (September 13, 2021): 48–55. http://dx.doi.org/10.17650/2222-8721-2021-11-2-48-55.
Full textAbstract:
Mutations in the PIEZO2 gene, which is involved in the formation of the mechanosensitive cation channel Piezo2, can cause distal arthrogryposis type 3 (Gordon’s syndrome), type 5, and Marden–Walker syndrome. Clinical and genetic characteristics of two patients with distal arthrogryposis with autosomal dominant inheritance and one with autosomal recessive inheritance are presented. Exome sequencing in one case revealed a de novo mutation in exon 52 of the PIEZO2gene c.8238G>A (p.Trp2746*, NM_022068.3), in the second, a known deletion of three nucleotides in exon 52 of the PIEZO2 gene c.8181_8183delAGA (p Glu2727del, NM_022068.3) was found, in the third, two mutations in the compound heterozygous state – a deletion of four nucleotides leading to a shift in the reading frame in c.1863_1866delTCAG(p.Ser621fs, NM_022068) and a deletion with putative coordinates 10785050–10789339 bp, spanning 15–16 exons of the PIEZO2 gene (NM_022068; LOD 2.40). The third patient was found to have two newly detected mutations in the compound heterozygous state – a deletion of four nucleotides, leading to a shift in the reading frame in exon 14, p.1863_1866delTCAG (p.Ser621fs, NM_022068) and a deletion with assumed coordinates 10785050–10789339 b. o., (NM_022068; LOD 2.40), spanning 15–16 exons of the PIEZO2 gene. The previous assumption was confirmed that heterozygous mutations are more often localized in exon 52 of the PIEZO2 gene and disrupt the amino acid sequence of the C‑terminal region of the protein molecule, while in patients with an autosomal recessive mode of inheritance of the mutation, the N‑terminal region is more often found.
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Shen-Ong, G. L., R. M. Skurla, J. D. Owens, and J. F. Mushinski. "Alternative splicing of RNAs transcribed from the human c-myb gene." Molecular and Cellular Biology 10, no. 6 (June 1990): 2715–22. http://dx.doi.org/10.1128/mcb.10.6.2715-2722.1990.
Full textAbstract:
An alternative splicing event in which a portion of the intron bounded by the vE6 and vE7 exons with v-myb homology is included as an additional 363-nucleotide coding exon (termed E6A or coding exon 9A) has been described for normal and tumor murine cells that express myb. We show here that this alternative splicing event is conserved in human c-myb transcripts. In addition, another novel exon (termed E7A or coding exon 10A) is identified in human c-myb mRNAs expressed in normal and tumor cells. Although the myb protein isoform encoded by murine E6A-containing mRNA is larger than the major c-myb protein, the predicted products of both forms of human alternatively spliced myb transcripts are 3'-truncated myb proteins that terminate in the alternative exons. These proteins are predicted to lack the same carboxy-terminal domains as the viral myb proteins encoded by avian myeloblastosis virus and E26 virus. The junction sequences that flank these exons closely resemble the consensus splice donor and splice acceptor sequences, yet the alternative transcripts are less abundant than is the major form of c-myb transcripts. The contribution that alternative splicing events in c-myb expression may make on c-myb function remains to be elucidated.
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Shen-Ong, G. L., R. M. Skurla, J. D. Owens, and J. F. Mushinski. "Alternative splicing of RNAs transcribed from the human c-myb gene." Molecular and Cellular Biology 10, no. 6 (June 1990): 2715–22. http://dx.doi.org/10.1128/mcb.10.6.2715.
Full textAbstract:
An alternative splicing event in which a portion of the intron bounded by the vE6 and vE7 exons with v-myb homology is included as an additional 363-nucleotide coding exon (termed E6A or coding exon 9A) has been described for normal and tumor murine cells that express myb. We show here that this alternative splicing event is conserved in human c-myb transcripts. In addition, another novel exon (termed E7A or coding exon 10A) is identified in human c-myb mRNAs expressed in normal and tumor cells. Although the myb protein isoform encoded by murine E6A-containing mRNA is larger than the major c-myb protein, the predicted products of both forms of human alternatively spliced myb transcripts are 3'-truncated myb proteins that terminate in the alternative exons. These proteins are predicted to lack the same carboxy-terminal domains as the viral myb proteins encoded by avian myeloblastosis virus and E26 virus. The junction sequences that flank these exons closely resemble the consensus splice donor and splice acceptor sequences, yet the alternative transcripts are less abundant than is the major form of c-myb transcripts. The contribution that alternative splicing events in c-myb expression may make on c-myb function remains to be elucidated.
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Bloemink, Marieke J., Karen H. Hsu, Michael A. Geeves, and Sanford I. Bernstein. "Alternative N-terminal regions of Drosophila myosin heavy chain II regulate communication of the purine binding loop with the essential light chain." Journal of Biological Chemistry 295, no. 42 (August 19, 2020): 14522–35. http://dx.doi.org/10.1074/jbc.ra120.014684.
Full textAbstract:
We investigated the biochemical and biophysical properties of one of the four alternative exon-encoded regions within the Drosophila myosin catalytic domain. This region is encoded by alternative exons 3a and 3b and includes part of the N-terminal β-barrel. Chimeric myosin constructs (IFI-3a and EMB-3b) were generated by exchanging the exon 3–encoded areas between native slow embryonic body wall (EMB) and fast indirect flight muscle myosin isoforms (IFI). We found that this exchange alters the kinetic properties of the myosin S1 head. The ADP release rate (k-D) in the absence of actin is completely reversed for each chimera compared with the native isoforms. Steady-state data also suggest a reciprocal shift, with basal and actin-activated ATPase activity of IFI-3a showing reduced values compared with wild-type (WT) IFI, whereas for EMB-3b these values are increased compared with wild-type (WT) EMB. In the presence of actin, ADP affinity (KAD) is unchanged for IFI-3a, compared with IFI, but ADP affinity for EMB-3b is increased, compared with EMB, and shifted toward IFI values. ATP-induced dissociation of acto-S1 (K1k+2) is reduced for both exon 3 chimeras. Homology modeling, combined with a recently reported crystal structure for Drosophila EMB, indicates that the exon 3–encoded region in the myosin head is part of the communication pathway between the nucleotide binding pocket (purine binding loop) and the essential light chain, emphasizing an important role for this variable N-terminal domain in regulating actomyosin crossbridge kinetics, in particular with respect to the force-sensing properties of myosin isoforms.
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Duthie, S. M., P. L. Taylor, and K. A. Eidne. "Characterization of the mouse thyrotrophin-releasing hormone receptor gene: an exon corresponds to a deletion in the rat cDNA." Journal of Molecular Endocrinology 11, no. 2 (October 1993): 141–49. http://dx.doi.org/10.1677/jme.0.0110141.
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ABSTRACT The cloning and characterization of the mouse TRH receptor (TRH-R) gene revealed an untranslated exon (exon 1), a single intron and an upstream dinucleotide repeat sequence (d(TG)16.d(AG)21) in the 5′ untranslated region (UTR). The coding region was contained almost entirely on a second exon (exon 2), with the final amino acid and stop codon at the COOH terminus of the gene encoded by a third exon (exon 3) flanked by two introns. The 3′ UTR was contained on the remainder of exon 3 and on the final exon (exon 4). Exon 3 (228 bp) corresponds exactly to a 228 bp deletion that exists in the rat TRH-R cDNA, but not in the mouse cDNA. The mouse TRH-R cDNA encodes a protein of 393 amino acids which is 96% homologous to the rat TRH-R protein of 412 amino acids, but is 19 amino acids shorter at its COOH terminus. The coding sequence for these 19 amino acids (plus 1 extra amino acid) does exist in the mouse TRH-R gene, but the sequence is encoded by exon 4, separated from the rest of the coding region by the stop codon and 223 bp of 3′ UTR on exon 3. Splicing of exon 3 in the mouse TRH-R gene would remove the last amino acid, the stop codon and the 223 bp of 3′ UTR, allowing transcription to continue into the 3′ UTR on exon 4, which encodes the 19 extra amino acids found in the rat cDNA. This would then result in an alternative 412 amino acid version of the mouse TRH-R protein, with 95% homology to the rat TRH-R. This study focused on the structural differences in the intracellular COOH-terminal tail of the receptor, which is known to be a functionally important domain in other members of the G protein-coupled receptor family. We have also recently characterized the human TRH-R cDNA, which revealed a third variant at the COOH terminus. Comparisons between mouse, rat and human TRH-Rs show that the amino acid sequences are virtually identical. However, significant differences between these species exist at the COOH terminus, with each TRH-R having a unique form of the COOH-terminal tail, beginning at exactly the same site and encoding 1, 20 and 6 amino acids in the mouse, rat and human respectively.
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LORENTSEN, Rikke H., Jonas H. GRAVERSEN, Nigel R. CATERER, Hans C. THØGERSEN, and Michael ETZERODT. "The heparin-binding site in tetranectin is located in the N-terminal region and binding does not involve the carbohydrate recognition domain." Biochemical Journal 347, no. 1 (March 27, 2000): 83–87. http://dx.doi.org/10.1042/bj3470083.
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Tetranectin is a homotrimeric plasma and extracellular-matrix protein that binds plasminogen and complex sulphated polysaccharides including heparin. In terms of primary and tertiary structure, tetranectin is related to the collectin family of Ca2+-binding C-type lectins. Tetranectin is encoded in three exons. Exon 3 encodes the carbohydrate recognition domain, which binds to kringle 4 in plasminogen at low levels of Ca2+. Exon 2 encodes an α-helix, which is necessary and sufficient to govern the trimerization of tetranectin by assembling into a triple-helical coiled-coil structural element. Here we show that the heparin-binding site in tetranectin resides not in the carbohydrate recognition domain but within the N-terminal region, comprising the 16 amino acid residues encoded by exon 1. In particular, the lysine residues in the decapeptide segment KPKKIVNAKK (tetranectin residues 6-15) are shown to be of primary importance in heparin binding.
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Hui, YF, SY Chan, and YL Lau. "Identification of mutations in seven Chinese patients with X-linked chronic granulomatous disease." Blood 88, no. 10 (November 15, 1996): 4021–28. http://dx.doi.org/10.1182/blood.v88.10.4021.bloodjournal88104021.
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X-linked chronic granulomatous disease (CGD) is due to mutations in the gp91phox gene on Xp21.1. Studies in white and Japanese X-linked CGD patients have shown mutations in nearly every exon. We studied the molecular defect of seven Chinese patients with X-linked CGD from six unrelated families. Mutations were located by single-strand conformation polymorphism and then defined by sequence analysis. The mutations were two different amino acid substitutions, a nonsense mutation, an in-frame trinucleotide deletion, a single A insertion causing a frameshift, and a premature stop. Lastly, a rare splice site mutation caused by G to A transition at the terminal nucleotide of exon 3, resulting in the skipping of exon 3, was found. The possible effects of these mutations on protein structure-function or splicing were discussed. Together with previous reports, the A insertion in the run of six As from nucleotide 749 to 754 and the G to A transition at the terminal position of exon 3 may be mutation hotspots of the gp91phox gene. The extreme heterogeneous mutations found in our patients suggest the absence of ethnic group-specific mutation.
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JIANG, Weiping, and Dwijendra GUPTA. "Structure of the carbonic anhydrase VI (CA6) gene: evidence for two distinct groups within the α-CA gene family." Biochemical Journal 344, no. 2 (November 24, 1999): 385–90. http://dx.doi.org/10.1042/bj3440385.
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The secreted carbonic anhydrase (CA VI) is believed to be one of the oldest mammalian CAs in evolutionary terms. To elucidate its gene structure and compare it with other members of the α-CA family, we cloned genomic fragments encoding the bovine CA6 gene and determined its exon/intron organization. The gene spans approx. 25 kb and consists of eight exons and seven introns. Exon 1 encodes the 5′ untranslated region, the signal peptide and the N-terminus of the mature enzyme. Exon 8 encodes the 3′ untranslated region and the C-terminal extension that is unique to CA VI. Exons 2-7 encode the CA domain, which shows significant sequence similarity to other CAs. Two distinct groups exist in the α-CA family on the basis of a comparison of the known gene structures. One group consists of the cytoplasmic (CA I, II, III and VII) and mitochondrial (CA V) members. The other group consists of the membrane-bound (CA IV and IX) and secreted (CA VI) members. In particular, the seven exon/intron boundaries in the CA domain of the CA6 gene are conserved in the CA9 gene, which encodes the multidomain protein CA IX that is overexpressed in tumours and has transforming potential.
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Yu, Lung-Chih, Yuh-Ching Twu, Ming-Lun Chou, Ching-Yi Chang, Chia-Ying Wu, and Marie Lin. "Molecular genetic analysis for the B3allele." Blood 100, no. 4 (August 15, 2002): 1490–92. http://dx.doi.org/10.1182/blood-2002-01-0188.
Full textAbstract:
Molecular genetic analysis of 14 samples from unrelated individuals with the B3 phenotype is reported here. Two different molecular changes in the blood group B gene were observed. One case was demonstrated to possess a 247G → T mutation, which predicts an Asp83Tyr alteration. The B genes of the other 13 cases were shown to have a G → A mutation at the +5 nucleotide of intron 3 (intervening sequence 3 [IVS3] + 5G → A). Reverse transcription polymerase chain reaction analysis showed that the complete exon 1–exon 7 B transcript was absent, and transcripts that skipped exon 3 were instead present in the RNA sample from the B3 individual with the IVS3 + 5G → A mutation. The result shows that the IVS3 + 5G → A mutation destroys the conserved sequence of the splice donor site and leads to the skipping of exon 3 during messenger RNA processing. TheB3 transcript without exon 3 predicts a B-transferase product that lacks 19 amino acids in the N-terminal segment.
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Green, C. J., R. S. Charles, B. F. Edwards, and P. H. Johnson. "Identification and characterization of PF4varl, a human gene variant of platelet factor 4." Molecular and Cellular Biology 9, no. 4 (April 1989): 1445–51. http://dx.doi.org/10.1128/mcb.9.4.1445-1451.1989.
Full textAbstract:
A synthetic DNA probe designed to detect coding sequences for platelet factor 4 and connective tissue-activating peptide III (two human platelet alpha-granule proteins) was used to identify several similar sequences in total human DNA. Sequence analysis of a corresponding 3,201-base-pair EcoRI fragment isolated from a human genomic library demonstrated the existence of a variant of platelet factor 4, designated PF4var1. The gene for PF4var1 consisted of three exons and two introns. Exon 1 coded for a 34-amino-acid hydrophobic leader sequence that had 70% sequence homology with the leader sequence for PF4 but, in contrast, contained a hydrophilic amino-terminal region with four arginine residues. Exon 2 coded for a 42-amino-acid segment that was 100% identical with the corresponding segment of the mature PF4 sequence containing the amino-terminal and disulfide-bonded core regions. Exon 3 coded for the 28-residue carboxy-terminal region corresponding to a domain specifying heparin-binding and cellular chemotaxis. However, PF4var1 had amino acid differences at three positions in the lysine-rich carboxy-terminal end that were all conserved among human, bovine, and rat PF4s. These differences should significantly affect the secondary structure and heparin-binding properties of the protein based on considerations of the bovine PF4 crystal structure. By comparing the PF4var1 genomic sequence with the known human cDNA and the rat genomic PF4-coding sequences, we identified potential genetic regulatory regions for PF4var1. Rat PF4 and human PF4var1 genes had identical 18-base sequences 5' to the promoter region. The intron positions appeared to correspond approximately to the boundaries of the protein functional domains.
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Green, C. J., R. S. Charles, B. F. Edwards, and P. H. Johnson. "Identification and characterization of PF4varl, a human gene variant of platelet factor 4." Molecular and Cellular Biology 9, no. 4 (April 1989): 1445–51. http://dx.doi.org/10.1128/mcb.9.4.1445.
Full textAbstract:
A synthetic DNA probe designed to detect coding sequences for platelet factor 4 and connective tissue-activating peptide III (two human platelet alpha-granule proteins) was used to identify several similar sequences in total human DNA. Sequence analysis of a corresponding 3,201-base-pair EcoRI fragment isolated from a human genomic library demonstrated the existence of a variant of platelet factor 4, designated PF4var1. The gene for PF4var1 consisted of three exons and two introns. Exon 1 coded for a 34-amino-acid hydrophobic leader sequence that had 70% sequence homology with the leader sequence for PF4 but, in contrast, contained a hydrophilic amino-terminal region with four arginine residues. Exon 2 coded for a 42-amino-acid segment that was 100% identical with the corresponding segment of the mature PF4 sequence containing the amino-terminal and disulfide-bonded core regions. Exon 3 coded for the 28-residue carboxy-terminal region corresponding to a domain specifying heparin-binding and cellular chemotaxis. However, PF4var1 had amino acid differences at three positions in the lysine-rich carboxy-terminal end that were all conserved among human, bovine, and rat PF4s. These differences should significantly affect the secondary structure and heparin-binding properties of the protein based on considerations of the bovine PF4 crystal structure. By comparing the PF4var1 genomic sequence with the known human cDNA and the rat genomic PF4-coding sequences, we identified potential genetic regulatory regions for PF4var1. Rat PF4 and human PF4var1 genes had identical 18-base sequences 5' to the promoter region. The intron positions appeared to correspond approximately to the boundaries of the protein functional domains.
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Scholl, Amanda, Alexander Muselman, and Dong-Er Zhang. "An Intronic Suppressor Element Regulates RUNX1 Alternative Polyadenylation." Blood 126, no. 23 (December 3, 2015): 3578. http://dx.doi.org/10.1182/blood.v126.23.3578.3578.
Full textAbstract:
Abstract Polyadenylation is a post-transcriptional modification where the 3' end of an mRNA is cleaved and 250-300 adenines are added. It is predicted that 70-75% of human genes have more than one polyadenylation sequence (PAS) and are subject to alternative polyadenylation (APA). APA events affect the coding sequence of a gene when a proximal PAS is located within an intron, constitutive exon, or alternative exon. Gene expression is also affected if there are multiple PAS within the distal 3' untranslated region (UTR); proximal PAS usage shortens the 3'UTR, which can remove cis-regulatory regions such as miRNA and RNA-binding protein (RBP) sites. Furthermore, global changes in APA are linked to cellular state-proximal PAS usage is associated with immature developmental phases, cell proliferation, and cancerous phenotypes. Consequently, APA is a pertinent post-transcriptional modification that regulates gene expression and isoform generation across developmental stages and tissue types. Despite its significance, there are few APA studies in the hematology field, and those that exist have focused on global shifts in PAS usage. In this study, we uniquely focus on the APA mechanism of a single gene, RUNX1, and how this event can alter hematopoietic stem cell (HSC) homeostasis and hematopoiesis. There are three main isoforms of RUNX1 that differ in promoter and/or PAS usage. RUNX1b/c use different promoters, but have identical C-terminal regions. RUNX1a utilizes the same promoter as RUNX1b, but differs from both RUNX1b/c due to usage of a proximal PAS located in alternative exon 7a. RUNX1b/c are robustly expressed in most progenitor populations and differentiated blood cell lineages, whereas RUNX1a is restricted to human CD34+ HSCs. Functionally, RUNX1b/c promote HSC differentiation and lineage commitment, whereas RUNX1a expands HSCs and their engraftment potential, a property with therapeutic advantages but leukemic potential. Due to the difference in expression pattern and distinct functionality of RUNX1a compared to RUNX1b/c, it is relevant to study the APA event that dictates isoform generation. Elucidating this mechanism could provide valuable insight into the transient control of the HSC population for therapeutic benefit and illuminate new leukemogenic pathways. To study RUNX1 APA, we cloned alternative terminal exon 7a (RUNX1a) and constitutive exon 7b (RUNX1b/c) in between the two exons of a split GFP minigene reporter, along with 500 bp of their upstream and downstream flanking introns. We hypothesized that exon 7a would be skipped during processing of the minigene construct because the proximal PAS is rarely used in vivo. Conversely, exon 7b, the penultimate exon in RUNX1b/c, would be spliced in between the GFP exons, disrupting the GFP protein. These constructs were tested in KG-1a and U937 cells. Flow cytometry for GFP fluorescence supported our hypothesis as the exon 7a minigene produced a robust GFP signal and the exon 7b minigene produced no GFP signal. We confirmed that the GFP changes were due to the hypothesized mRNA processing events by performing RT-PCR using primers specific to the two GFP exons. These data show that important cis-regulatory elements that determine RUNX1 APA are located within exon 7a, 7b, and the cloned intronic regions. Next, we altered these minigenes by strategically making chimeric constructs that consist of either exon 7a or 7b with all combinations of upstream/downstream flanking introns. We discovered that replacing the intron upstream of exon 7a confers 2-5 fold greater splicing and polyadenylation of exon 7a, indicative of RUNX1a isoform generation. Therefore, a suppressor cis-element is located in this upstream intronic region. However, placing this intron upstream of exon 7b is not sufficient to reduce its inclusion between the GFP exons. Instead, both the upstream and downstream intronic regions flanking exon 7a are required. This suggests an RNA-looping mechanism that prevents splicing and usage of the exon 7a proximal PAS. Cleavage factor (CFIm) and Polypyrimidine-tract binding protein 1 (PTBP1) are RBPs involved in splicing and polyadenylation that alter mRNA processing by RNA-looping. We aim to narrow down the suppressor region upstream of exon 7a to identify a consensus sequence and the respective RBP that diminishes RUNX1 proximal PAS usage. This knowledge can be leveraged to enhance RUNX1a production and expand HSCs for therapeutic benefit. Disclosures No relevant conflicts of interest to declare.
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Sellers, V. M., T. A. Dailey, and H. A. Dailey. "Examination of Ferrochelatase Mutations That Cause Erythropoietic Protoporphyria." Blood 91, no. 10 (May 15, 1998): 3980–85. http://dx.doi.org/10.1182/blood.v91.10.3980.3980_3980_3985.
Full textAbstract:
Ferrochelatase (E.C. 4.99.1.1), the enzyme that catalyzes the terminal step in the heme biosynthetic pathway, is the site of defect in the human inherited disease erythropoietic protoporphyria (EPP). Previously it has been demonstrated that patients with EPP may have missense mutations leading to amino acid substitutions, early chain termination, or exon deletions. While it has been clearly demonstrated that two missense mutations result in lowered enzyme activity, it has never been shown what effect specific exon deletions may have. In the current work, recombinant human ferrochelatase has been engineered to have individual exon deletions corresponding to exons 3 through 11. When expressed in Escherichia coli, none of these possesses significant enzyme activity and all lack the [2Fe-2S] cluster. One of the human missense mutations, F417S, and a series of amino acid replacements at this site (ie, F417W, F417Y, and F417L) were examined. With the exception of F417L, all lacked enzyme activity and did not contain the [2Fe-2S] cluster in vivo or as isolated in vitro.
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Mercado, Adriana, Norma Vázquez, Luyan Song, Rosa Cortés, Alissa H. Enck, Rick Welch, Eric Delpire, Gerardo Gamba, and David B. Mount. "NH2-terminal heterogeneity in the KCC3 K+-Cl− cotransporter." American Journal of Physiology-Renal Physiology 289, no. 6 (December 2005): F1246—F1261. http://dx.doi.org/10.1152/ajprenal.00464.2004.
Full textAbstract:
The SLC12A6 gene encoding the K+-Cl− cotransporter KCC3 is expressed in multiple tissues, including kidney. Here, we report the molecular characterization of several NH2-terminal isoforms of human and mouse KCC3, along with intrarenal localization and functional characterization in Xenopus laevis oocytes. Two major isoforms, KCC3a and KCC3b, are generated by transcriptional initiation 5′ of two distinct first coding exons. Northern blot analysis of mouse tissues indicates that KCC3b expression is particularly robust in the kidney, which also expresses KCC3a. Western blotting of mouse tissue using an exon 3-specific antibody reveals that the kidney is also unique in expressing immunoreactive protein of a lower mass, suggestive evidence that the shorter KCC3b protein predominates in kidney. Immunofluorescence reveals basolateral expression of KCC3 protein along the entire length of the proximal tubule, in both the mouse and rat. Removal of the 15-residue exon 2 by alternative splicing generates the KCC3a-x2M and KCC3b-x2M isoforms; other splicing events at an alternative acceptor site within exon 1a generate the KCC3a-S isoform, which is 60 residues shorter than KCC3a. This variation in sequence of NH2-terminal cytoplasmic domains occurs proximal to a stretch of highly conserved residues and affects the content of putative phosphorylation sites. Kinetic characterization of KCC3a in X. laevis oocytes reveals apparent Kms for Rb+ and Cl− of 10.7 ± 2.5 and 7.3 ± 1.2 mM, respectively, with an anion selectivity of Br− > Cl− > PO4 = I− = SCN− = gluconate. All five NH2-terminal isoforms are activated by cell swelling (hypotonic conditions), with no activity under isotonic conditions. Although the isoforms do not differ in the osmotic set point of swelling activation, this activation is more rapid for the KCC3a-x2M and KCC3a-S proteins. In summary, there is significant NH2-terminal heterogeneity of KCC3, with particularly robust expression of KCC3b in the kidney. Basolateral swelling-activated K+-Cl− cotransport mediated by KCC3 likely functions in cell volume regulation during the transepithelial transport of both salt and solutes by the proximal tubule.
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van Zalen, Sebastiaan, Grace R. Jeschke, Elizabeth Hexner, and J. Eric Russell. "Cytoplasmic Regulators of β-Globin mRNA Are Structurally Modified During Erythroid Terminal Differentiation." Blood 118, no. 21 (November 18, 2011): 1093. http://dx.doi.org/10.1182/blood.v118.21.1093.1093.
Full textAbstract:
Abstract Abstract 1093 The high-level accumulation of β globin in mature erythrocytes requires a correspondingly high level of its encoding mRNA in terminally differentiating erythroid progenitors. We recently identified two RNA-binding proteins–AUF1 and YB1–that appear to regulate levels of β-globin mRNA in these cells by assembling a cytoplasm-restricted RNA-protein 'β-complex' on its 3'UTR. The function of the β-complex was predicted by in vitro analyses mapping it to a cis-acting determinant of β-globin mRNA stability, and subsequently validated by in vivo siRNA studies demonstrating that simultaneous knockdown of AUF1 and YB1 ablated the β-complex and coordinately reduced the accumulation of β-globin mRNA in K562 cells. Although both AUF1 and YB1 are ubiquitously expressed, studies in cultured cells and in Epo-induced CD34+ primary cells indicated that their β-globin mRNA-specific regulatory properties are restricted to erythroid cells during later stages of terminal differentiation. Based upon these observations, we reasoned that AUF1 and YB1 undergo erythroid and differentiation stage-restricted alterations that permit their assembly into the mRNA-regulatory β-complex. Our analyses of AUF1 focused on three structural isoforms, observed in K562 cells, resulting from alternative pre-mRNA processing events that retain or exclude exon 7. GST-AUF1 fusion isoforms that retain exon 7 fail to bind the β-globin 3'UTR in vitro, while related isoforms that exclude exon 7 bind the 3'UTR with high efficiency. These results, which implicate the importance of exon 7 exclusion to AUF1 function, were subsequently validated in intact K562 cells using an AUF1 isotype-specific siRNA knockdown strategy. In these in vivo experiments, a reduction in exon 7-excluded AUF1 effected a two-fold decrease in steady-state β-globin mRNA, while similar reductions in exon 7-retained AUF1 isoforms had no measurable effect. The isotype-specific mRNA-binding characteristics of AUF1 may be particularly important during terminal differentiation: Epo-induced CD34+ cells display an increase in exon 7-excluded AUF1, paralleling their capacity to assemble a regulatory β-complex in vitro. Among several possible mechanisms, we asked whether the isoform-specific function of AUF1 might relate to the unusually high number (20) of phosphorylation-capable residues encoded by exon 7. In vitro analyses were fully consistent with this possibility, demonstrating that the β-globin mRNA-binding activity of exon 7-retained AUF1 could be restored by prior dephosphorylation. These experiments suggest that post-transcriptional regulation of β-globin mRNA during erythroid differentiation is likely to be effected by alternative splicing of AUF1 pre-mRNA that eliminates phosphorylation-active exon 7 amino-acids in the translated protein. Based upon these results, we reasoned that related post-translational processes might similarly regulate the β-globin mRNA-binding specificity of YB1. Our analyses focused on a specific residue (Ser102) that is a known target for regulatory phosphorylation and can be experimentally identified using a Ser102 phospho-specific YB1 antibody. In in vitro studies with K562 cytoplasmic extract, which contains both phospho- and dephospho- forms of YB1, we observed that only dephospho-YB1 adheres to the β-globin 3'UTR; likewise, in in vivo studies of CD34+ cells we noted a substantial increase in the ratio of dephospho:phospho-YB1 following Epo induction. Both experiments indicate the likely importance of this post-translational process to the function of YB1 during terminal differentiation. Confirmatory studies are currently being conducted in vivo using an epitope-tagged YB1 containing a position 102 Ser->Ala substitution. Collectively, our analyses indicate that the β-globin mRNA-binding specificities of AUF1 and YB1–and, hence, their corresponding regulatory activities–are determined by post-transcriptional and -translational events. This work suggests mechanisms through which erythroid progenitors can maintain dynamic regulatory control during an interval when transcriptional processes are beginning to silence, and identifies new pathways that can be therapeutically targeted in patients with congenital disorders of β-globin gene expression. Disclosures: No relevant conflicts of interest to declare.
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Parra, Marilyn, Jeff Tan, Narla Mohandas, and John G. Conboy. "Mechanisms That Link Promoter Choice with Downstream Alternative Splicing in the Erythroid Protein 4.1R Gene." Blood 108, no. 11 (November 16, 2006): 1562. http://dx.doi.org/10.1182/blood.v108.11.1562.1562.
Full textAbstract:
Abstract The protein 4.1R gene is a large transcription unit (240kb) that utilizes complex RNA processing pathways to encode distinct protein isoforms, both during erythropoiesis and also in nonerythroid cells. Proper regulation of these pathways is essential for stage-specific synthesis of the 80-kDa isoforms of 4.1R protein during terminal erythroid differentiation. The 5′ region of the gene contains multiple alternative first exons that map far upstream of the coding exons, and we have shown previously that promoter choice is coupled to alternative splicing decisions 100kb downstream in exon 2′/2. Transcripts that initiate at exon 1A predominate in late stages of erythropoiesis and splice only to a weak internal 3′ splice acceptor site in exon 2, skipping translation start site AUG1 and ensuring proper translation initiation at AUG2 in exon 4 for synthesis of the 80-kDa isoforms. In contrast, 4.1 transcripts initiated at exons 1B or 1C exclusively splice to the strong first 3′ splice acceptor site at exon 2′ to include AUG1 and encode a higher molecular weight 135-kDa isoform known to interact with different affinity to major erythroid membrane proteins in earlier stages of erythropoiesis. Our studies show that this linkage between transcription and splicing is (a) cell type independent; (b) conserved in the 4.1R gene from fish to man; and (c) conserved in the paralogous 4.1B gene. Our recent functional studies suggest that a novel re-splicing mechanism, reminiscent of recursive splicing of large introns previously described in the Drosophila ubx gene, may couple promoter choice with downstream splicing in the 4.1R gene. Using minigenes that reproduce the differential splicing patterns in transfected mammalian cells, we have shown that accurate splicing of exon 1A requires a unique downstream regulatory element. This element maps several kilobases downstream of exon 1A and is conserved among mammals. Analysis of wild type and mutated minigenes suggests a two step splicing model in which this element behaves as a temporary “intra-exon” that is present in a splicing intermediate but eliminated from the mature mRNA. According to this model, the regulatory element behaves as an exon in the first step as its consensus 5′ donor site splices to the strong 3′ splice site of exon 2′, removing this splice site pair and joining the intra-exon directly to exon 2′. In the second step, the juxtaposed region of the intra-exon then behaves as an intron, contributing to the activation of the weak internal splice acceptor at exon 2. This second splicing event joins exon 1A to exon 2, thus deleting the intra-exon, the 2′ region (and AUG1) and generating a mature 5′ end capable of encoding 80-kDa 4.1R. Importantly, pre-mRNA constructs that lack the intra-exon, or have a mutated intra-exon 5′ splice donor site, are uncoupled and exhibit inappropriate splicing of exon 1A to the first acceptor site at exon 2′. In support of the generality of this model, we have identified a candidate intra-exon with similar sequence properties in the long 5′ region of the human 4.1B gene, and have demonstrated that this element successfully rescues proper splicing of 4.1R exon 1A in our minigenes. Detailed molecular analysis is under way to identify the specific cis and trans elements required to effect this unusual, long-distance coupling between RNA processing events which have implications for detailed mechanistic understanding of membrane assembly during erythropoiesis.
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KEATES, Andrew C., David P. NUNES, Nezam H. AFDHAL, Robert F. TROXLER, and Gwynneth D. OFFNER. "Molecular cloning of a major human gall bladder mucin: complete C-terminal sequence and genomic organization of MUC5B." Biochemical Journal 324, no. 1 (May 15, 1997): 295–303. http://dx.doi.org/10.1042/bj3240295.
Full textAbstract:
Gall bladder mucin has been shown to play a central role in the pathogenesis of cholesterol gallstone disease. While cloning and sequencing studies have provided a wealth of information on the structure of other gastrointestinal and respiratory mucins, nothing is known about the primary structure of human gall bladder mucin. In this study, we show that the tracheobronchial mucin MUC5B is a major mucin gene product expressed in the gall bladder. Antibodies directed against deglycosylated human gall bladder mucin were used to screen a gall bladder cDNA expression library, and most of the isolated clones contained repetitive sequences nearly identical with those in the tandem repeat region of MUC5B. An additional clone (hGBM2-3) contained an open reading frame coding for a 389 residue cysteine-rich sequence. The arrangement of cysteine residues in this sequence was very similar to that in the C-terminal regions of MUC2, MUC5AC and human von Willebrand factor. This cysteine-rich sequence was connected to a series of degenerate MUC5B tandem repeats in a 7.5 kb HincII genomic DNA fragment. This fragment, with ten exons and nine introns, contained MUC5B repeats in exon 1 and a 469 residue cysteine-rich sequence in exons 2–10 that provided a 152 nucleotide overlap with cDNA clone hGBM2-3. Interestingly, the exon–intron junctions in the MUC5B genomic fragment occurred at positions equivalent to those in the D4 domain of human von Willebrand factor, suggesting that these proteins evolved from a common evolutionary ancestor through addition or deletion of exons encoding functional domains.
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RASTEGAR, Mojgan, Claude SZPIRER, Guy G. ROUSSEAU, and Frédéric P. LEMAIGRE. "Hepatocyte nuclear factor 6: organization and chromosomal assignment of the rat gene and characterization of its promoter." Biochemical Journal 334, no. 3 (September 15, 1998): 565–69. http://dx.doi.org/10.1042/bj3340565.
Full textAbstract:
Hepatocyte nuclear factor 6 (HNF-6) is the prototype of a family of tissue-specific transcription factors characterized by a bipartite DNA-binding domain consisting of a single cut domain and a novel type of homeodomain. We have previously cloned rat cDNA species coding for two isoforms, HNF-6α (465 residues) and β (491 residues), which differ only by the length of the spacer between the two DNA-binding domains. We have now localized the rat Hnf6 gene to chromosome 8q24–q31 by Southern blotting of DNA from somatic cell hybrids and by fluorescence in situhybridization. Cloning and sequencing of the rat gene showed that the two HNF-6 isoforms are generated by alternative splicing of three exons that are more than 10 kb apart from each other. Exon 1 codes for the N-terminal part and the cut domain, exon 2 codes for the 26 HNF-6β-specific amino acids, and exon 3 codes for the homeodomain and the C-terminal amino acids. The transcription initiation site was mapped by ribonuclease protection and 5´ rapid amplification of cDNA ends. Transfection experiments showed that promoter activity was contained within 0.75 kb upstream of the transcription initiation site. This activity was detected by the transfection of liver-derived HepG2 cells, but not of Rat-1 fibroblasts, suggesting that the promoter is sufficient to confer liver-specific expression.
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Saito, A., M. Sakatsume, H. Yamazaki, F. Ogata, Y. Hirasawa, and M. Arakawa. "A deletion mutation in the 3' end of the alpha 5(IV) collagen gene in juvenile-onset Alport syndrome." Journal of the American Society of Nephrology 4, no. 9 (March 1994): 1649–53. http://dx.doi.org/10.1681/asn.v491649.
Full textAbstract:
Alport syndrome is a hereditary progressive glomerular basement membrane disorder in which juvenile-or adult-onset renal failure is often accompanied by sensorineural deafness and ocular abnormalities. Recently, mutations have been found in the type IV collagen alpha 5 chain gene in patients with X-linked Alport syndrome. This study searched for gene mutations in seven unrelated Japanese patients by the use of conventional Southern blot analysis with cDNA probes for the carboxyl-terminal noncollagenous domain that is encoded by exons 46 to 51. A deletion mutation was found in a patient who developed juvenile-onset (age 15) ESRD with typical ultrastructural glomerular basement membrane destruction and sensorineural hearing loss but no characteristic ocular abnormalities. His mother showed hematuria and proteinuria with normal renal function, suggesting that she may be the heterozygous carrier. Exon-specific polymerase chain reaction amplified the coding sequence of exon 48 but not exons 49 to 51. Analysis with pulsed-field gel electrophoresis revealed that the deletion is approximately 10 kb in length and does not involve the CpG island, which is located in the 3' distal site of the gene. Identification of this novel deletion causing juvenile-type Alport syndrome would contribute to elucidating the mechanisms of renal failure progression in the syndrome.
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Chen, Bohao, Elena Yates, Yong Huang, Paul Kogut, Lan Ma, Jerrold R. Turner, Yun Tao, et al. "Alternative promoter and GATA5 transcripts in mouse." American Journal of Physiology-Gastrointestinal and Liver Physiology 297, no. 6 (December 2009): G1214—G1222. http://dx.doi.org/10.1152/ajpgi.00165.2009.
Full textAbstract:
GATA5 is a member of the GATA zinc finger transcription factor family involved in tissue-specific transcriptional regulation during cell differentiation and embryogenesis. Previous reports indicate that null mutation of the zebrafish GATA5 gene results in embryonic lethality, whereas deletion of exon 1 from the mouse GATA5 gene causes only derangement of female urogenital development. Here, we have identified an alternate promoter within intron 1 of the mouse GATA5 gene that transcribes a 2.5-kb mRNA that lacks exon 1 entirely but includes 82 bp from intron 1 and all of exons 2–6. The alternative promoter was active during transient transfection in cultured airway myocytes and bronchial epithelial cells, and it drove reporter gene expression in gastric epithelial cells in transgenic mice. The 2.5-kb alternative transcript encodes an NH2-terminally truncated “short GATA5” comprising aa 226–404 with a single zinc finger, which retains ability to transactivate the atrial natriuretic factor promoter (albeit less efficiently than full-length GATA5). Another new GATA5 transcript contains all of exons 1–5 and the 5′ portion of exon 6 but lacks the terminal 1143 bp of the 3′-untranslated region from exon 6. These findings extend current understanding of the tissue distribution of GATA5 expression and suggests that GATA5 expression and function are more complex than previously appreciated.
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Zeng, Changqing, and Susan M. Berget. "Participation of the C-Terminal Domain of RNA Polymerase II in Exon Definition during Pre-mRNA Splicing." Molecular and Cellular Biology 20, no. 21 (November 1, 2000): 8290–301. http://dx.doi.org/10.1128/mcb.20.21.8290-8301.2000.
Full textAbstract:
ABSTRACT Interaction between transcription and pre-mRNA processing via binding of polymerase II (Pol II) to factors involved in capping, splicing, and polyadenylation has recently been demonstrated. The C-terminal domain (CTD), a highly phosphorylated repeat sequence of the largest subunit of Pol II, has been implicated in this interaction because deletion of this domain affects downstream RNA processing events and because it is the binding site for numerous processing factors. Here we show that recombinant CTD, free of other components of Pol II, activated in vitro splicing and assembly of the spliceosome in nuclear extracts if, and only if, the assayed precursor RNA was recognized via exon definition, i.e., if the substrates contained complete exons with both 3′ and 5′ splice sites. Furthermore, depletion of intact Pol II inactivated splicing of this set of precursor RNAs and addition of recombinant CTD restored activity. The added recombinant CTD was quickly hyper- and hypophosphorylated in extract, became associated with the precursor RNA, and stimulated the association of U1 snRNPs but not ASF/SF2 with substrate RNA. These observations suggest that the mode of interaction between the CTD and splicing factors is integrally tied to exon definition and the mechanism whereby distal exons can be recognized and brought into juxtaposition during assembly of the spliceosome.
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Rodriguez de Cordoba, S., P. Sanchez-Corral, and J. Rey-Campos. "Structure of the gene coding for the alpha polypeptide chain of the human complement component C4b-binding protein." Journal of Experimental Medicine 173, no. 5 (May 1, 1991): 1073–82. http://dx.doi.org/10.1084/jem.173.5.1073.
Full textAbstract:
The human gene coding for the 70-kD polypeptide of the complement regulatory component C4b-binding protein (C4BP alpha) spans over 40 kb of DNA and is composed of twelve exons. Upon transcription in liver, or in Hep-G2 cells, this gene produces a single transcript of 2,262 nucleotides, excepting the poly A tail, that presents an unusually long 5' untranslated region (5' UTR) of 223 nucleotides. The C4BP alpha gene is organized as follows: the first exon codes for the first 198 nucleotides of the 5' UTR. It is separated by a large intron from the second exon including the remaining of the 5' UTR and the coding region for the signal peptide. Each of the eight 60-amino acid repeats (short consensus repeats [SCRs]) that compose the C4BP alpha polypeptide chain is encoded by a single exon, except for the second SCR, which is split in two exons. At the 3' end of the C4BP alpha gene, the twelfth exon codes for the COOH-terminal 57 amino acids of the mature protein, which have no similarities to the SCRs, and the 245 nucleotides of the 3' UTR. Examination of the nucleotide sequence of the first exon revealed an interesting characteristic, strongly suggesting that this exon may specify a functional domain of the C4BP alpha transcript. It includes two in-phase ATG codons, in a different frame respect to that coding the C4BP alpha polypeptide, followed by an in-frame termination codon, also within the first exon. Comparison between mouse and human C4BP alpha transcripts indicates conservation of this structure within the 5' UTR. C4BP is expressed in the liver and is an acute phase protein. A computer search of the genomic sequences upstream the transcription start site demonstrates the presence of potential cis-acting regulatory elements similar to those found in the promoters of other liver-expressed and/or acute phase genes.
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von Brandenstein, Melanie, Katharina Puetz, Monika Schlosser, Heike Löser, Joachim P. Kallinowski, Daniel Gödde, Reinhard Buettner, Stefan Störkel, and Jochen W. U. Fries. "Vimentin 3, the New Hope, Differentiating RCC versus Oncocytoma." Disease Markers 2015 (2015): 1–8. http://dx.doi.org/10.1155/2015/368534.
Full textAbstract:
Vimentin is currently used to differentiate between malignant renal carcinomas and benign oncocytomas. Recent reports showing Vimentin positive oncocytomas seriously question the validity of this present diagnostic approach. Vimentin 3 is a spliced variant and ends with a unique C-terminal ending after exon 7 which differentiates it from the full length version that has 9 exons. Therefore, the protein size is different; the full length Vimentin version has a protein size of ~57 kDa and the truncated version of ~47 kDa. We designed an antibody, called Vim3, against the unique C-terminal ending of the Vimentin 3 variant. Using immune histology, immune fluorescence, Western blot, and qRT-PCR analysis, a Vim3 overexpression was detectable exclusively in oncocytoma, making the detection of Vim3 a potential specific marker for benign kidney tumors. This antibody is the first to clearly differentiate benign oncocytoma and the mimicking eosinophilic variants of the RCCs. This differentiation between malignant and benign RCCs is essential for operative planning, follow-up therapy, and patients’ survival. In the future the usage of Vimentin antibodies in routine pathology has to be applied with care. Consideration must be given to Vimentin specific binding epitopes otherwise a misdiagnosis of the patients’ tumor samples may result.