Journal articles on the topic 'Intronic polyadenylation'
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Tikhonov, M. V., P. G. Georgiev, and O. G. Maksimenko. "Competition within Introns: Splicing Wins over Polyadenylation via a General Mechanism." Acta Naturae 5, no. 4 (December 15, 2013): 52–61. http://dx.doi.org/10.32607/20758251-2013-5-4-52-61.
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Most eukaryotic messenger RNAs are capped, spliced, and polyadenylated via co-transcriptional processes that are coupled to each other and to the transcription machinery. Coordination of these processes ensures correct RNA maturation and provides for the diversity of the transcribed isoforms. Thus, RNA processing is a chain of events in which the completion of one event is coupled to the initiation of the next one. In this context, the relationship between splicing and polyadenylation is an important aspect of gene regulation. We have found that cryptic polyadenylation signals are widely distributed over the intron sequences of Drosophila melanogaster. As shown by analyzing the distribution of genes arranged in a nested pattern, where one gene is fully located within an intron of another gene, overlapping of putative polyadenylation signals is a fairly common event affecting about 17% of all genes. Here we show that polyadenylation signals are silenced within introns: the poly(A) signal is utilized in the exonic but not in the intronic regions of the transcript. The transcription does not end within the introns, either in a transient reporter system or in the genomic context, while deletion of the 5'-splice site restores their functionality. According to a full Drosophila transcriptome analysis, utilization of intronic polyadenylation signals occurs very rarely and such events are likely to be inducible. These results confirm that the transcription apparatus ignores premature polyadenylation signals for as long as they are intronic.
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Wang, Xiuye, Liang Liu, Adam W. Whisnant, Thomas Hennig, Lara Djakovic, Nabila Haque, Cindy Bach, et al. "Mechanism and consequences of herpes simplex virus 1-mediated regulation of host mRNA alternative polyadenylation." PLOS Genetics 17, no. 3 (March 8, 2021): e1009263. http://dx.doi.org/10.1371/journal.pgen.1009263.
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Eukaryotic gene expression is extensively regulated by cellular stress and pathogen infections. We have previously shown that herpes simplex virus 1 (HSV-1) and several cellular stresses cause widespread disruption of transcription termination (DoTT) of RNA polymerase II (RNAPII) in host genes and that the viral immediate early factor ICP27 plays an important role in HSV-1-induced DoTT. Here, we show that HSV-1 infection also leads to widespread changes in alternative polyadenylation (APA) of host mRNAs. In the majority of cases, polyadenylation shifts to upstream poly(A) sites (PAS), including many intronic PAS. Mechanistically, ICP27 contributes to HSV-1-mediated APA regulation. HSV-1- and ICP27-induced activation of intronic PAS is sequence-dependent and does not involve general inhibition of U1 snRNP. HSV1-induced intronic polyadenylation is accompanied by early termination of RNAPII. HSV-1-induced mRNAs polyadenylated at intronic PAS (IPA) are exported into the cytoplasm while APA isoforms with extended 3’ UTRs are sequestered in the nuclei, both preventing the expression of the full-length gene products. Finally we provide evidence that HSV-induced IPA isoforms are translated. Together with other recent studies, our results suggest that viral infection and cellular stresses induce a multi-faceted host response that includes DoTT and changes in APA profiles.
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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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Spraggon, Lee, and Luca Cartegni. "U1 snRNP-Dependent Suppression of Polyadenylation: Physiological Role and Therapeutic Opportunities in Cancer." International Journal of Cell Biology 2013 (2013): 1–10. http://dx.doi.org/10.1155/2013/846510.
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Pre-mRNA splicing and polyadenylation are critical steps in the maturation of eukaryotic mRNA. U1 snRNP is an essential component of the splicing machinery and participates in splice-site selection and spliceosome assembly by base-pairing to the 5′ splice site. U1 snRNP also plays an additional, nonsplicing global function in 3′ end mRNA processing; it actively suppresses the polyadenylation machinery from using early, mostly intronic polyadenylation signals which would lead to aberrant, truncated mRNAs. Thus, U1 snRNP safeguards pre-mRNA transcripts against premature polyadenylation and contributes to the regulation of alternative polyadenylation. Here, we review the role of U1 snRNP in 3′ end mRNA processing, outline the evidence that led to the recognition of its physiological, general role in inhibiting polyadenylation, and finally highlight the possibility of manipulating this U1 snRNP function for therapeutic purposes in cancer.
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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.
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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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Duan, Cheng-Guo, Xingang Wang, Lingrui Zhang, Xiansong Xiong, Zhengjing Zhang, Kai Tang, Li Pan, et al. "A protein complex regulates RNA processing of intronic heterochromatin-containing genes in Arabidopsis." Proceedings of the National Academy of Sciences 114, no. 35 (August 14, 2017): E7377—E7384. http://dx.doi.org/10.1073/pnas.1710683114.
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In several eukaryotic organisms, heterochromatin (HC) in the introns of genes can regulate RNA processing, including polyadenylation, but the mechanism underlying this regulation is poorly understood. By promoting distal polyadenylation, the bromo-adjacent homology (BAH) domain-containing and RNA recognition motif-containing protein ASI1 and the H3K9me2-binding protein EDM2 are required for the expression of functional full-length transcripts of intronic HC-containing genes in Arabidopsis. Here we report that ASI1 and EDM2 form a protein complex in vivo via a bridge protein, ASI1-Immunoprecipitated Protein 1 (AIPP1), which is another RNA recognition motif-containing protein. The complex also may contain the Pol II CTD phosphatase CPL2, the plant homeodomain-containing protein AIPP2, and another BAH domain protein, AIPP3. As is the case with dysfunction of ASI1 and EDM2, dysfunction of AIPP1 impedes the use of distal polyadenylation sites at tested intronic HC-containing genes, such as the histone demethylase gene IBM1, resulting in a lack of functional full-length transcripts. A mutation in AIPP1 causes silencing of the 35S-SUC2 transgene and genome-wide CHG hypermethylation at gene body regions, consistent with the lack of full-length functional IBM1 transcripts in the mutant. Interestingly, compared with asi1, edm2, and aipp1 mutations, mutations in CPL2, AIPP2, and AIPP3 cause the opposite effects on the expression of intronic HC-containing genes and other genes, suggesting that CPL2, AIPP2, and AIPP3 may form a distinct subcomplex. These results advance our understanding of the interplay between heterochromatic epigenetic modifications and RNA processing in higher eukaryotes.
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Wang, Ruijia, and Bin Tian. "APAlyzer: a bioinformatics package for analysis of alternative polyadenylation isoforms." Bioinformatics 36, no. 12 (April 22, 2020): 3907–9. http://dx.doi.org/10.1093/bioinformatics/btaa266.
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Abstract Summary Most eukaryotic genes produce alternative polyadenylation (APA) isoforms. APA is dynamically regulated under different growth and differentiation conditions. Here, we present a bioinformatics package, named APAlyzer, for examining 3′UTR APA, intronic APA and gene expression changes using RNA-seq data and annotated polyadenylation sites in the PolyA_DB database. Using APAlyzer and data from the GTEx database, we present APA profiles across human tissues. Availability and implementation APAlyzer is freely available at https://bioconductor.org/packages/release/bioc/html/APAlyzer.html as an R/Bioconductor package. Supplementary information Supplementary data are available at Bioinformatics online.
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Lee, Shih-Han, Irtisha Singh, Sarah Tisdale, Omar Abdel-Wahab, Christina S. Leslie, and Christine Mayr. "Widespread intronic polyadenylation inactivates tumour suppressor genes in leukaemia." Nature 561, no. 7721 (August 27, 2018): 127–31. http://dx.doi.org/10.1038/s41586-018-0465-8.
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Dubbury, Sara J., Paul L. Boutz, and Phillip A. Sharp. "CDK12 regulates DNA repair genes by suppressing intronic polyadenylation." Nature 564, no. 7734 (November 28, 2018): 141–45. http://dx.doi.org/10.1038/s41586-018-0758-y.
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Wang, Hong-Wei. "A Link between Intronic Polyadenylation and HR Maintenance Discovered." Biochemistry 58, no. 14 (March 28, 2019): 1835–36. http://dx.doi.org/10.1021/acs.biochem.9b00202.
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Kan, J. "Intronic polyadenylation in the human glycinamide ribonucleotide formyltransferase gene." Nucleic Acids Research 25, no. 15 (August 1, 1997): 3118–23. http://dx.doi.org/10.1093/nar/25.15.3118.
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Lepennetier, Gildas, and Francesco Catania. "Exploring the Impact of Cleavage and Polyadenylation Factors on Pre-mRNA Splicing Across Eukaryotes." G3 Genes|Genomes|Genetics 7, no. 7 (July 1, 2017): 2107–14. http://dx.doi.org/10.1534/g3.117.041483.
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Abstract In human, mouse, and Drosophila, the spliceosomal complex U1 snRNP (U1) protects transcripts from premature cleavage and polyadenylation at proximal intronic polyadenylation signals (PAS). These U1-mediated effects preserve transcription integrity, and are known as telescripting. The watchtower role of U1 throughout transcription is clear. What is less clear is whether cleavage and polyadenylation factors (CPFs) are simply patrolled or if they might actively antagonize U1 recruitment. In addressing this question, we found that, in the introns of human, mouse, and Drosophila, and of 14 other eukaryotes, including multi- and single-celled species, the conserved AATAAA PAS—a major target for CPFs—is selected against. This selective pressure, approximated using DNA strand asymmetry, is detected for peripheral and internal introns alike. Surprisingly, it is more pronounced within—rather than outside—the action range of telescripting, and particularly intense in the vicinity of weak 5′ splice sites. Our study uncovers a novel feature of eukaryotic genes: that the AATAAA PAS is universally counter-selected in spliceosomal introns. This pattern implies that CPFs may attempt to access introns at any time during transcription. However, natural selection operates to minimize this access. By corroborating and extending previous work, our study further indicates that CPF access to intronic PASs might perturb the recruitment of U1 to the adjacent 5′ splice sites. These results open the possibility that CPFs may impact the splicing process across eukaryotes.
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Liu, Liang, Elizabeth Forbes, and Wei Zhang. "Abstract 5646: Altered intronic polyadenylation by mutant p53 impairs transcription of DNA repair genes in lung cancer." Cancer Research 84, no. 6_Supplement (March 22, 2024): 5646. http://dx.doi.org/10.1158/1538-7445.am2024-5646.
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Abstract Introduction: Changes in alternative pre-mRNA processing have been found in many cancers. Aberrant intronic polyadenylation (IPA) causes a stop of normal transcription resulting in the production of truncated mRNA isoform or lncRNA. We sought to examine the role of mutant p53 in pre-mRNA processing, especially in IPA regulation in lung cancer. Methods: We established stable cell lines transfected with mutant p53 (R273H, R175H, H179Q, C238Y, and C242F) or vector control using the NSCLC cell line H1299 with a homozygous deletion of the endogenous p53. Total RNA was used to build libraries enriched in sequences near polyadenylation sites (PASs) using the 3’mRNA-Seq REV library kit (Lexogen; Greenland, NH). Sequencing reads were mapped to the human genome (GRCh37), and PASs with ≥2 supporting reads and ≥0.05 usage in any experiment were identified. Counts for each Intronic Polyadenylation (IPA) and total reads in the terminal exon were compiled into matrices for each condition. DEXseq was applied to detect changes in the relative abundance of each exon part normalized to all exons within the gene. Gene Ontology (GO) analysis was implemented using the enrichR package. Results: We identified 32,752 IPA sites in 5,718 genes, and 26,416 distal PASs (dPASs) in the 3’UTRs of 10,298 genes. Across all five cell lines, when compared individually to the vector control, a comprehensive shift in IPA events was observed, with a concurrent decrease in dPASs. Many of IPA isoforms exhibited consistent patterns of increased or decreased abundance changes. Upon closer examination of each individual cell line, such as the p53-R273H cell line (vs. vector), significant usage changes were identified for 2,389 IPA sites, of which 1,210 were increased and 1,128 terminal exons were altered, with 650 being suppressed. At the gene level, a total of 1,183 genes had at least one significantly increased IPA isoform, a significantly decreased distal isoform, or both. GO analysis showed that these genes were highly enriched in DNA repair-related functional terms such as DNA repair, double-strand break repair, and nucleotide excision repair. The observed IPA changes in DNA-repair genes were consistently observed across the other 4 mutant lines (vs. vector). This observation was further supported by analyzing data from patients with lung adenocarcinoma (LUAD) in the TCGA cohort, where we selected samples carrying nonsense/frameshift mutations, and/or a deep deletion as the p53-null control group, and those carrying missense mutations as the p53-mutant group. Conclusions: Our study illustrated a novel role of p53 mutants in the regulation of intronic polyadenylation processing that contributes to deficient DNA repair. These observations offer a fresh perspective on the multifaceted roles of p53 in cellular homeostasis. Acknowledgment: This work was supported by NCI Cancer Center Support Grant P30CA012197 and NIH/NCI grant R03CA256100. Citation Format: Liang Liu, Elizabeth Forbes, Wei Zhang. Altered intronic polyadenylation by mutant p53 impairs transcription of DNA repair genes in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5646.
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Winstanley-Zarach, Phaedra, Gregor Rot, Shweta Kuba, Aibek Smagul, Mandy J. Peffers, and Simon R. Tew. "Analysis of RNA Polyadenylation in Healthy and Osteoarthritic Human Articular Cartilage." International Journal of Molecular Sciences 24, no. 7 (April 1, 2023): 6611. http://dx.doi.org/10.3390/ijms24076611.
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Polyadenylation (polyA) defines the 3′ boundary of a transcript’s genetic information. Its position can vary and alternative polyadenylation (APA) transcripts can exist for a gene. This causes variance in 3′ regulatory domains and can affect coding sequence if intronic events occur. The distribution of polyA sites on articular chondrocyte transcripts has not been studied so we aimed to define their transcriptome-wide location in age-matched healthy and osteoarthritic knee articular cartilage. Total RNA was isolated from frozen tissue samples and analysed using the QuantSeq-Reverse 3′ RNA sequencing approach, where each read runs 3′ to 5′ from within the polyA tail into the transcript and contains a distinct polyA site. Differential expression of transcripts was significant altered between healthy and osteoarthritic samples with enrichment for functionalities that were strongly associated with joint pathology. Subsequent examination of polyA site data allowed us to define the extent of site usage across all the samples. When comparing healthy and osteoarthritic samples, we found that differential use of polyadenylation sites was modest. However, in the genes affected, there was potential for the APA to have functional relevance. We have characterised the polyadenylation landscape of human knee articular chondrocytes and conclude that osteoarthritis does not elicit a widespread change in their polyadenylation site usage. This finding differentiates knee osteoarthritis from pathologies such as cancer where APA is more commonly observed.
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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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Mueller, Alisa A., Cindy T. van Velthoven, Kathryn D. Fukumoto, Tom H. Cheung, and Thomas A. Rando. "Intronic polyadenylation of PDGFRα in resident stem cells attenuates muscle fibrosis." Nature 540, no. 7632 (November 28, 2016): 276–79. http://dx.doi.org/10.1038/nature20160.
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Sommer, Jan, Christoph Garbers, Janina Wolf, Ahmad Trad, Jens M. Moll, Markus Sack, Rainer Fischer, et al. "Alternative Intronic Polyadenylation Generates the Interleukin-6 Trans-signaling Inhibitor sgp130-E10." Journal of Biological Chemistry 289, no. 32 (June 27, 2014): 22140–50. http://dx.doi.org/10.1074/jbc.m114.560938.
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Wang, Ruijia, Dinghai Zheng, Lu Wei, Qingbao Ding, and Bin Tian. "Regulation of Intronic Polyadenylation by PCF11 Impacts mRNA Expression of Long Genes." Cell Reports 26, no. 10 (March 2019): 2766–78. http://dx.doi.org/10.1016/j.celrep.2019.02.049.
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Castelo-Branco, Pedro, Andre Furger, Matthew Wollerton, Christopher Smith, Alexandra Moreira, and Nick Proudfoot. "Polypyrimidine Tract Binding Protein Modulates Efficiency of Polyadenylation." Molecular and Cellular Biology 24, no. 10 (May 15, 2004): 4174–83. http://dx.doi.org/10.1128/mcb.24.10.4174-4183.2004.
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ABSTRACT Polypyrimidine tract binding protein (PTB) is a major hnRNP protein with multiple roles in mRNA metabolism, including regulation of alternative splicing and internal ribosome entry site-driven translation. We show here that a fourfold overexpression of PTB results in a 75% reduction of mRNA levels produced from transfected gene constructs with different polyadenylation signals (pA signals). This effect is due to the reduced efficiency of mRNA 3′ end cleavage, and in vitro analysis reveals that PTB competes with CstF for recognition of the pA signal's pyrimidine-rich downstream sequence element. This may be analogous to its role in alternative splicing, where PTB competes with U2AF for binding to pyrimidine-rich intronic sequences. The pA signal of the C2 complement gene unusually possesses a PTB-dependent upstream sequence, so that knockdown of PTB expression by RNA interference reduces C2 mRNA expression even though PTB overexpression still inhibits polyadenylation. Consequently, we show that PTB can act as a regulator of mRNA expression through both its negative and positive effects on mRNA 3′ end processing.
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Tian, Shuye, Bin Zhang, Yuhao He, Zhiyuan Sun, Jun Li, Yisheng Li, Hongyang Yi, et al. "CRISPR-iPAS: a novel dCAS13-based method for alternative polyadenylation interference." Nucleic Acids Research 50, no. 5 (February 22, 2022): e26-e26. http://dx.doi.org/10.1093/nar/gkac108.
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Abstract Alternative polyadenylation (APA) plays an important role in gene regulation. With the recent application of novel sequencing technology in APA profiling, an ever-increasing number of APA genes/sites have been identified. However, the phenotypic relevance of most of these APA isoforms remains elusive, which is largely due to the lack of a convenient genetics tool for APA interference. To address this issue, herein, an efficient method is developed based on the CRISPR-dCas13 system, termed as CRISPR-iPAS. Out of eight different dCas13 proteins, Porphyromonas gulae (Pgu) dCas13b, is identified as the most effective one in blocking the usage of the polyadenylation site (PAS). With guide RNAs targeting at core regulatory elements, dPguCas13b enabled APA regulation of endogenous genes with different APA types, including tandem 3′UTR, alternative terminal exon, as well as intronic PAS. Finally, we demonstrated that the proposed APA perturbation tool could be used to investigate the functional relevance of APA isoforms.
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Kaer, Kristel, Jelena Branovets, Anni Hallikma, Pilvi Nigumann, and Mart Speek. "Intronic L1 Retrotransposons and Nested Genes Cause Transcriptional Interference by Inducing Intron Retention, Exonization and Cryptic Polyadenylation." PLoS ONE 6, no. 10 (October 13, 2011): e26099. http://dx.doi.org/10.1371/journal.pone.0026099.
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Elton, Terry S., Victor A. Hernandez, Jessika Carvajal-Moreno, Xinyi Wang, Deborah Ipinmoroti, and Jack C. Yalowich. "Intronic Polyadenylation in Acquired Cancer Drug Resistance Circumvented by Utilizing CRISPR/Cas9 with Homology-Directed Repair: The Tale of Human DNA Topoisomerase IIα." Cancers 14, no. 13 (June 27, 2022): 3148. http://dx.doi.org/10.3390/cancers14133148.
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Intronic polyadenylation (IPA) plays a critical role in malignant transformation, development, progression, and cancer chemoresistance by contributing to transcriptome/proteome alterations. DNA topoisomerase IIα (170 kDa, TOP2α/170) is an established clinical target for anticancer agents whose efficacy is compromised by drug resistance often associated with a reduction of nuclear TOP2α/170 levels. In leukemia cell lines with acquired resistance to TOP2α-targeted drugs and reduced TOP2α/170 expression, variant TOP2α mRNA transcripts have been reported due to IPA that resulted in the translation of C-terminal truncated isoforms with altered nuclear-cytoplasmic distribution or heterodimerization with wild-type TOP2α/170. This review provides an overview of the various mechanisms regulating pre-mRNA processing and alternative polyadenylation, as well as the utilization of CRISPR/Cas9 specific gene editing through homology directed repair (HDR) to decrease IPA when splice sites are intrinsically weak or potentially mutated. The specific case of TOP2α exon 19/intron 19 splice site editing is discussed in etoposide-resistant human leukemia K562 cells as a tractable strategy to circumvent acquired TOP2α-mediated drug resistance. This example supports the importance of aberrant IPA in acquired drug resistance to TOP2α-targeted drugs. In addition, these results demonstrate the therapeutic potential of CRISPR/Cas9/HDR to impact drug resistance associated with aberrant splicing/polyadenylation.
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Druhan, Lawrence J., Amanda Lance, Alicia Hamilton, Nury M. Steuerwald, Elise Tjaden, and Belinda R. Avalos. "Alternative Splicing and Intronic Polyadenylation Post-Transcriptionally Regulate CSF3R Via a Cryptic Exon." Blood 134, Supplement_1 (November 13, 2019): 2462. http://dx.doi.org/10.1182/blood-2019-129102.
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Increasing evidence suggests that alternative splicing of CSF3R occurs during normal granulopoiesis and is altered in certain pathologic conditions. To further examine CSF3R splicing, Sashimi plots from RNA-seq data were generated from CD34+ cells and neutrophils isolated from healthy donors (Figure 1A). The majority of splicing events were found to follow a pattern of enforced exon order resulting in production of CSF3R-V1, and to a lesser extent production of variants 3 and 4 that arise from alternative splicing of distal exonic sequences. However, a significant number of splicing events also mapped to a region within intron 3 (Figure 1A, black hashed box), suggesting a putative cryptic exon (CE). RT-PCR was performed to amplify the region containing the putative CE and Sanger sequencing of the amplification products confirmed the presence of a 73 base pair CE between exons 3 and 4 (E3 and E4). The CE adds an in-frame stop codon that is far removed from the terminal exon; transcripts containing the CE would be predicted to be degraded by nonsense-mediated decay (NMD). We detected transcripts containing the CE sequence in HL-60 cells as well as in CD34+ cells, neutrophils, and monocytes from healthy donors. Cycloheximide had no appreciable effect on the CE-containing transcript, indicating this transcript does not undergo NMD. One mechanism by which the CSF3R CE-containing transcripts could evade NMD would be through alternative polyadenylation (ApA). ApA commonly occurs in the 3'UTRs of mRNAs and is a mechanism for regulating differential transcript expression. ApA can also occur in introns and lead to production of either non-coding RNA or transcripts that produce truncated proteins. Using RT-PCR with a forward primer positioned across the E3:CE junction and a reverse primer positioned within the intronic sequence following the CE and Sanger sequencing of the amplified product, we detected the ApA transcript. The presence of the ApA transcript was confirmed by custom TaqMan assays capable of identifying each of the two predicted CE-containing CSF3R transcripts (Figure 1B). Transcription of either of the CE-containing transcripts would generate mRNAs with a very short open reading frame, and, hence, a protein of only 28 amino acids that is predicted to be signaling-incompetent. Production of the CE-containing CSF3R transcripts could quantitatively alter the amount of full-length CSF3R (V1) transcripts available for production of CSF3R protein, and therefore alter cellular responses to G-CSF. We next examined cells from patients with AML to determine whether expression of the signaling-incompetent CE-containing CSF3R transcripts was altered. Real-time RT-PCR was performed on total RNA isolated from CD34+ cells from healthy donors and from patients with acute myeloid leukemia (AML) to quantitate "productive" CSF3R transcripts (signaling-competent CSF3R-V1 transcripts arising from correct splicing of E3 to E4) versus "non-productive" CSF3R transcripts (signaling-incompetent CSF3R+CE and CSF3R+CEApA transcripts) arising from inclusion of the CE. Significant changes in the relative amounts of CSF3R-V1, CSF3R+CE, and CSF3R+CEApA, and in overall CSF3R transcription were detected in AML samples compared to normal samples. We are currently investigating the effects of mutations in different splicing genes on the output of "productive" versus "non-productive" CSF3R transcripts. In conclusion, we have identified two additional CSF3R transcripts generated by a CE following exon 3 that are expressed in human CD34+ cells and myeloid cells (Figure 1B). Notably, the expression of both CE-containing transcripts effectively decreases expression of the mature CSF3R mRNA, and by extension, of mature full-length CSF3R protein. Our data support a previously unrecognized mechanism of post-transcriptional regulation of CSF3R expression involving a CE and alternative splicing. Recently, widespread intronic polyadenylation of multiple genes including tumor suppressor genes was detected in cells from patients with CLL that resulted in expression of truncated mRNAs and proteins that functionally mimic somatic mutations, which was proposed as a novel mechanism for tumorigenesis. We suggest a similar mechanism involving CSF3R may play a role in AML. Disclosures Avalos: Best Practice-Br Med J: Patents & Royalties: receives royalties from a coauthored article on evaluation of neutropenia; Juno: Membership on an entity's Board of Directors or advisory committees.
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Shulman, Eldad David, and Ran Elkon. "Cell-type-specific analysis of alternative polyadenylation using single-cell transcriptomics data." Nucleic Acids Research 47, no. 19 (September 10, 2019): 10027–39. http://dx.doi.org/10.1093/nar/gkz781.
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AbstractAlternative polyadenylation (APA) is emerging as an important layer of gene regulation because the majority of mammalian protein-coding genes contain multiple polyadenylation (pA) sites in their 3′ UTR. By alteration of 3′ UTR length, APA can considerably affect post-transcriptional gene regulation. Yet, our understanding of APA remains rudimentary. Novel single-cell RNA sequencing (scRNA-seq) techniques allow molecular characterization of different cell types to an unprecedented degree. Notably, the most popular scRNA-seq protocols specifically sequence the 3′ end of transcripts. Building on this property, we implemented a method for analysing patterns of APA regulation from such data. Analyzing multiple datasets from diverse tissues, we identified widespread modulation of APA in different cell types resulting in global 3′ UTR shortening/lengthening and enhanced cleavage at intronic pA sites. Our results provide a proof-of-concept demonstration that the huge volume of scRNA-seq data that accumulates in the public domain offers a unique resource for the exploration of APA based on a very broad collection of cell types and biological conditions.
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Tsuchiya, T., and T. Eulgem. "An alternative polyadenylation mechanism coopted to the Arabidopsis RPP7 gene through intronic retrotransposon domestication." Proceedings of the National Academy of Sciences 110, no. 37 (August 12, 2013): E3535—E3543. http://dx.doi.org/10.1073/pnas.1312545110.
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Gong, Qiuming, Matthew R. Stump, and Zhengfeng Zhou. "Upregulation of functional Kv11.1 isoform expression by inhibition of intronic polyadenylation with antisense morpholino oligonucleotides." Journal of Molecular and Cellular Cardiology 76 (November 2014): 26–32. http://dx.doi.org/10.1016/j.yjmcc.2014.08.007.
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Druhan, Lawrence J., Amanda Lance, Alicia Hamilton, Nury M. Steuerwald, Elise Tjaden, and Belinda R. Avalos. "Altered splicing and intronic polyadenylation of CSF3R via a cryptic exon in acute myeloid leukemia." Leukemia Research 92 (May 2020): 106349. http://dx.doi.org/10.1016/j.leukres.2020.106349.
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Winchester, Joni S., Eric C. Rouchka, Naomi S. Rowland, and Nancy A. Rice. "In Silico characterization of phosphorylase kinase: Evidence for an alternate intronic polyadenylation site in PHKG1." Molecular Genetics and Metabolism 92, no. 3 (November 2007): 234–42. http://dx.doi.org/10.1016/j.ymgme.2007.06.015.
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Wang, Pingzhang, Peng Yu, Peng Gao, Taiping Shi, and Dalong Ma. "Discovery of novel human transcript variants by analysis of intronic single-block EST with polyadenylation site." BMC Genomics 10, no. 1 (2009): 518. http://dx.doi.org/10.1186/1471-2164-10-518.
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Pan, Zhenhua, Haibo Zhang, Lisa K. Hague, Ju Youn Lee, Carol S. Lutz, and Bin Tian. "An intronic polyadenylation site in human and mouse CstF-77 genes suggests an evolutionarily conserved regulatory mechanism." Gene 366, no. 2 (February 2006): 325–34. http://dx.doi.org/10.1016/j.gene.2005.09.024.
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Zhang, Ganggang, Bin Lan, Xin Zhang, Mengyao Lin, Yi Liu, Junsong Chen, and Fang Guo. "AR-A014418 regulates intronic polyadenylation and transcription of PD-L1 through inhibiting CDK12 and CDK13 in tumor cells." Journal for ImmunoTherapy of Cancer 11, no. 5 (May 2023): e006483. http://dx.doi.org/10.1136/jitc-2022-006483.
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BackgroundImmune checkpoint molecules, especially programmed death 1 (PD-1) and its ligand, programmed death ligand 1 (PD-L1), protect tumor cells from T cell-mediated killing. Immune checkpoint inhibitors, designed to restore the antitumor immunosurveillance, have exhibited significant clinical benefits for patients with certain cancer types. Nevertheless, the relatively low response rate and acquisition of resistance greatly limit their clinical applications. A deeper understanding of the regulatory mechanisms of PD-L1 protein expression and activity will help to develop more effective therapeutic strategies.MethodsThe effects of AR-A014418 and THZ531 on PD-L1 expression were detected by western blot, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and flow cytometry. In vitro kinase assays with recombinant proteins were performed to confirm that AR-A014418 functioned as a CDK12 and CDK13 dual inhibitor. The roles of CDK12 and CDK13 in intronic polyadenylation (IPA) and transcription of PD-L1 were determined via RNA interference or protein overexpression. T-cell cytotoxicity assays were used to validate the activation of antitumor immunity by AR-A014418 and THZ531.ResultsAR-A014418 inhibits CDK12 to enhance the IPA, and inhibits CDK13 to repress the transcription of PD-L1. IPA generates a secreted PD-L1 isoform (PD-L1-v4). The extent of IPA was not enough to reduce full-length PD-L1 expression obviously. Only the superposition of enhancing IPA and repressing transcription (dual inhibition of CDK12 and CDK13) dramatically suppresses full-length PD-L1 induction by interferon-γ. AR-A014418 and THZ531 could potentiate T-cell cytotoxicity against tumor cells.ConclusionsOur work identifies a new regulatory pathway for PD-L1 expression and discovers CDK12 and CDK13 as promising drug targets for immune modulation and combined therapeutic strategies.
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Pawlicki, Jan M., and Joan A. Steitz. "Primary microRNA transcript retention at sites of transcription leads to enhanced microRNA production." Journal of Cell Biology 182, no. 1 (July 14, 2008): 61–76. http://dx.doi.org/10.1083/jcb.200803111.
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MicroRNAs (miRNAs) are noncoding RNAs with important roles in regulating gene expression. In studying the earliest nuclear steps of miRNA biogenesis, we observe that primary miRNA (pri-miRNA) transcripts retained at transcription sites due to the deletion of 3′-end processing signals are converted more efficiently into precursor miRNAs (pre-miRNAs) than pri-miRNAs that are cleaved, polyadenylated, and released. Flanking exons, which also increase retention at transcription sites, likewise contribute to increased levels of intronic pri-miRNAs. Consistently, efficiently processed endogenous pri-miRNAs are enriched in chromatin-associated nuclear fractions. In contrast, pri-miRNAs that accumulate to high nuclear levels after cleavage and polyadenylation because of the presence of a viral RNA element (the ENE of the Kaposi's sarcoma–associated herpes virus polyadenylated nuclear RNA) are not efficiently processed to precursor or mature miRNAs. Exogenous pri-miRNAs unexpectedly localize to nuclear foci containing splicing factor SC35; yet these foci are unlikely to represent sites of miRNA transcription or processing. Together, our results suggest that pri-miRNA processing is enhanced by coupling to transcription.
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Urbanczyk, Andreas, Anselm Jünemann, and Ralf Enz. "PKCζ-interacting protein ZIP3 is generated by intronic polyadenylation, and is expressed in the brain and retina of the rat." Biochemical Journal 433, no. 1 (December 15, 2010): 43–50. http://dx.doi.org/10.1042/bj20101111.
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Scaffold proteins contain multiple protein–protein interaction modules that physically assemble functionally related proteins into larger complexes. ZIPs [PKC (protein kinase C) ζ-interacting proteins] link the enzymatic activity of the atypical PKC isoforms PKCλ/ι or PKCζ to target proteins and are associated with neurodegenerative disorders. In the rat, alternative splicing generates three ZIP variants. Previously, we identified the ZIP3 transcript, containing 13 C-terminal amino acids encoded by intron 4, in the rat CNS (central nervous system). In the present study, we identified intronic polyadenylation signals in rat and human ZIP genes [known as SQSTM1 (sequestosome-1) in humans] and detected the corresponding ZIP3-like transcripts. In addition, we generated ZIP3-specific immune sera and observed expression of the protein in the brain and retina of the adult rat. In the retina, ZIP3 is present in nuclear layers where it co-localizes with PKCζ. An immune serum recognizing all three ZIP isoforms labelled the same cells as the newly generated ZIP3-specific antibodies and, in addition, stained both synaptic layers of the retina. There, ZIPs are localized in axon terminals of rod bipolar cells that also contain ZIP-interacting PKCζ and GABAC (γ-aminobutyric acid type C) receptors. In summary, we detected ZIP3-like transcripts in rat- and human-derived samples and describe the expression of ZIP3 in the rat CNS.
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Wu, Zhe, Robert Ietswaart, Fuquan Liu, Hongchun Yang, Martin Howard, and Caroline Dean. "Quantitative regulation of FLC via coordinated transcriptional initiation and elongation." Proceedings of the National Academy of Sciences 113, no. 1 (December 22, 2015): 218–23. http://dx.doi.org/10.1073/pnas.1518369112.
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The basis of quantitative regulation of gene expression is still poorly understood. In Arabidopsis thaliana, quantitative variation in expression of FLOWERING LOCUS C (FLC) influences the timing of flowering. In ambient temperatures, FLC expression is quantitatively modulated by a chromatin silencing mechanism involving alternative polyadenylation of antisense transcripts. Investigation of this mechanism unexpectedly showed that RNA polymerase II (Pol II) occupancy changes at FLC did not reflect RNA fold changes. Mathematical modeling of these transcriptional dynamics predicted a tight coordination of transcriptional initiation and elongation. This prediction was validated by detailed measurements of total and chromatin-bound FLC intronic RNA, a methodology appropriate for analyzing elongation rate changes in a range of organisms. Transcription initiation was found to vary ∼25-fold with elongation rate varying ∼8- to 12-fold. Premature sense transcript termination contributed very little to expression differences. This quantitative variation in transcription was coincident with variation in H3K36me3 and H3K4me2 over the FLC gene body. We propose different chromatin states coordinately influence transcriptional initiation and elongation rates and that this coordination is likely to be a general feature of quantitative gene regulation in a chromatin context.
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Kan, Julie L. C., and Richard G. Moran. "Analysis of a Mouse Gene Encoding Three Steps of Purine Synthesis Reveals Use of an Intronic Polyadenylation Signal without Alternative Exon Usage." Journal of Biological Chemistry 270, no. 4 (January 27, 1995): 1823–32. http://dx.doi.org/10.1074/jbc.270.4.1823.
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Thomas, Christie P., Janet I. Andrews, and Kang Z. Liu. "Intronic polyadenylation signal sequences and alternate splicing generate human soluble Fltl variants and regulate the abundance of soluble Flt1 in the placenta." FASEB Journal 21, no. 14 (July 5, 2007): 3885–95. http://dx.doi.org/10.1096/fj.07-8809com.
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Samur, Mehmet K., Irtisha Singh, Lee Shih-Han, Adam Samuel Sperling, Mariateresa Fulciniti, Yu-Tzu Tai, Giovanni Parmigiani, Christina S. Leslie, Christine Mayr, and Nikhil C. Munshi. "3' Untranslated Region (UTR) Alterations Are Frequently Targeted By MM-Related Mirnas and Affects the Clinical Outcome." Blood 128, no. 22 (December 2, 2016): 4447. http://dx.doi.org/10.1182/blood.v128.22.4447.4447.
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Abstract More than half of human genes use alternative cleavage and polyadenylation to generate alternative 3' untranslated region (3'UTR) isoforms that play a role in gene expression regulation. The 3' untranslated region (3'UTR) of mRNA contains elements that play regulatory roles in polyadenylation, localization, translation efficiency, and mRNA stability. Although relative contributions of different regulatory mechanisms remain poorly understood, several recent studies showed that alterations in 3'UTRs might affect protein localization as well as their interactions with other proteins. Here, we obtained global measurements of 3' UTR sequencing and RNA-seq in MM and normal plasma cells to study the effects of 3'UTR alterations in MM. Whole transcriptome sequencing (RNAseq) data from 420 uniformly treated newly diagnosed MM samples were compared with 18 normal plasma cell by using dynamic analyses of alternative PolyAdenylation and results were compared with alternative 3'UTR isoforms measured using 3'-seq, an established quantitative 3' end sequencing method from 15 samples pilot study. We observed ~10000 different isoforms with a median frequency of 297 (range 1-2495) altered 3'UTRs per sample. We observed 563 isoforms that have distal alternative poly A (APA) site and 449 isoforms that have proximal APA site compared to normal plasma cells in at least 10% of our patients. Enrichment analysis showed that short UTR genes are significantly targeted by miR-506, miR-133, miR-130, miR-27, miR-181 and miR-200 (FDR q-value < 0.05). We also observed that expression of 90 genes significantly increased between those samples with shorter UTR vs. regular UTR. Longer UTR genes are enriched in mir-124, mir-186, mir-200, mir-302, mir-495 and expression of 123 genes were significantly downregulated between long UTR and others. There were no tumor suppressor genes and 8 oncogenes in short UTR genes and 4 tumor suppressor and 14 oncogenes in longer UTR genes. Our results were confirmed using the 3'seq study which showed that 170 genes have longer UTR in MM compared to normal plasma cells and 187 genes have shorter UTR. ~60% of longer and ~65% of shorter UTR genes reported by 3'seq were also observed with RNAseq. Furthermore, we focused on common genes reported by both high-throughput sequencing methods and analyzed any connection between outcome and UTR shortening or lengthening. We observed 26 genes with altered UTR regions have impact on PFS or OS. In conclusion, we report significant alternate UTR usage, including intronic UTRs in MM affecting the disease biology and clinical outcome. Our data suggests the need to further investigate the molecular impact of alternate UTR usage and its relationship with miR in myeloma. Disclosures No relevant conflicts of interest to declare.
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Thomas, Christie P., Nandita S. Raikwar, Elizabeth A. Kelley, and Kang Z. Liu. "Alternate processing of Flt1 transcripts is directed by conserved cis -elements within an intronic region of FLT1 that reciprocally regulates splicing and polyadenylation." Nucleic Acids Research 38, no. 15 (April 10, 2010): 5130–40. http://dx.doi.org/10.1093/nar/gkq198.
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Lian, Jin, Zheng Lian, Alexander Karpikov, Milind Mahajan, Mark Gerstein, Michael Snyde, and Sherman Weissman. "Genomic Distribution of Transcripts and DNA Associated Proteins in One Percent of the Genome of Erythroid and Myeloid Cells." Blood 108, no. 11 (November 16, 2006): 4201. http://dx.doi.org/10.1182/blood.v108.11.4201.4201.
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Abstract The NIHGRI has sponsored a multi-group enterprise to use a variety of approaches to map DNA elements in one per cent of the human genome-the ENCODE Project. As part of this effort we have mapped sites of active transcription (transcriptionally active regions or TARS) for myeloid cells including the undifferentiated and retinoic acid treated promyelocytic NB4 cell line and normal neutrophils, as well as the erythroid cell line K562. The results show a large number of intergenic and intronic transcripts, particularly in neutrophils. One feature of note is the amount of cell type specific intergenic transcription in the HoxA cluster. In addition we have been developing simple methods to map the sites of polyadenylation of RNAs and applied this to several cell types. This method also indicates the presence of polyadenylated transcripts derived from the multiple introns of known genes as well as intergenic regions. In parallel with the transcript mapping, we have been performing microarray based chromatin immunoprecipitation studies (Chip-chip) on several factors, analyzing them on oligonucleotide arrays tiling the ENCODE selected genomic regions. These include various phosphorylated forms of RNA polymerase, various methylated forms of histone H3, certain DNA sequence specific transcription factors, and some other factors either broadly involved in chromatin remodeling or specifically found to be associated with certain sequences in the beta globin cluster. Among other things, our results as well as those of other members of the ENCODE consortium, provide a signature motif commonly associated with promoters.
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Luo, Wenting, Zhe Ji, Zhenhua Pan, Bei You, Mainul Hoque, Wencheng Li, Samuel I. Gunderson, and Bin Tian. "The Conserved Intronic Cleavage and Polyadenylation Site of CstF-77 Gene Imparts Control of 3′ End Processing Activity through Feedback Autoregulation and by U1 snRNP." PLoS Genetics 9, no. 7 (July 11, 2013): e1003613. http://dx.doi.org/10.1371/journal.pgen.1003613.
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Ganaie, Safder S., Aaron Yun Chen, Chun Huang, Peng Xu, Steve Kleiboeker, Aifang Du, and Jianming Qiu. "RNA Binding Protein RBM38 Regulates Expression of the 11-Kilodalton Protein of Parvovirus B19, Which Facilitates Viral DNA Replication." Journal of Virology 92, no. 8 (February 7, 2018): e02050-17. http://dx.doi.org/10.1128/jvi.02050-17.
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ABSTRACTHuman parvovirus B19 (B19V) expresses a single precursor mRNA (pre-mRNA), which undergoes alternative splicing and alternative polyadenylation to generate 12 viral mRNA transcripts that encode two structural proteins (VP1 and VP2) and three nonstructural proteins (NS1, 7.5-kDa protein, and 11-kDa protein). Splicing at the second 5′ donor site (D2 site) of the B19V pre-mRNA is essential for the expression of VP2 and the 11-kDa protein. We previously identified thatcis-acting intronic splicing enhancer 2 (ISE2) that lies immediately after the D2 site facilitates the recognition of the D2 donor for its efficient splicing. In this study, we report that ISE2 is critical for the expression of the 11-kDa viral nonstructural protein. We found that ISE2 harbors a consensus RNA binding motif protein 38 (RBM38) binding sequence, 5′-UGUGUG-3′. RBM38 is expressed during the middle stage of erythropoiesis. We first confirmed that RBM38 binds specifically with the ISE2 elementin vitro. The knockdown of RBM38 significantly decreases the level of spliced mRNA at D2 that encodes the 11-kDa protein but not that of the D2-spliced mRNA that encodes VP2. Importantly, we found that the 11-kDa protein enhances viral DNA replication and virion release. Accordingly, the knockdown of RBM38 decreases virus replication via downregulating 11-kDa protein expression. Taken together, these results suggest that the 11-kDa protein facilitates B19V DNA replication and that RBM38 is an essential host factor for B19V pre-mRNA splicing and for the expression of the 11-kDa protein.IMPORTANCEB19V is a human pathogen that can cause fifth disease, arthropathy, anemia in immunocompromised patients and sickle cell disease patients, myocarditis, and hydrops fetalis in pregnant women. Human erythroid progenitor cells (EPCs) are most susceptible to B19V infection and fully support viral DNA replication. The exclusive tropism of B19V for erythroid-lineage cells is dependent not only on the expression of viral receptors and coreceptors on the cell surface but also on the intracellular host factors that support B19V replication. Our present study shows that B19V uses a host factor, RNA binding motif protein 38 (RBM38), for the processing of its pre-mRNA during virus replication. Specifically, RBM38 interacts with the intronic splicing enhancer 2 (ISE2) element of B19V pre-mRNA and promotes 11-kDa protein expression, thereby regulating the 11-kDa protein-mediated augmentation of B19V replication. The identification of this novel host-pathogen interaction will provide mechanistic insights into B19V replication and aid in finding new targets for anti-B19V therapeutics.
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Ameri, Afshin, Deepa K. Machiah, Darlene Livingston, Thuy T. Tran, Cynthia Channell, Kristen L. Toren, Valerie Crenshaw, and Tom Howard. "A Novel 5bp Deletion Mutation in the Factor X (FX) Gene, Designated FX-Augusta, Causes Severe FX Deficiency Possibly by a Unique Mechanism Involving mRNAs that Lack Inframe Stop Codons." Blood 104, no. 11 (November 16, 2004): 1045. http://dx.doi.org/10.1182/blood.v104.11.1045.1045.
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Abstract Due to its position in the coagulation cascade, factor X (fX), a circulating zymogen is activated during hemostatic challenges to its serine protease fXa first via the extrinsic pathway’s tissue factor/factor VIIa complex and next by the intrinsic Xase complex of factors Ixa and VIIIa on phospholipids membranes. Among the congenital factor abnormalities manifesting as hemorrhagic predispositions, fX deficiency states, which are often autosomal recessive and associated with consanguinity, are among the rarest. Since concurrent studies on the unrelated Stuart and Prower kindreds in the southeastern United States and London (England), respectively, led to the discovery of fX, it is also known as the Stuart-Prower factor. Only 59 distinct fX gene mutations have been described worldwide since its discovery in the 1950s. We report a novel mutation within the encoding structural locus that we have designated FX-Augusta. The phenotype and genotype of the proband, a 14 year-old African-American boy, were studied and we are currently in the process of obtaining samples from the patient’s parents and siblings. The proband has experienced severe bleeding throughout his life with his first episode occurring three days after birth following circumcision. He experiences ~30 bleeds per year, mainly into joints and muscles, which occur spontaneously or following minor trauma. Although the patient appears to be mildly mentally retarded a formal workup has not occurred. On laboratory testing, a severely prolonged PT of 137.4 seconds and aPTT of 112.8 seconds were observed. While clotting-based fX activity (fX:C) levels were consistently <1% when using fX-deficient plasma and the PT-reagent, chromogenic factor X activity levels were ~5%. In light of these findings, we suspect the patient may have a CRM-reduced, or less-likely a CRM-positive, deficiency. However, the patient’s fX antigen level (fX:Ag) has not yet been measured. Molecular analysis of the patient’s fX gene by direct automated sequencing of PCR-amplicons representing ~200-bp of the promoter, all exonic and flanking intronic regions and ~200-bp of the flanking 3′-genomic DNA, disclosed a novel 5-bp deletion mutation (ATGCC) in exon 8 (del[1350–1354]; 1 is the transcription start site) that disrupts codons 477 and 478, and results in a frameshift. The patient is homozygous for this deletion and an additional 8-bp insertion mutation (TGCCGCCA) located downstream in the 3′-flanking genomic DNA between nucleotides 28376 and 28377 (GenBank #: AF503510). Previously, no insertions and only 4 small deletions have been reported for this gene. Interestingly, the wildtype fX mRNA has a 14-base 3′-UTR as the AUUAAA hexanucleotide polyadenylation element in the fX gene is located 1-bp upstream of the stop codon. Because the resultant frameshift predicts the use of an alternative stop codon 75 nucleotides downstream, 3′ of the in vivo polyadenylation site, this mutation may result in mature cytoplasmic transcripts that lack in-frame stop codons. To our knowledge this has not been reported. Since non-sense mediated decay has not been described for mutations that necessitate the use of downstream stop codons, if we are correct, translation of the mutant mRNAs would not terminate normally and the ribosome would translate the poly(A)-tail into a C-terminal poly-lysine stretch. We hypothesize that the mutant fX pre-mRNAs will be cleaved and polyadenylated immediately 3′ of nucleotide 28346, the same site utilized in vivo during 3′-end processing of the wildtype pre-mRNAs for the following reasons. First, there are no downstream signals capable of supporting 3′-end formation. Specifically, an examination of >1kb of the flanking 3′-genomic DNA failed to identify consensus AAUAAA polyadenylation signals or variants of this highly conserved element. Second, the native fX 3′-end signals appear to be “strong” or efficiently utilized, as no alternative polyadenylation was found in a comprehensive review of the fX cDNAs in the NCBI database, which included 6 full-length cDNAs and >20 3′-ESTs containing polyA-stretches on the 3′-end of high-quality fX mRNA sequence. Finally, neither the causative 5-bp deletion nor the 8-bp insertion disrupts these native 3′-end signals. Nevertheless, the molecular studies underway, including fX:Ag measurements, 3′-end characterization of the mutant mRNA via 3′-RACE and western blotting, should help to distinguish between the possible molecular mechanisms.
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Hernandez, Victor A., Jessika Carvajal-Moreno, Xinyi Wang, Maciej Pietrzak, Jack C. Yalowich, and Terry S. Elton. "Use of CRISPR/Cas9 with homology-directed repair to silence the human topoisomerase IIα intron-19 5’ splice site: Generation of etoposide resistance in human leukemia K562 cells." PLOS ONE 17, no. 5 (May 26, 2022): e0265794. http://dx.doi.org/10.1371/journal.pone.0265794.
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DNA Topoisomerase IIα (TOP2α/170) is an enzyme essential for proliferating cells. For rapidly multiplying malignancies, this has made TOP2α/170 an important target for etoposide and other clinically active anticancer drugs. Efficacy of these agents is often limited by chemoresistance related to alterations in TOP2α/170 expression levels. Our laboratory recently demonstrated reduced levels of TOP2α/170 and overexpression of a C-terminal truncated 90-kDa isoform, TOP2α/90, due to intronic polyadenylation (IPA; within intron 19) in an acquired etoposide-resistant K562 clonal cell line, K/VP.5. We previously reported that this isoform heterodimerized with TOP2α/170 and was a determinant of acquired resistance to etoposide. Optimization of the weak TOP2α exon 19/intron 19 5′ splice site in drug-resistant K/VP.5 cells by gene-editing restored TOP2α/170 levels, diminished TOP2α/90 expression, and circumvented drug resistance. Conversely, in the present study, silencing of the exon 19/intron 19 5′ splice site in parental K562 cells by CRISPR/Cas9 with homology-directed repair (HDR), and thereby forcing intron 19 retention, was used to induce resistance by disrupting normal RNA processing (i.e., gene knockout), and to further evaluate the role of TOP2α/170 and TOP2α/90 isoforms as resistance determinants. Gene-edited clones were identified by quantitative polymerase chain reaction (qPCR) and verified by Sanger sequencing. TOP2α/170 mRNA/protein expression levels were attenuated in the TOP2α gene-edited clones which resulted in resistance to etoposide as assessed by reduced etoposide-induced DNA damage (γH2AX, Comet assays) and growth inhibition. RNA-seq and qPCR studies suggested that intron 19 retention leads to decreased TOP2α/170 expression by degradation of the TOP2α edited mRNA transcripts. Forced expression of TOP2α/90 in the gene-edited K562 cells further decreased etoposide-induced DNA damage in support of a dominant negative role for this truncated isoform. Together results support the important role of both TOP2α/170 and TOP2α/90 as determinants of sensitivity/resistance to TOP2α-targeting agents.
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Millar, R., D. Conklin, C. Lofton-Day, E. Hutchinson, B. Troskie, N. Illing, SC Sealfon, and J. Hapgood. "A novel human GnRH receptor homolog gene: abundant and wide tissue distribution of the antisense transcript." Journal of Endocrinology 162, no. 1 (July 1, 1999): 117–26. http://dx.doi.org/10.1677/joe.0.1620117.
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Gonadotropin releasing hormone (GnRH) regulates the reproductive system through a specific G-protein-coupled receptor (GPCR) in pituitary gonadotropes. The existence of two (or more) forms of GnRH in most vertebrates suggested the existence of GnRH receptor subtypes (I and II). Using sequence information for extracellular loop 3 of a putative Type II GnRH receptor from a reptile species, we have looked for a Type II GnRH receptor gene in the human genome EST (expressed sequence tag) database. A homolog was identified which has 45% and 41% amino acid identity with exons 2 and 3 of the known human GnRH pituitary receptor (designated Type I) and much lower homology with all other GPCRs. A total of 27 contiguous ESTs was found and comprised a continuous sequence of 1642 nucleotides. The EST sequences were confirmed in the cloned human gene and in PCR products of cDNA from several tissues. All EST transcripts detected were in the antisense orientation with respect to the novel GnRH receptor sequence and were highly expressed in a wide range of human brain and peripheral tissues. PCR of cDNA from a wide range of tissues revealed that intronic sequence equivalent to intron 2 of the Type I GnRH receptor was retained. The failure to splice out putative intron sequences in transcripts which spanned exon-intron boundaries is expected in antisense transcripts, as candidate donor and acceptor sites were only present in the gene when transcribed in the orientation encoding the GnRH receptor homolog. No transcripts extended 5' to the sequence corresponding to intron 2 of the Type I GnRH as the antisense transcripts terminated in poly A due to the presence of a polyadenylation signal sequence in the putative intron 2 when transcribed in the antisense orientation. These findings suggest that a Type II GnRH receptor gene has arisen during vertebrate evolution and is also present in the human. However, the receptor may have become vestigial in the human, possibly due to the abundant and universal tissue transcription of the opposite DNA strand to produce antisense RNA.
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Frank, Sander, Ilsa Coleman, Navonil De Sarkar, Dmytro Rudoy, Valeri Vasioukhin, and Pete Nelson. "Abstract B060: Characterization of DNA repair defects in CDK12 mutant prostate cancer and the identification of differential vulnerabilities." Cancer Research 83, no. 11_Supplement (June 2, 2023): B060. http://dx.doi.org/10.1158/1538-7445.prca2023-b060.
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Abstract Background: CDK12 expression is lost in ~5% of metastatic castration-resistant prostate cancer (mCRPC) and associates with aggressive disease. Previous literature, based on acute CDK12-loss models, proposes a mechanism where CDK12 loss leads to a homologous recombination deficiency (HRD) phenotype via premature intronic polyadenylation of classic HR pathway genes, including BRCA1 and BRCA2. Cyclin K is an essential gene and functions in a heterodimer with CDK12 or CDK13. We sought to test if CDK12 loss: (1) confers HRD and a corresponding sensitivity to platinum and poly-ADP ribose polymerase (PARP) inhibitors and/or (2) confers synthetic lethality to other cellular targets and pathways. Methods: To investigate the role of CDK12 loss we used a combination of acute (Tet-inducible shRNA, CDK12/13 inhibitors) and adapted (naturally mutant LuCaP 189.4, CRISPR KO clones) models. DNA repair gene expression and polyadenylation site usage was measured by RNA-seq, qPCR, and western blot while HR function was measured by immunofluorescence for RAD51 foci following irradiation. Stable CDK12-negative (CDK12-neg) cell lines were treated with sgRNAs, PARP inhibitors, carboplatin, and CDK inhibitors to test for potential targeted vulnerabilities. Results: Acute CDK12 inhibition (6h, SR4835) confirmed the reported mechanism of 3’ transcript loss, but the effects were minimal in long term shCDK12 or CRISRP KO lines as measured by RNA-seq, qPCR, and western blot. CDK12-neg lines showed slight decreases in ATR and ATM protein but minimal changes in BRCA1 or BRCA2 and no decrease in RAD51 foci formation. Furthermore, these lines did not show increased sensitivity to carboplatin and only partial sensitivity to PARPi. Human tumor data were analyzed and showed that CDK12-neg tumors lack genomic signatures of HRD and do not show significant de-enrichment of long genes. However, CDK12-neg cell lines did show sensitivity to CDK13 KO or pharmacologic inhibition with CDK12/13 inhibitors (SR4835, THZ531, dinaciclib). In vivo treatment of LuCaP xenograft lines showed that 189.4 responded to SR4835, while two CDK12-intact lines (35 and 136) did not. Conclusions: Our data support a model where HRD is primarily a result of acute CDK12 loss, while the downregulation of long DNA repair genes is largely compensated for (via currently unknown mechanisms) in cells that have adapted to CDK12 loss. We suspect that the partial PARPi sensitivity observed in CDK12-neg lines is due to other (non-HRD) DNA repair deficiencies and potential mechanisms are under investigation. CDK12-neg cells exhibited dependency on CDK13, which can be targeted with semi-selective pharmacologic compounds, though our data support the development of specific CDK13 inhibitors to maximize synthetic lethality and minimize off-target effects. Finally, additional work is ongoing with CRISPR and drug screens to identify additional vulnerabilities in adapted CDK12-neg models and provide future clinical targets for this aggressive subset of mCRPC. Citation Format: Sander Frank, Ilsa Coleman, Navonil De Sarkar, Dmytro Rudoy, Valeri Vasioukhin, Pete Nelson. Characterization of DNA repair defects in CDK12 mutant prostate cancer and the identification of differential vulnerabilities [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B060.
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Tian, B., Z. Pan, and J. Y. Lee. "Widespread mRNA polyadenylation events in introns indicate dynamic interplay between polyadenylation and splicing." Genome Research 17, no. 2 (January 8, 2007): 156–65. http://dx.doi.org/10.1101/gr.5532707.
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Cooke, Charles, and James C. Alwine. "Characterization of Specific Protein-RNA Complexes Associated with the Coupling of Polyadenylation and Last-Intron Removal." Molecular and Cellular Biology 22, no. 13 (July 1, 2002): 4579–86. http://dx.doi.org/10.1128/mcb.22.13.4579-4586.2002.
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ABSTRACT Polyadenylation and splicing are highly coordinated on substrate RNAs capable of coupled polyadenylation and splicing. Individual elements of both splicing and polyadenylation signals are required for the in vitro coupling of the processing reactions. In order to understand more about the coupling mechanism, we examined specific protein-RNA complexes formed on RNA substrates, which undergo coupled splicing and polyadenylation. We hypothesized that formation of a coupling complex would be adversely affected by mutations of either splicing or polyadenylation elements known to be required for coupling. We defined three specific complexes (AC′, AC, and BC) that form rapidly on a coupled splicing and polyadenylation substrate, well before the appearance of spliced and/or polyadenylated products. The AC′ complex is formed by 30 s after mixing, the AC complex is formed between 1 and 2 min after mixing, and the BC complex is formed by 2 to 3 min after mixing. AC′ is a precursor of AC, and the AC′ and/or AC complex is a precursor of BC. Of the three complexes, BC appears to be a true coupling complex in that its formation was consistently diminished by mutations or experimental conditions known to disrupt coupling. The characteristics of the AC′ complex suggest that it is analogous to the spliceosomal A complex, which forms on splicing-only substrates. Formation of the AC′ complex is dependent on the polypyrimidine tract. The transition from AC′ to AC appears to require an intact 3′-splice site. Formation of the BC complex requires both splicing elements and the polyadenylation signal. A unique polyadenylation-specific complex formed rapidly on substrates containing only the polyadenylation signal. This complex, like the AC′ complex, formed very transiently on the coupled splicing and polyadenylation substrate; we suggest that these two complexes coordinate, resulting in the BC complex. We also suggest a model in which the coupling mechanism may act as a dominant checkpoint in which aberrant definition of one exon overrides the normal processing at surrounding wild-type sites.
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Ismail, Said I., Jonathan B. Rohll, Susan M. Kingsman, Alan J. Kingsman, and Mark Uden. "Use of Intron-Disrupted Polyadenylation Sites To Enhance Expression and Safety of Retroviral Vectors." Journal of Virology 75, no. 1 (January 1, 2001): 199–204. http://dx.doi.org/10.1128/jvi.75.1.199-204.2001.
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ABSTRACT Normal mRNA polyadenylation signals are composed of an AAUAAA motif and G/U box spaced 20 to 30 bp apart. If this spacing is increased further, then polyadenylation is disrupted. Previously it has been demonstrated that insertion of an intron will similarly disrupt this signal even though such introns are removed during a nuclear splicing reaction (X. Liu and J. Mertz, Nucleic Acids Res. 21:5256–5263, 1993). This observation has led to the suggestion that polyadenylation site selection is undertaken prior to intron excision. We now present results that both support and extend these observations and in doing so create a novel class of retroviral expression vector with improved qualities. We found that when an intron-disrupted polyadenylation signal is inserted within a retroviral expression vector, such a signal, although reformed in the producer cell, remains benign until transduction, where it is then preferentially used. Thus, we demonstrate that upon transduction these vectors now produce a majority of shortened subgenomic species and as a consequence have a reduced tendency for subsequent mobilization from transduced cells. In addition, we demonstrate that the use of this internal signal leads to enhanced expression from such vectors and that this is achieved without any loss in titer. Therefore, split polyadenylation signals confer enhanced performance and improved safety upon retroviral expression vectors into which they are inserted. Such split signals may prove useful for the future optimization of retroviral vectors in gene therapy.
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Yamakawa, Hiroko, Shunsuke Ebara, Akio Mizutani, Misaki Yoshida, Midori Sugiyama, Koji Yamamoto, Daisuke Komura, et al. "Abstract 3301: Translational research of CDK12/13 inhibitor, CTX-439, informing clinical trial strategy." Cancer Research 84, no. 6_Supplement (March 22, 2024): 3301. http://dx.doi.org/10.1158/1538-7445.am2024-3301.
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Abstract Gene dysregulation is a hallmark of cancer, arising from genetic alterations that invariably lead to dysregulated transcriptional programs. This dysregulation renders cancer cells highly dependent on certain gene expression regulators, leading to a potential for novel therapeutic strategies. Consequently, ongoing research aims to develop small molecules targeting RNA transcriptional processes. Cyclin-dependent kinase 12/13 (CDK12/13) is a member of the cyclin-dependent kinase family of serine/threonine protein kinases. CDK12/13 regulates the RNA transcription elongation by phosphorylating RNA polymerase II and promotes the elongation of transcripts, specifically those involved in DNA damage responses, such as BRCA1/2. The inhibition of CDK12/13 is expected to exert a synergistic effect when combined with PARP inhibitors and chemotherapeutic reagents. Therefore, we developed a novel small molecule inhibitor, CTX-439 (former name: CRD-1835439). Additionally, we demonstrated that CTX-439 exhibits a preclinical efficacy in vitro and in vivo [Cancer Res. (2022) 82 (12_Supplement): 5485]. To identify potential strategies for a clinical trial of CTX-439, in the current study, we conducted translational research to elucidate (1) pharmacodynamics (PD) markers to monitor the suppression of CDK12/13 in humans, (2) patient selection biomarkers to determine sensitivity to CTX-439, and (3) combination strategies with other key medications. Firstly, to identify PD markers of CTX-439, we performed a comprehensive analysis employing RNA-seq, Pol II-ChIP-seq, and Poly(A)-seq. We confirmed that CTX-439 induced the usage of intronic polyadenylation sites by inhibiting CDK12, resulting in the production of shortened mRNA. Of these transcripts, PCF11 emerged as a potential PD marker for clinical trials, which was confirmed using qPCR analysis of human blood. Secondly, to clarify the indication of CTX-439, we performed an in vitro survey involving 400 cell lines and 100 PDX-derived organoids (PDXOs) to identify biomarkers displaying sensitivity and insensitivity to CTX-439. Furthermore, a significant anti-tumor effect of CTX-439 was revealed in PDX models that showed sensitivity in the survey. CTX-439 exhibited a synergistic effect in combination with PARP inhibitors both in vitro and in vivo, highlighting its potential for use in combination strategies. Additionally, CTX-439 showed an anti-tumor effect when combined with other chemotherapy and molecular-targeted drugs. Collectively, the results from these translational research studies provide a foundation for developing a clinical trial by clearly defining PD monitoring, patient stratification biomarkers, and combination strategies. These findings highlight the therapeutic potential of CTX-439 as a single-agent and in combination with inhibitors, such as PARP, for the treatment of cancers characterized by transcriptional addiction. Citation Format: Hiroko Yamakawa, Shunsuke Ebara, Akio Mizutani, Misaki Yoshida, Midori Sugiyama, Koji Yamamoto, Daisuke Komura, Miwako Kakiuchi, Shumpei Ishikawa, Kosuke Yusa, Daisuke Morishita. Translational research of CDK12/13 inhibitor, CTX-439, informing clinical trial strategy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3301.
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Qiu, Jianming, Ramnath Nayak, and David J. Pintel. "Alternative Polyadenylation of Adeno-Associated Virus Type 5 RNA within an Internal Intron Is Governed by both a Downstream Element within the Intron 3′ Splice Acceptor and an Element Upstream of the P41 Initiation Site." Journal of Virology 78, no. 1 (January 1, 2004): 83–93. http://dx.doi.org/10.1128/jvi.78.1.83-93.2004.
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ABSTRACT Adeno-associated virus type 5 (AAV5) has a linear, single-stranded DNA genome of ca. 5 kb and an overlapping transcription profile featuring multiple promoters and a single intron in the center of the genome. Unlike the situation for the prototype AAV2, AAV5 RNAs transcribed from upstream promoters at map units 7 (P7) and 19 (P19), which encode the viral Rep proteins, are predominantly polyadenylated at a site within the intron [(pA)p]. RNAs generated from the AAV5 capsid gene promoter P41, which is only 78 nucleotides (nt) upstream of the intron donor, and 281 nt upstream of (pA)p, primarily readthrough (pA)p, are polyadenylated at a more distal site at the 3′ end of the genome [(pA)d] and ultimately spliced. The intron contains the core sequences sufficient for polyadenylation at (pA)p, which is governed by a G/U-rich downstream element that overlaps with the intron 3′ A2 splice acceptor. In addition, polyadenylation of P7- and P19-generated RNAs at (pA)p is influenced by an upstream element that lies 5′ to the start of the P41 transcript. Our results also suggest that splicing and polyadenylation of P41-generated RNA can compete for the same pool of precursor pre-mRNA molecules. The cis-acting signals within the A2 3′ splice site that govern polyadenylation and splicing of AAV5 RNAs must be optimized to program both (i) the levels of polyadenylation of P7- and P19-generated RNA at (pA)p required to generate the proper levels of the essential Rep proteins and (ii) the splicing of P41-generated RNAs to generate the proper ratio of capsid proteins during AAV5 infection.