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Interplay Between Splicing and Transcriptional Pausing Exerts Genome-Wide Control Over Alternative Polyadenylation

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Interplay Between Splicing and Transcriptional Pausing Exerts Genome-Wide Control Over Alternative Polyadenylation Transcription ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ktrn20 Interplay between splicing and transcriptional pausing exerts genome-wide control over alternative polyadenylation Carmen Mora Gallardo, Ainhoa Sánchez de Diego, Carlos Martínez-A & Karel H.M. van Wely To cite this article: Carmen Mora Gallardo, Ainhoa Sánchez de Diego, Carlos Martínez-A & Karel H.M. van Wely (2021): Interplay between splicing and transcriptional pausing exerts genome-wide control over alternative polyadenylation, Transcription, DOI: 10.1080/21541264.2021.1959244 To link to this article: https://doi.org/10.1080/21541264.2021.1959244 © 2021 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group. View supplementary material Published online: 07 Aug 2021. Submit your article to this journal View related articles View Crossmark data The Version of Record of this manuscript has been published and is freely available in "Transcription", 07 august, 2021 https://www.tandfonline.com/doi/full/1 0.1080/21541264.2021.1959244 Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=ktrn20 TRANSCRIPTION https://doi.org/10.108021541264.2021.1959244 RESEARCH PAPER Interplay between splicing and transcriptional pausing exerts genome-wide control over alternative polyadenylation Carmen Mora Gallardo , Ainhoa Sánchez de Diego , Carlos Martínez-A , and Karel H.M. van Wely Department of Immunology and Oncology Centro Nacional De Biotecnología (CNB)/, CSIC Darwin 3, Campus UAM Cantoblanco, Madrid, Spain ABSTRACT ARTICLE HISTORY Recent studies have identified multiple polyadenylation sites in nearly all mammalian genes. Received 19 May 2021 Although these are interpreted as evidence for alternative polyadenylation, our knowledge of Revised 18 July 2021 the underlying mechanisms is still limited. Most studies only consider the immediate surroundings Accepted 19 July 2021 of gene ends, even though in vitro experiments have uncovered the involvement of external KEWORDS factors such as splicing. Whereas in vivo splicing manipulation was impracticable until recently, we RNA splicing; now used mutants in the Death Inducer Obliterator (DIDO) gene to study their impact on 3ʹ end polyadenylation; processing. We observe multiple rounds of readthrough and gene fusions, suggesting that no transcription pausing; arbitration between polyadenylation sites occurs. Instead, a window of opportunity seems to sequence analysis; control end processing. Through the identification of T-rich sequence motifs, our data indicate mammalian gene expression that splicing and transcriptional pausing interact to regulate alternative polyadenylation. We propose that 3ʹ splice site activation comprises a variable timer, which determines how long transcription proceeds before polyadenylation signals are recognized. Thus, the role of core polyadenylation signals could be more passive than commonly believed. Our results provide new insights into the mechanisms of alternative polyadenylation and expand the catalog of related aberrations. Abbreviations APA: alternative polyadenylation; bp: basepair; MEF: mouse embryonic fibroblasts; PA: polyadenylation; PAS: polyadenylation site; Pol II: (RNA) polymerase II ; RT-PCR:reverse-tran- scriptase PCR; SF:splicing factor; SFPQ:splicing factor rich in proline and glutamine; SS:splice site; TRSM:Thymidine rich sequence motif; UTR:untranslated terminal region Introduction proliferation [5]. Altered APA patterns also char- Formation of the 3ʹ tail of messenger RNA by acterize several pathological states, for example cleavage and polyadenylation (PA) is an essential shortening of 3ʹ untranslated regions (UTRs) dur- step that aides export from the nucleus and trans- ing oncogenic dedifferentiation [6,7]. Much effort lation in the cytoplasm. PA requires recognition of has recently gone into cataloging polyadenylation the 3' gene end by a multiprotein complex that sites (PAS), which has given rise to catalyzes cleavage of the precursor, its release from a comprehensive census and has identified APA RNA Polymerase II (Pol II), and poly(A) tail addi- in more than half of all mammalian genes [8]. tion by a dedicated polymerase [1]. Finally, an While the mechanisms of RNA cleavage and poly- exonuclease promotes release of the remaining (A) tail addition are currently being elucidated, transcription complex from the chromatin tem- many details of polyadenylation site (PAS) selec- plate [2]. Most of the genes transcribed by RNA tion and APA remain poorly understood. Pol II lack a defined 3ʹ border and produce multi- The majority of recent studies on polyadenyla- ple ends [3]. This phenomenon, termed alternative tion focused on cis-acting elements [3,9]. For polyadenylation (APA) is an important regulator example, gradual decrease of 3ʹ end processing of mRNA isoform expression [4]. APA has been factors during early embryonic development pro- associated with important biological processes duces slippage toward downstream PAS [6]. More such as development, cell differentiation, and distant genomic features are considered important, CONTACT Karel H.M. van Wely [email protected] Department of Immunology and Oncology Centro Nacional De Biotecnología (CNB)/CSIC Darwin 3, Campus UAM Cantoblanco, Madrid 28049, Spain Supplemental data for this article can be accessed here 2 C. MORA GALLARDO et AL. too, since PA is coordinated with processing of the Elimination of a DIDO3-specific exon (E16) allows upstream RNA splice signals [10,11]. In vitro for production of DIDO2, an isoform that binds experiments indicate that recognition of the Pol II but fails to enrich SFPQ [19,24]. While the upstream 3ʹ splice site (SS) is of particular impor- E16 mutant suffers from an overall decrease of tance, and that binding of the splicing apparatus splicing capacity, a group of SS that are flanked defines the constraints of PA [12,13]. Analyses by TRSM are processed with increased efficiency. in vivo have shown that terminal 3ʹ SS processing A second DIDO mutant (E3+4), which lacks his- is sufficiently rapid for selection of canonical PAS tone binding but retains the domains for RNA Pol for most genes [11]. PA is assisted further by Pol II II and SFPQ interaction, also has been generated pausing at the 3ʹ gene end, again observed for [27]. Compared to E16, E3+4 shows mild splicing a large subset of genes [14]. This Pol II pausing defects but is more responsive to sequence bias. E3 frequently occurs some distance downstream of +4 is particularly sensitive to TRSM, which in this the actual gene body, so a stretch of RNA is mutant greatly increase RNA processing [19]. exposed to promote assembly of the actual 3ʹ end Here, we combine our prior knowledge of processing factors [15,16]. Finally, Pol II pausing is DIDO mutants with a statistical analysis of APA. thought to have an integrating role, allowing dis- We identified distant TRSM that stimulate PAS tant regions on the precursor RNA to be processed usage, and compared these to our data on 3ʹ SS together [17]. Notwithstanding the widespread usage [19]. Where prior publications reported only observations of Pol II pausing and processing fac- PAS usage frequencies [28,29], the RNA sequen- tor recruitment around PAS, the flexibility of APA cing of our mutants allowed us to establish is understood incompletely. We show here that Pol a statistical correlation between sequence motifs, II pausing occurs frequently, but may not be Pol II pausing, and PAS readthrough. Instead of a universal feature of 3ʹ gene ends. arbitration between adjacent PAS, a window of Transcriptional pausing is enforced by T-rich opportunity appears to determine the use or read- sequence motifs (TRSM) in the template DNA through of individual signals. Terminal 3ʹ SS and in vitro [18]. However, a genome-wide analysis of sequence motifs have an important role in this TRSM at 3ʹ gene ends has not yet been carried out. process, indicating that TRSM comprise a timer Our prior work has identified an interplay between to resolve terminal exon splicing and thereby con- TRSM and mutants of the Death Inducer trol PAS selection. Differential positioning of cri- Obliterator (DIDO) gene. DIDO has a role in tical pause sites suggests that multiple routes may recruiting splicing factors (SF) to 3ʹ SS, and dele- establish coupling between splicing and tion of different sections of DIDO lead to splicing polyadenylation. defects that are differentially modulated by TRSM [19]. DIDO is the vertebrate homolog to yeast BYE1 Results and insect PPS genes [20,21], and produces T-rich sequences are abundant but a family of transcription factors that bind to his- heterogeneously distributed tone H3 trimethylated on lysine 4 [22,23]. The protein family also binds to the jaw and funnel Our recent data uncovered a role for DIDO3 in domains of RNA Pol II [21], leading to a bivalent transcription, and indicated that the interplay genomic distribution at both gene ends [24,25]. between DIDO3 levels and intronic Thymidine- The vertebrate DIDO gene produces several pro- rich sequence motifs controls exon skipping or tein products (DIDO1, −2, and −3) by alternative inclusion [19]. In model transcripts, T-rich splicing. Among these, only DIDO3 catalyzes sequences with a length of 5 to 10 basepairs (bp) recruitment of the splicing factor SFPQ to RNA are efficient and selective inducers of RNA pol II precursors [19]. Since SFPQ binds upstream of 3ʹ pausing; such pause sites are thought to promote
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