Journal articles on the topic '’ terminal exon'
To see the other types of publications on this topic, follow the link: ’ terminal exon.
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the top 50 journal articles for your research on the topic '’ terminal exon.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Browse journal articles on a wide variety of disciplines and organise your bibliography correctly.
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.
Full textAbstract:
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.
2
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.
Full textAbstract:
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.
3
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.
Full textAbstract:
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.
4
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.
Full textAbstract:
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.
5
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.
Full textAbstract:
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.
6
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.
Full textAbstract:
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.
7
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.
Full textAbstract:
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.
8
Tan, Jeff, Marilyn K. Parra, Narla Mohandas, and John G. Conboy. "Evolutionarily Conserved Coupling of Transcription and Alternative Splicing in the Protein 4.1R and 4.1B Genes Regulates N-Terminal Protein Structure." Blood 106, no. 11 (November 16, 2005): 1664. http://dx.doi.org/10.1182/blood.v106.11.1664.1664.
Full textAbstract:
Abstract The protein 4.1R gene is regulated by complex pre-mRNA processing events that facilitate the synthesis of protein isoforms with different structure, function, and subcellular localization in red cells and various nucleated cell types. One of these events involves the stage-specific activation of exon 16 inclusion in erythroblasts, which mechanically stabilizes the membrane skeleton by increasing the protein’s affinity for spectrin and actin. Some of the splicing factor proteins and RNA regulatory elements responsible for this tissue-specific alternative splicing event have been defined. Here we focus on another RNA processing event, in the 5′ end of the transcript that can affect the structure and function of the membrane binding domain of protein 4.1R. We have shown that 4.1R transcripts originating at three far upstream alternative promoters/first exons splice differentially to alternative acceptor sites in exon 2′/2 in a manner that suggests strict coupling between transcription and alternative splicing events. A precisely analogous gene organization and RNA processing pattern has also been shown to occur in the paralogous 4.1B gene. Now we demonstrate that this coupling is evolutionarily conserved among several vertebrate classes from fish to mammals. The 4.1R and 4.1B genes from fish, bird, amphibian, and mammal genomes exhibit shared features including alternative first exons and differential splice acceptors in exon 2. In all cases, the 5′-most exon (exon 1A) splices exclusively to a weaker internal acceptor site in exon 2, skipping a short sequence designated as exon 2′ (17-33nt). Conversely, alternative first exons 1B and/or 1C always splice to the stronger first acceptor site, retaining exon 2′. These correlations are independent of tissue type or species of origin. Since exon 2′ contains a translation initiation site, this regulated splicing event generate protein isoforms with distinct N-termini. We propose that these 4.1 genes represent a physiologically relevant model system for mechanistic analysis of transcription-coupled alternative splicing. We have recently constructed a 9kb “minigene” that successfully reproduces the differential splicing patterns of exons 1A and 1B to exon 2′/2 in transfected cells. This minigene will facilitate identification of the determinants that guide coupling. Current experiments are testing the importance for proper splicing of the transcriptional promoter, first exon sequences, length and sequence of the intron, and sequence of a conserved element within exon 2′. Ultimately these studies should provide new insights into the mechanisms of coupling between far upstream, transcription-related processes and downstream alternative splicing.
9
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.
Full textAbstract:
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.
10
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.
Full textAbstract:
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.
11
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.
Full textAbstract:
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.
12
Kobayashi, Eri, Ritsuko Shimizu, Yuko Kikuchi, Satoru Takahashi, and Masayuki Yamamoto. "Loss of the Gata1 Gene IE Exon Leads to Variant Transcript Expression and the Production of GATA1 Protein Lacking the N-Terminal Domain." Blood 114, no. 22 (November 20, 2009): 3648. http://dx.doi.org/10.1182/blood.v114.22.3648.3648.
Full textAbstract:
Abstract Abstract 3648 Poster Board III-584 GATA1 is a transcription factor essential for the differentiation of erythroid cells and megakaryocytes. Since GATA1 regulates genes related to the survival, proliferation and differentiation of hematopoietic cells, regulation of the Gata1 gene expression is critically important for the understanding of hematopoiesis. The Gata1 locus contains multiple untranslated first exons plus five common coding exons. Of these first exons, erythroid first exon (IE exon) is important for the Gata1 gene expression in the hematopoietic lineages. However, due to the embryonic lethality of this IE exon knockdown mice, less is understood about the contribution of the IE exon to adult hematopoiesis. Here, we achieved specific deletion of the IE exon in adulthood by crossing the IE-floxed mice with the interferon-inducible Mx1-Cre transgenic mice. This conditional IE-deletion mouse (ΔIE mouse) showed severe thrombocytopenia with increased premature megakaryocytes similarly to the phenotypes reported in the conditional Gata1 knockout mice in which the entire Gata1 gene was deleted in adulthood. In addition, the ΔIE mice showed severe anemia with skewed erythroid maturation, and importantly this erythroid phenotypes substantially differed from those observed in the conditional Gata1 knockout mice. Further analyses revealed that the Gata1 mRNA level in the megakaryocytic lineage was significantly downregulated. By contrast, in the erythroid lineage, Gata1 mRNA was retained at a comparable level to that in control mice utilizing two alternative first exons; one was the IEb/c, which was previously reported as a first exon rarely used in hematopoietic cells, and the other was newly identified IEd exon located within the second intron. Surprisingly, in the ΔIE mice these transcripts failed to produce full-length GATA1 protein, but instead inefficiently yielded GATA1 lacking the N-terminal 83 amino acids. This form of GATA1 is often observed in Down syndrome-associated transient myeloproliferative disorder and acute megakaryoblastic leukemia. Of note, the transcript derived from exon IEb/c preserved the first translation initiation codon in exon 2 but lost the potential to select the first translation initiation codon or failed to produce full-length GATA1. The present study demonstrates that the IE exon is instrumental to adult erythropoiesis by regulating the proper level of transcription and by selecting the correct translation start site for production of adequate full-length GATA1 protein. Disclosures: No relevant conflicts of interest to declare.
13
Humphrey, M. B., J. Bryan, T. A. Cooper, and S. M. Berget. "A 32-nucleotide exon-splicing enhancer regulates usage of competing 5' splice sites in a differential internal exon." Molecular and Cellular Biology 15, no. 8 (August 1995): 3979–88. http://dx.doi.org/10.1128/mcb.15.8.3979.
Full textAbstract:
Large alternatively spliced internal exons are uncommon in vertebrate genes, and the mechanisms governing their usage are unknown. In this report, we examined alternative splicing of a 1-kb internal exon from the human caldesmon gene containing two regulated 5' splice sites that are 687 nucleotides apart. In cell lines normally splicing caldesmon RNA via utilization of the exon-internal 5' splice site, inclusion of the differential exon required a long purine-rich sequence located between the two competing 5' splice sites. This element consisted of four identical 32-nucleotide purine-rich repeats that resemble exon-splicing enhancers (ESE) identified in other genes. One 32-nucleotide repeat supported exon inclusion, repressed usage of the terminal 5' splice site, and functioned in a heterologous exon dependent on exon enhancers for inclusion, indicating that the caldesmon purine-rich sequence can be classified as an ESE. The ESE was required for utilization of the internal 5' splice site only in the presence of the competing 5' splice site and had no effect when placed downstream of the terminal 5' splice site. In the absence of the internal 5' splice site, the ESE activated a normally silent cryptic 5' splice site near the natural internal 5' splice site, indicating that the ESE stimulates upstream 5' splice site selection. We propose that the caldesmon ESE functions to regulate competition between two 5' splice sites within a differential internal exon.
14
Borysov, Sergiy I., Brook S. Nepon-Sixt, and Mark G. Alexandrow. "The N Terminus of the Retinoblastoma Protein Inhibits DNA Replication via a Bipartite Mechanism Disrupted in Partially Penetrant Retinoblastomas." Molecular and Cellular Biology 36, no. 5 (December 28, 2015): 832–45. http://dx.doi.org/10.1128/mcb.00636-15.
Full textAbstract:
The N-terminal domain of the retinoblastoma (Rb) tumor suppressor protein (RbN) harbors in-frame exon deletions in partially penetrant hereditary retinoblastomas and is known to impair cell growth and tumorigenesis. However, how such RbN deletions contribute to Rb tumor- and growth-suppressive functions is unknown. Here we establish that RbN directly inhibits DNA replication initiation and elongation using a bipartite mechanism involving N-terminal exons lost in cancer. Specifically, Rb exon 7 is necessary and sufficient to target and inhibit the replicative CMG helicase, resulting in the accumulation of inactive CMGs on chromatin. An independent N-terminal loop domain, which forms a projection, specifically blocks DNA polymerase α (Pol-α) and Ctf4 recruitment without affecting DNA polymerases ε and δ or the CMG helicase. Individual disruption of exon 7 or the projection in RbN or Rb, as occurs in inherited cancers, partially impairs the ability of Rb/RbN to inhibit DNA replication and block G1-to-S cell cycle transit. However, their combined loss abolishes these functions of Rb. Thus, Rb growth-suppressive functions include its ability to block replicative complexes via bipartite, independent, and additive N-terminal domains. The partial loss of replication, CMG, or Pol-α control provides a potential molecular explanation for how N-terminal Rb loss-of-function deletions contribute to the etiology of partially penetrant retinoblastomas.
15
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.
Full text
16
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.
Full textAbstract:
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.
17
Witzel-Schlömp, K., P. J. Späth, M. J. Hobart, B. A. Fernie, C. Rittner, T. Kaufmann, and P. M. Schneider. "The human complement C9 gene: identification of two mutations causing deficiency and revision of the gene structure." Journal of Immunology 158, no. 10 (May 15, 1997): 5043–49. http://dx.doi.org/10.4049/jimmunol.158.10.5043.
Full textAbstract:
Abstract The ninth component of human complement (C9) is the last of the terminal complement components creating the membrane attack complex. C9 is a single-chain serum protein that is encoded by a gene located on chromosome 5p. Deficiency of terminal complement components is generally associated with recurrent neisseria infections. We studied a previously described Swiss family with inherited C9 deficiency. To identify the genetic basis of C9 deficiency, we developed an approach using exon-specific PCR and direct DNA sequencing. As a cause of C9 deficiency, we found two different point mutations, both generating TGA stop codons in the coding sequence. One mutation, a C to A exchange, was detected in exon 2 at cDNA position 166, the other, a C to T exchange, was located in exon 4 (cDNA position 464). In family studies of three first-degree relatives with heterozygous C9 deficiency, we demonstrated that the two mutations are segregating independently. Therefore, these mutations are sufficient to explain the complete deficiency of both the probands studied. DNA sequencing of the exon-intron junctions revealed a number of revisions regarding the boundaries between exons 4, 5, and 6 as well as between exons 10 and 11. No additional introns were detected in exons 6 and 10. Furthermore, DNA marker studies were conducted using known polymorphisms of the C6, C7, and C9 genes, confirming the linkage of the observed C9 mutations with defined haplotypes.
18
Zhang-Keck, Z. Y., M. Srivastava, C. A. Kozak, H. Caohuy, A. Shirvan, A. L. Burns, and H. B. Pollard. "Genomic organization and chromosomal localization of the mouse synexin gene." Biochemical Journal 301, no. 3 (August 1, 1994): 835–45. http://dx.doi.org/10.1042/bj3010835.
Full textAbstract:
We have isolated and characterized the gene encoding mouse synexin, which consists of 14 exons and spans approximately 30 kbp of genomic DNA. The protein's unique N-terminal domain is encoded by six exons, and the C-terminal tetrad repeat, the site of the membrane-fusion and ion-channel domain, is encoded by seven exons. The first exon encodes the 5′-untranslated region. Analysis of synexin-gene expression in different mouse tissues shows that mRNA with exon 6 is only present in brain, heart and skeletal muscle. mRNA lacking exon 6 is expressed in all tissues we have examined. The initiation site for transcription was determined by primer-extension analysis and S1 nuclease mapping. Sequence analysis of the 1.3 kb 5′-flanking region revealed that the promoter has a TATA box located at position -25 and a number of potential promoter and regulatory elements. A CCAAT motif was not observed but CCATT is located in an appropriate position for the CCAAT motif upstream from the transcription-initiation start site. In addition, the 5′-flanking region contains two sets of palindromic sequences. Finally, we have determined that the functional synexin gene (Anx7) is located on mouse chromosome 14 and that a pseudogene (Anx7-ps1) is located on chromosome 10.
19
Hobart, M. J., B. A. Fernie, and R. G. DiScipio. "Structure of the human C7 gene and comparison with the C6, C8A, C8B, and C9 genes." Journal of Immunology 154, no. 10 (May 15, 1995): 5188–94. http://dx.doi.org/10.4049/jimmunol.154.10.5188.
Full textAbstract:
Abstract The seventh component of complement is a single chain plasma glycoprotein that is involved in the cytolytic phase of complement activation. We have determined the structure of the C7 gene, which is encoded by 18 exons whose sizes vary from 56 to 244 bp. For the most part, the exons do not correspond to the protein homology units. However, two intron/exon boundaries occur at junctions between different functional parts of the protein. The first is at a site between the end of the C9 homology unit and the carboxyl-terminal extension which is also a feature of C6. The second of these boundaries occurs between the regions encoding two pairs of cysteine-rich modules (the short consensus repeats and the factor I modules) located in the carboxyl-terminal part of C7. In contrast to the exons, the introns range considerably in size from 0.5 to 8.5 kbp. The complete analysis indicates that the gene encoding C7 is approximately 80 kbp in length. We show here that the C7 gene is highly homologous to that for C6, and also to C8A, C8B, and C9, confirming and extending the published data. With the exception of exon 1, all intron/exon boundaries are preserved with respect to phase when compared with C6.
20
Putt, W., J. H. Ives, M. Hollyoake, D. A. Hopkinson, D. B. Whitehouse, and Y. H. Edwards. "Phosphoglucomutase 1: a gene with two promoters and a duplicated first exon." Biochemical Journal 296, no. 2 (December 1, 1993): 417–22. http://dx.doi.org/10.1042/bj2960417.
Full textAbstract:
In view of its central role in glycolysis and gluconeogenesis and its polymorphic genetic variability, the phosphoglucomutase 1 (PGM1) gene in man has been the target of protein structural studies and genetic analysis for more than 25 years. We have now isolated genomic clones containing the complete PGM1 gene and have shown that it spans over 65 kb and contains 11 exons. We have also shown that the sites of the two mutations which form the molecular basis for the common PGM1 protein polymorphism lie in exons 4 and 8 and are 18 kb apart. Within this region there is a site of intragenic recombination. We have discovered two alternatively spliced first exons, one of which, exon 1A, is transcribed in a wide variety of cell types; the other, exon 1B, is transcribed in fast muscle. Exon 1A is transcribed from a promoter which has the structural hallmarks of a housekeeping promoter but lies more than 35 kb upstream of exon 2. Exon 1B lies 6 kb upstream of exon 2 within the large first intron of the ubiquitously expressed PGM1 transcript. The fast-muscle form of PGM1 is characterized by 18 extra amino acid residues at its N-terminal end. Sequence comparisons show that exons 1A and 1B are structurally related and have arisen by duplication.
21
Mayr, Simon J., Juliane Röper, and Guenter Schwarz. "Alternative splicing of the bicistronic gene molybdenum cofactor synthesis 1 (MOCS1) uncovers a novel mitochondrial protein maturation mechanism." Journal of Biological Chemistry 295, no. 10 (January 29, 2020): 3029–39. http://dx.doi.org/10.1074/jbc.ra119.010720.
Full textAbstract:
Molybdenum cofactor (Moco) biosynthesis is a highly conserved multistep pathway. The first step, the conversion of GTP to cyclic pyranopterin monophosphate (cPMP), requires the bicistronic gene molybdenum cofactor synthesis 1 (MOCS1). Alternative splicing of MOCS1 within exons 1 and 9 produces four different N-terminal and three different C-terminal products (type I–III). Type I splicing results in bicistronic transcripts with two open reading frames, of which only the first, MOCS1A, is translated, whereas type II/III splicing produces MOCS1AB proteins. Here, we first report the cellular localization of alternatively spliced human MOCS1 proteins. Using fluorescence microscopy, fluorescence spectroscopy, and cell fractionation experiments, we found that depending on the alternative splicing of exon 1, type I splice variants (MOCS1A) either localize to the mitochondrial matrix (exon 1a) or remain cytosolic (exon 1b). MOCS1A proteins required exon 1a for mitochondrial translocation, but fluorescence microscopy of MOCS1AB variants (types II and III) revealed that they were targeted to mitochondria independently of exon 1 splicing. In the latter case, cell fractionation experiments displayed that mitochondrial matrix import was facilitated via an internal motif overriding the N-terminal targeting signal. Within mitochondria, MOCS1AB underwent proteolytic cleavage resulting in mitochondrial matrix localization of the MOCS1B domain. In conclusion, MOCS1 produces two functional proteins, MOCS1A and MOCS1B, which follow different translocation routes before mitochondrial matrix import for cPMP biosynthesis involving both proteins. MOCS1 protein maturation provides a novel alternative splicing mechanism that ensures the coordinated mitochondrial targeting of two functionally related proteins encoded by a single gene.
22
TABISH, Mohammad, Roger A. CLEGG, Huw H. REES, and Michael J. FISHER. "Organization and alternative splicing of the Caenorhabditis elegans cAMP-dependent protein kinase catalytic-subunit gene (kin-1)." Biochemical Journal 339, no. 1 (March 25, 1999): 209–16. http://dx.doi.org/10.1042/bj3390209.
Full textAbstract:
The cAMP-dependent protein kinase (protein kinase A, PK-A) is multifunctional in nature, with key roles in the control of diverse aspects of eukaryotic cellular activity. In the case of the free-living nematode, Caenorhabditis elegans, a gene encoding the PK-A catalytic subunit has been identified and two isoforms of this subunit, arising from a C-terminal alternative-splicing event, have been characterized [Gross, Bagchi, Lu and Rubin (1990) J. Biol. Chem. 265, 6896–6907]. Here we report the occurrence of N-terminal alternative-splicing events that, in addition to generating a multiplicity of non-myristoylatable isoforms, also generate the myristoylated variant(s) of the catalytic subunit that we have recently characterized [Aspbury, Fisher, Rees and Clegg (1997) Biochem. Biophys. Res. Commun. 238, 523–527]. The gene spans more than 36 kb and is divided into a total of 13 exons. Each of the mature transcripts contains only 7 exons. In addition to the already characterized exon 1, the 5´-untranslated region and first intron actually contain 5 other exons, any one of which may be alternatively spliced on to exon 2 at the 5´ end of the pre-mRNA. This N-terminal alternative splicing occurs in combination with either of the already characterized C-terminal alternative exons. Thus, C. elegans expresses at least 12 different isoforms of the catalytic subunit of PK-A. The significance of this unprecedented structural diversity in the family of PK-A catalytic subunits is discussed.
23
Ponissery Saidu, Samsudeen, Aaron B. Stephan, Anna K. Talaga, Haiqing Zhao, and Johannes Reisert. "Channel properties of the splicing isoforms of the olfactory calcium-activated chloride channel Anoctamin 2." Journal of General Physiology 141, no. 6 (May 13, 2013): 691–703. http://dx.doi.org/10.1085/jgp.201210937.
Full textAbstract:
Anoctamin (ANO)2 (or TMEM16B) forms a cell membrane Ca2+-activated Cl− channel that is present in cilia of olfactory receptor neurons, vomeronasal microvilli, and photoreceptor synaptic terminals. Alternative splicing of Ano2 transcripts generates multiple variants with the olfactory variants skipping exon 14 and having alternative splicing of exon 4. In the present study, 5′ rapid amplification of cDNA ends analysis was conducted to characterize the 5′ end of olfactory Ano2 transcripts, which showed that the most abundant Ano2 transcripts in the olfactory epithelium contain a novel starting exon that encodes a translation initiation site, whereas transcripts of the publically available sequence variant, which has an alternative and longer 5′ end, were present in lower abundance. With two alternative starting exons and alternative splicing of exon 4, four olfactory ANO2 isoforms are thus possible. Patch-clamp experiments in transfected HEK293T cells expressing these isoforms showed that N-terminal sequences affect Ca2+ sensitivity and that the exon 4–encoded sequence is required to form functional channels. Coexpression of the two predominant isoforms, one with and one without the exon 4 sequence, as well as coexpression of the two rarer isoforms showed alterations in channel properties, indicating that different isoforms interact with each other. Furthermore, channel properties observed from the coexpression of the predominant isoforms better recapitulated the native channel properties, suggesting that the native channel may be composed of two or more splicing isoforms acting as subunits that together shape the channel properties.
24
Del Gatto-Konczak, Fabienne, Michelle Olive, Marie-Claude Gesnel, and Richard Breathnach. "hnRNP A1 Recruited to an Exon In Vivo Can Function as an Exon Splicing Silencer." Molecular and Cellular Biology 19, no. 1 (January 1, 1999): 251–60. http://dx.doi.org/10.1128/mcb.19.1.251.
Full textAbstract:
ABSTRACT Some exons contain exon splicing silencers. Their activity is frequently balanced by that of splicing enhancers, and this is important to ensure correct relative levels of alternatively spliced mRNAs. Using an immunoprecipitation and UV-cross-linking assay, we show that RNA molecules containing splicing silencers from the human immunodeficiency virus type 1 tat exon 2 or the human fibroblast growth factor receptor 2 K-SAM exon bind to hnRNP A1 in HeLa cell nuclear extracts better than the corresponding RNA molecule without a silencer. Two different point mutations which abolish the K-SAM exon splicing silencer’s activity reduce hnRNP A1 binding twofold. Recruitment of hnRNP A1 in the form of a fusion with bacteriophage MS2 coat protein to a K-SAM exon whose exon splicing silencer has been replaced by a coat binding site efficiently represses splicing of the exon in vivo. Recruitment of only the glycine-rich C-terminal domain of hnRNP A1, which is capable of interactions with other proteins, is sufficient to repress exon splicing. Our results show that hnRNP A1 can function to repress splicing, and they suggest that at least some exon splicing silencers could work by recruiting hnRNP A1.
25
Birkeland, M. L., P. Johnson, I. S. Trowbridge, and E. Puré. "Changes in CD45 isoform expression accompany antigen-induced murine T-cell activation." Proceedings of the National Academy of Sciences 86, no. 17 (September 1989): 6734–38. http://dx.doi.org/10.1073/pnas.86.17.6734.
Full textAbstract:
Leukocytes express a family of plasma membrane proteins called CD45 or the leukocyte common antigen. Isoforms of various molecular masses, 180-240 kDa, are produced by alternative splicing and usage of three exons, named A, B, and C, that encode the N-terminal portion of the external domain. By using monoclonal antibodies that precipitate B exon-dependent and B exon-independent isoforms we find that both murine CD4+ and murine CD8+ T cells selectively down-regulate the B exon-dependent forms of CD45 during an immune response. This change was monitored by using fluorescence-activated cell sorter (FACS) analysis and immunoprecipitation from surface radioiodinated and metabolically labeled cells. The loss of the 190-kDa B exon-dependent isoform during T-cell activation is accompanied by an increased production of a 180-kDa form, which does not contain the B exon-encoded sequence. This accounts for our observation that the overall expression of CD45, as assessed by FACS analysis, does not change.
26
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.
Full textAbstract:
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.
27
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.
Full textAbstract:
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.
28
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.
29
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.
Full text
30
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.
Full textAbstract:
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.
31
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.
32
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.
33
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.
Full textAbstract:
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.
34
Hall, GW, and S. Thein. "Nonsense codon mutations in the terminal exon of the beta-globin gene are not associated with a reduction in beta-mRNA accumulation: a mechanism for the phenotype of dominant beta-thalassemia." Blood 83, no. 8 (April 15, 1994): 2031–37. http://dx.doi.org/10.1182/blood.v83.8.2031.2031.
Full textAbstract:
Abstract We present in vivo evidence that there is no reduction in beta-mRNA accumulation in patients with nonsense codons in the terminal exon of the beta-globin gene. Using reverse transcriptase/polymerase chain reaction (RT-PCR), beta-globin cDNA was isolated from the reticulocytes of individuals heterozygous for nonsense codon mutations in exons II and III of the beta-globin gene. Clinically asymptomatic individuals heterozygous for mutations causing premature termination of translation in exon II [beta(0)39(C-T) and F/S71/72(+A)] were found to have almost no mutant beta-cDNA, whereas patients with nonsense codon mutations in exon III [beta 121(G-T) and beta 127(C-T)] with the clinical phenotype of thalassemia intermedia had comparable levels of mutant and normal beta-cDNA. Translation of the mutant beta-mRNA from patients with nonsense codon mutations in exon III would give rise to truncated beta- globin chains, which could explain the more severe phenotype seen in these individuals.
35
Hall, GW, and S. Thein. "Nonsense codon mutations in the terminal exon of the beta-globin gene are not associated with a reduction in beta-mRNA accumulation: a mechanism for the phenotype of dominant beta-thalassemia." Blood 83, no. 8 (April 15, 1994): 2031–37. http://dx.doi.org/10.1182/blood.v83.8.2031.bloodjournal8382031.
Full textAbstract:
We present in vivo evidence that there is no reduction in beta-mRNA accumulation in patients with nonsense codons in the terminal exon of the beta-globin gene. Using reverse transcriptase/polymerase chain reaction (RT-PCR), beta-globin cDNA was isolated from the reticulocytes of individuals heterozygous for nonsense codon mutations in exons II and III of the beta-globin gene. Clinically asymptomatic individuals heterozygous for mutations causing premature termination of translation in exon II [beta(0)39(C-T) and F/S71/72(+A)] were found to have almost no mutant beta-cDNA, whereas patients with nonsense codon mutations in exon III [beta 121(G-T) and beta 127(C-T)] with the clinical phenotype of thalassemia intermedia had comparable levels of mutant and normal beta-cDNA. Translation of the mutant beta-mRNA from patients with nonsense codon mutations in exon III would give rise to truncated beta- globin chains, which could explain the more severe phenotype seen in these individuals.
36
Liu, Hong, Hao Wang, Matthew Peterson, Wen Zhang, Guoqiang Hou, and Zhong-wei Zhang. "N-terminal alternative splicing of GluN1 regulates the maturation of excitatory synapses and seizure susceptibility." Proceedings of the National Academy of Sciences 116, no. 42 (September 30, 2019): 21207–12. http://dx.doi.org/10.1073/pnas.1905721116.
Full textAbstract:
The majority of NMDA receptors (NMDARs) in the brain are composed of 2 GluN1 and 2 GluN2 subunits. The inclusion or exclusion of 1 N-terminal and 2 C-terminal domains of GluN1 results in 8 splicing variants that exhibit distinct temporal and spatial patterns of expression and functional properties. However, previous functional analyses of Grin1 variants have been done using heterologous expression and the in vivo function of Grin1 splicing is unknown. Here we show that N-terminal splicing of GluN1 has important functions in the maturation of excitatory synapses. The inclusion of exon 5 of Grin1 is up-regulated in several brain regions such as the thalamus and neocortex. We find that deletion of Grin1 exon 5 disrupts the developmental remodeling of NMDARs in thalamic neurons and the effect is distinct from that of Grin2a (GluN2A) deletion. Deletion of Grin2a or exon 5 of Grin1 alone partially attenuates the shortening of NMDAR-mediated excitatory postsynaptic currents (NMDAR-EPSCs) during early life, whereas deletion of both Grin2a and exon 5 of Grin1 completely abolishes the developmental change in NMDAR-EPSC decay time. Deletion of exon 5 of Grin1 leads to an overproduction of excitatory synapses in layer 5 pyramidal neurons in the cortex and increases seizure susceptibility in adult mice. Our findings demonstrate that N-terminal splicing of GluN1 has important functions in synaptic maturation and neuronal network excitability.
37
Shilovsky, Gregory A., Oleg A. Zverkov, Alexandr V. Seliverstov, Vasily V. Ashapkin, Tatyana S. Putyatina, Lev I. Rubanov, and Vassily A. Lyubetsky. "New C-Terminal Conserved Regions of Tafazzin, a Catalyst of Cardiolipin Remodeling." Oxidative Medicine and Cellular Longevity 2019 (October 24, 2019): 1–13. http://dx.doi.org/10.1155/2019/2901057.
Full textAbstract:
Cardiolipin interacts with many proteins of the mitochondrial inner membrane and, together with cytochrome C and creatine kinase, activates them. It can be considered as an integrating factor for components of the mitochondrial respiratory chain, which provides for an efficient transfer of electrons and protons. The major, if not the only, factor of cardiolipin maturation is tafazzin. Variations of isoform proportions of this enzyme can cause severe diseases such as Barth syndrome. Using bioinformatic methods, we have found conserved C-terminal regions in many tafazzin isoforms and identified new mammalian species that acquired exon 5 as well as rare occasions of intron retention between exons 8 and 9. The regions in the C-terminal part arise from frameshifts relative to the full-length TAZ transcript after skipping exon 9 or retention of the intron between exons 10 and 11. These modifications demonstrate specific distribution among the orders of mammals. The dependence of the species maximum lifespan, body weight, and mitochondrial metabolic rate on the modifications has been demonstrated. Arguably, unconventional tafazzin isoforms provide for the optimal balance between the increased biochemical activity of mitochondria (resulting from specific environmental or nutritional conditions) and lifespan maintenance; and the functional role of such isoforms is linked to the modification of the primary and secondary structures at their C-termini.
38
Chen, Mingchen, and Peter G. Wolynes. "Aggregation landscapes of Huntingtin exon 1 protein fragments and the critical repeat length for the onset of Huntington’s disease." Proceedings of the National Academy of Sciences 114, no. 17 (April 11, 2017): 4406–11. http://dx.doi.org/10.1073/pnas.1702237114.
Full textAbstract:
Huntington’s disease (HD) is a neurodegenerative disease caused by an abnormal expansion in the polyglutamine (polyQ) track of the Huntingtin (HTT) protein. The severity of the disease depends on the polyQ repeat length, arising only in patients with proteins having 36 repeats or more. Previous studies have shown that the aggregation of N-terminal fragments (encoded by HTT exon 1) underlies the disease pathology in mouse models and that the HTT exon 1 gene product can self-assemble into amyloid structures. Here, we provide detailed structural mechanisms for aggregation of several protein fragments encoded by HTT exon 1 by using the associative memory, water-mediated, structure and energy model (AWSEM) to construct their free energy landscapes. We find that the addition of the N-terminal 17-residue sequence (NT17) facilitates polyQ aggregation by encouraging the formation of prefibrillar oligomers, whereas adding the C-terminal polyproline sequence (P10) inhibits aggregation. The combination of both terminal additions in HTT exon 1 fragment leads to a complex aggregation mechanism with a basic core that resembles that found for the aggregation of pure polyQ repeats using AWSEM. At the extrapolated physiological concentration, although the grand canonical free energy profiles are uphill for HTT exon 1 fragments having 20 or 30 glutamines, the aggregation landscape for fragments with 40 repeats has become downhill. This computational prediction agrees with the critical length found for the onset of HD and suggests potential therapies based on blocking early binding events involving the terminal additions to the polyQ repeats.
39
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.
40
Ikeda-Iwabu, Yuka, Yoshiaki Taniyama, Naruto Katsuragi, Fumihiro Sanada, Nobutaka Koibuchi, Kana Shibata, Kenzo Shimazu, Hiromi Rakugi, and Ryuichi Morishita. "Periostin Short Fragment with Exon 17 via Aberrant Alternative Splicing Is Required for Breast Cancer Growth and Metastasis." Cells 10, no. 4 (April 14, 2021): 892. http://dx.doi.org/10.3390/cells10040892.
Full textAbstract:
Background: Periostin (POSTN) is a 93 kDa matrix protein that helps to regulate collagen gene expression in the extracellular matrix. POSTN overexpression is a prognostic factor in malignant cancers; however, some researchers have observed it in the stroma, whereas others have reported it on tumors. Objective: This study aimed to investigate the function of POSTN on tumors. Methods and Results: We found that POSTN in cancer cells can be detected by using an antibody against the POSTN C-terminal region exon 17 (Ex17 antibody), but not with an antibody against the POSTN N-terminal region exon 12 (Ex12 antibody) in patients with breast cancer. In a fraction secreted from fibroblasts, LC–MS/MS analysis revealed a short fragment of POSTN of approximately 40 kDa with exon 17. In addition, molecular interaction analysis showed that POSTN with exon 17, but not POSTN without exon 17, bound specifically to wnt3a, and the Ex17 antibody inhibited the binding. Conclusion: A short fragment of POSTN with exon 17, which originates in the fibroblasts, is transported to cancer cells, whereas POSTN fragments without exon 17 are retained in the stroma. The Ex17 antibody inhibits the binding between POSTN exon 17 and wnt3a.
41
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.
42
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.
43
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.
Full textAbstract:
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.
44
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.
45
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.
Full textAbstract:
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.
46
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.
Full textAbstract:
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.
47
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.
Full textAbstract:
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.
48
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.
Full textAbstract:
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.
49
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.
50
Leroy, E. "Intron-exon Structure of Ubiquitin C-terminal Hydrolase-L1." DNA Research 5, no. 6 (January 1, 1998): 397–400. http://dx.doi.org/10.1093/dnares/5.6.397.
Full text