Poster Session 9: Splicing & Polyadenylation 20:00 - 21:00 Friday, 29th May, 2020 Poster

36 Activation of Prp28 ATPase by Phosphorylated Npl3 at a Critical Step of Spliceosome Remodeling

Fu-lung Yeh1, Shang-Lin Chang2, Golam Rizvee Ahmed2, Luh Tung2, Leah Stands Lanier3, Corina Maeder4, Che- Min Lin5, Shu-Chun Tsai2, Wei-Hau Chang6, Tien-Hsien Chang2 1Genomics Research Center, Academia Sinicaa, Taipei, Taiwan. 2Genomics Research Center, Academia Sinica, Taipei, Taiwan. 3Department of Biology, Washington and Lee University, Lexington, VA 24450, USA. 4Department of Chemistry, Trinity University, San Antonio, TX 78212, USA. 5Department of Chemical Engineering, National Taiwan University, Taipei, Taiwan. 6Institute of Chemistry, Academia Sinica, Taipei, Taiwan

Abstract

Splicing, a key step in the eukaryotic gene-expression pathway, converts precursor messenger RNA (pre- mRNA) into mRNA by excising introns and ligating exons. This task, which demands single-nucleotide precision, is accomplished by the spliceosome, a macromolecular machine made of five small nuclear RNAs (snRNAs) and numerous proteins. Unique among ribonucleoprotein (RNP) machines, the spliceosome is assembled anew upon each intron and undergoes sequential conformational changes to establish its active site. Each of these major changes requires a dedicated DExD/H-box ATPase, but how these enzymes are rigorously regulated to trigger specific conformational changes remain obscure. To probe this issue, we site- specifically placed a photo-activatable unnatural-amino-acid into a yeast DEAD-box ATPase Prp28 that dissociates U1 snRNP from the 5’splice-site (5’SS) in the fully assembled pre-B complex. This strategy enabled us to“capture”Prp28 in action within the assembling splicing complexes by UV crosslinking. Here we show that Prp28 transiently interacts with the conserved 5’SS GU dinucleotide and also makes splicing-dependent contacts with the U1 snRNP protein U1C, and U4/U6.U5 tri-snRNP proteins, Prp8, Brr2, and Snu114. Unexpectedly, Prp28 also contacts the phosphorylated form of Npl3, a yeast SR-like protein. Genetic perturbations of these interactions compromise cellular fitness and delay U1 snRNP departure. Finally, we showed that phosphorylated Npl3 at a single site (Serine 411) by Sky1, but not its unphosphorylated form, potentiates the otherwise feeble ATPase activity of Prp28, suggesting a novel strategy for regulating DExD/H- box ATPases via phosphorylating their cofactors. We propose that Npl3 is a functional counterpart of the metazoan-specific Prp28 N-terminal RS domain, which can be phosphorylated and serves as an anchor to human spliceosome.

Presenting author email [email protected]

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Splicing Mechanism 59 Diverse regulatory layers in 3’-splice site recognition

Hyun-Seo Kang1,2, Luca Sperotto1,2, Clara Hipp1,2, Nitin Kachariya1,2, Michael Sattler1,2 1Technische Universität München, Garching, Germany. 2Helmholtz Zentrum München, Neuherberg, Germany

Abstract

Pre-mRNA splicing is an essential mechanism in eukaryotic mRNA processing and greatly contributes to proteome diversity by alternative splicing. A critical early step involves defining the exon/intron boundaries in the pre-mRNA transcripts, such as the branch-point sequence (BPS), polypyrimidine (Py) tract and 3’-AG dinucleotide in the 3’-splice site. In our previous structural studies, we have revealed layers of diverse regulatory switches, involving inter-/intra-molecular interactions and post-translational modification, for selecting bona fide 3’-splice sites, employing integrative structural and biophysical methods. Conformational dynamics of RNA-binding domains U2AF2-RRM1,2 was shown to play a key role in recognizing loosely defined Py-tracts, where the presence of U2AF1 triggers the conformational population shift to accommodate weak Py- tracts. More recently, we have shown that the dynamic interaction of domain linker to RRM1,2 domains acts as a gate keeper to reduce non-productive bindings in the pre-mRNA sequences and thereby enhancing its specificity for the stronger Py-tract at the 3’-splice site in a more cellular context. Phosphorylation at the helix- hairpin domain of SF1 may also play a role in the 3’-splice site recognition complex. Additional splicing regulators, hnRNP A1 and FUBP1, were shown to helps proof-reading the 3’-splice site and selecting weak Py- tract sites, respectively. Our current work focuses on understanding the structural role of hnRNP A1 in the context of 3’-splice site recognition by U2AF heterodimer. And further, we are pursuing the challenging task of unveiling the structural role of U2AF1 zinc-fingers.

Presenting author email [email protected]

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Splicing Mechanism 251 Prp22 Remodeling of Pre-mRNA During the P-To-ILS Spliceosome Transition

Elizabeth Duran, Nils Walter University of Michigan, Ann Arbor, MI, USA

Abstract

The spliceosome is a eukaryotic multi-megadalton macromolecular machine that catalyzes the removal of introns from newly transcribed precursor messenger RNA (pre-mRNA) and ligation of exons to form messenger RNA (mRNA). Once the catalytic core of the enzyme is formed, the spliceosome transitions through a series of eight complexes to precisely juxtapose splice sites. The ATP- or GTP- dependent helicase activity of seven essential DExD/H-box helicases is essential to complete each splicing cycle. Despite large amounts of splicing structural and biochemical data, the precise roles of the essential DExD/H-box helicases and the mechanisms by which they transition the spliceosome through the catalytic stage of the cycle remain unclear. To help bridge this knowledge gap, we are developing a single molecule fluorescence approach, guided by recent structural work, to investigate spliceosome complex transitions. Our assay aims to probe the role of the DEAH- box ATP-dependent helicase Prp22 during the post-spliceosome to intron-lariat spliceosome (P-to-ILS) transition. By incorporating a FRET fluorophore pair on U5 snRNA loop 1 and the 5’ exon of a surface immobilized pre-mRNA, we interrogate the role of Prp22 in displacing the ligated mRNA from the splicing machinery. This work represents a substantial step toward the application of single-molecule spectroscopy to elucidate the molecular mechanisms of complex biological machines.

Presenting author email [email protected]

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Splicing Mechanism 308 Spliceosomal diversity and a transcriptomic analysis of Rozella allomycis

Thomas A. Whelan1, C. Alisha Quandt2, Timothy Y. James3, Naomi M. Fast1 1University of British Columbia, Vancouver, Canada. 2University of Colorado Boulder, Boulder, USA. 3University of Michigan, Ann Arbor, USA

Abstract

Microsporidia are obligate intracellular parasites whose genomes are the most reduced among eukaryotes. With this reduction has come the loss of many, and in some cases all, of the spliceosomal introns. This loss of introns has been mirrored by a loss of spliceosomal components. As an example, Encephalitozoon cuniculi has 24 of the approximately 90 spliceosomal proteins in Saccharomyces cerevisiae. In E. cuniculi, the splicing levels for the majority of the 36 introns were <20%, which we attributed to this reduced spliceosome. The extremely derived nature of loss in microsporidia made drawing overarching conclusions about intron gain or loss difficult. Rozella allomycis is in the sister group to microsporidia and is also an intracellular parasite. With ~6300 protein coding genes and >10,000 introns, R. allomycis has not seen the same genome reduction as the microsporidia. This makes R. allomycis a promising candidate to study the pressures of genome reduction as well as the mechanisms of intron gain and loss. We sequenced transcriptomes across different stages of the life history of R. allomycis and have analyzed intron density, splicing levels, and spliceosomal motifs. Unlike microsporidia, introns in R. allomycis do not have conserved spliceosomal motifs, yet they are quite consistent in length (~30) and are spliced at much higher levels than those seen in microsporidia. We reconstructed the spliceosome of R. allomycis and found that while it had more spliceosomal components than any microsporidian, it had fewer components than even the reduced model system of S. cerevisiae. With high levels of splicing, despite a reduced spliceosome, we concluded that either (1) there are R. allomycis-specific spliceosomal proteins that have replaced canonical components of the spliceosome or (2) after initial intron loss there was a proliferation of the small introns we identified that are effectively spliced by a reduced spliceosome. If this is the case, R. allomycis is an exceptional candidate to study the process of intron loss and gain.

Presenting author email [email protected]

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Splicing Mechanism 356 Co-transcriptional splicing modulates gene expression

Tucker Carrocci, Diana Ottoz, Korinna Straube, Tara Alpert, Karla Neugebauer Yale University, New Haven, CT, USA

Abstract

Splicing and transcription occur simultaneously during the synthesis of eukaryotic pre-messenger RNA. We previously developed single-molecule nascent RNA sequencing methods (Single Molecule Intron Tracking; SMIT and long-read sequencing) to monitor the progression of the splicing reaction relative to the position of RNA polymerase II (Pol II)1. For endogenous yeast genes, spliced RNA was observed when Pol II was just downstream of each intron for both consensus and nonconsensus splice site sequences, suggesting that splicing can occur rapidly as Pol II transcribes past the intron and that splice site variation does not cause a delay. Here, we have constructed a novel, modular HA-YFP reporter gene that allows us to quantify the contribution of a wide array of intronic features to both co-transcriptional splicing and protein levels. Substitution of the consensus yeast GUAUGU 5’ splice site (SS) sequence with a minor variant (GUAcGU) results in a significant decrease in the amount of spliced RNA formed but has no effect on the onset of splicing. Further testing of all other possible single nucleotide variants of the 5’SS revealed significant differences in gene output but similar levels of co-transcriptional splicing. To determine if these findings can be applied more generally to regulate endogenous gene expression, we used CRISPR/Cas9-based editing to introduce single nucleotide changes to three genes and probed for their effects on co-transcriptional splicing using targeted long-read sequencing. Again, we observed similarities in the onset of splicing regardless of splice site sequence. Together, these data support a model wherein transcription and splicing cooperate within a “window of opportunity” to tune gene expression.

1. Carrillo-Oesterreich, F., Herzel, L., Straube, K., Hujer, K., Howard, J., Neugebauer, K.M., 2016. Splicing of Nascent RNA Coincides with Intron Exit from RNA Polymerase II. Cell 165, 372-381.

Presenting author email [email protected]

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Splicing Mechanism 388 Hyperstabilizing the extended duplex between U2 snRNA and the pre- mRNA branchpoint region influences branchpoint usage

Stephanie Nystrom, Jason Talkish, Haller Igel, Manuel Ares, Jr. University of California, Santa Cruz, Santa Cruz, CA, USA

Abstract

Branchpoint (BP) recognition is a critical step in spliceosome assembly that influences 3’ splice site selection, with consequences for mRNA function. One surprise of cryo-EM structures of yeast and human spliceosomes is that pairing between U2 snRNA and the BP is extended almost a full helical turn despite the absence of significant Watson-Crick-Franklin base pairing in the extension1,2. Neither human nor yeast intron sequences are conserved to pair with U2 in the extended part of the helix, yet some information may be present in mammalian introns at these positions3. To address questions raised by this unusual structure, we tested the effect on splicing of adding Watson-Crick-Franklin base pairing to the helix upstream of the BP. Using a dual branchpoint reporter in yeast that allows evaluation of relative branchpoint use within the same intron, we find that addition of 8 intron nucleotides complementary to U2 just upstream of either BP strongly shifts branchpoint use to that BP. Pairing of fewer nucleotides than 8 has a position dependent ability to increase the use of the nearby BP, suggesting that the effect is due to increased stability of the U2-branchpoint helix. We tested the ability of 8 paired nucleotides to increase use of adjacent mutant BPs that are poorly recognized and find that although some mutations are susceptible to partial rescue by the upstream pairing, other mutations result in a loss of any spliced products. These results suggest that although the region upstream of the BP can increase wild type BP use by the U2 snRNA when complementarity is high, the ability of the spliceosome to reject incorrect branchpoint choices may be compromised by the increased stability, explaining its evolutionary absence. The loss of any spliced products for certain mutant branchpoints suggests failure of a fidelity mechanism that releases U2 from incorrect branchpoints and allows re-engagement of the pre-mRNA with the splicing machinery. If hyperstabilization prevents release, a heretofore unknown decay mechanism may recognize the failed complex and destroy the pre-mRNA.

1. Betram et al. PMID: 28781166 2. Plashka et al. PMID: 28530653 3. Paggi and Bejerano. PMID:30224349

Presenting author email [email protected]

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Splicing Mechanism 406 Humanizing the yeast spliceosome to understand branchpoint selection and the action of splicing inhibitors

Oarteze Hunter1,2, Jason Talkish1,2, Haller Igel1,2, Manuel Ares, Jr.1,2 1University of California, Santa Cruz, Santa Cruz, California, USA. 2Center for the Molecular Biology of RNA, Santa Cruz, California, USA

Abstract

Pre-mRNA splicing constitutes a major post-transcriptional gene regulation step. How spliceosomal proteins and RNAs correctly recognize key intron RNA sites in favor of the of many competing sites remains unclear. The branchpoint sequence (BPS) is one site whose accurate selection is critical for success of the first splicing step, and has important consequences for identifying the 3’ splice site. Cryo-EM structures show that both SF3B1 and its budding yeast homolog HSH155 contact the branchpoint adenosine (BPA) residue through highly conserved HEAT repeats 15 and 16 (of 22 total). Moreover, spliceostatin-like small drugs such as pladienolide-B (Plad-B) and herboxidiene (HB) bind in the same pocket as the BPA in human SF3B1 and inhibit splicing in human cells and splicing extracts, but do not inhibit yeast splicing. To investigate the details of the action of this growing class of splicing inhibitors, we have humanized yeast by exchanging yeast HSH155 HEAT repeats 15 and 16 with the corresponding HEAT repeats from human SF3B1 using CRISPR/Cas9. We find that a yeast HSH155 protein carrying a swap of the human amino acids from the C-terminal half of HEAT15 and all of HEAT16 grows well at all temperatures. Inhibition of splicing occurs within minutes after Plad-B addition as detected by RT-PCR of the rapidly synthesized and degraded MATa1 mRNA. Using RT-PCR we find that splicing of different introns show different levels of sensitivity to Plad-B. In vitro extracts from the humanized yeast strain are sensitive to both Plad-B and HB at concentrations similar to those reported for HeLa cell extracts. Splicing is blocked before the first catalytic step, and the formation of ATP-dependent U2 complexes (prespliceosomes or A-complex) is inhibited by the drug. We hypothesize that the same region responsible for drug sensitivity play a role in BPS choice. We are testing this idea with high throughput sequencing and a novel dual branchpoint reporter. The availability of a yeast strain in which splicing can be blocked provides a useful tool for studying the relationship between splicing and other cellular events.

Presenting author email [email protected]

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Splicing Mechanism 430 Ecm2 Functions to Support U6 snRNA Structure and Dynamics during Spliceosome Catalysis

Charles W. Schneider, Clarisse van der Feltz, Aaron A. Hoskins University of Wisconsin-Madison, Madison, WI, USA

Abstract

Spliceosomes catalyze the removal of non-coding regions, or introns, from pre-mRNA. Assembly of the small nuclear RNA (snRNA) core of spliceosomes occurs anew on each intron via a series of carefully coordinated conformational rearrangements. However, the functional importance of many of the snRNA-protein interactions guiding these rearrangements remain unknown. The splicing factor Ecm2 integrates into the spliceosome adjacent to the active site, where it interacts near a region of the U6 snRNA forming the catalytic RNA core of the spliceosome. By combining mutations in the U6 snRNA that perturb splicing catalysis with Ecm2 deletion in yeast, we have determined that Ecm2 functions during the chemical steps of splicing. While Ecm2 deletion or U6 mutations can be tolerated by yeast individually, we demonstrate strong synthetic interactions between these factors. We have further characterized the impact of Ecm2 deletion on splicing in vivo using a reporter pre-mRNA. Neither Ecm2 deletion nor the U6 mutations impact splicing of pre-mRNAs containing strong splice sites. However, both Ecm2 deletion and U6 mutation change splicing when the pre-mRNAs contain weak, non- consensus splice sites. Moreover, many changes observed with U6 and Ecm2 are dependent on both factors. For example, Ecm2 deletion promotes splicing of pre-mRNAs containing a substitution at the +5 position of the 5'SS but mutation of U6 eliminates this effect. We propose that Ecm2 stabilizes U6 conformation so that it may re-arrange to support the catalytic steps of splicing.

Presenting author email [email protected]

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Splicing Mechanism 475 Using Mango RNA aptamers to watch U4 snRNA release from the spliceosome

Karli Lipinski1, Peter Unrau2, Aaron Hoskins1 1University of Wisconsin - Madison, Madison, WI, USA. 2Simon Fraser University, Burnaby, British Columbia, Canada

Abstract

The spliceosome undergoes a large conformational change during spliceosome activation where dozens of protein factors are exchanged and the U4 snRNA is released along with its associated proteins. However, it is not known if the U4 snRNA is released before, after, or concertedly with U4 snRNP proteins such as Prp3. In particular, release of Prp3 prior to U4 snRNA may be required for destabilization of the U4/U6 snRNA duplex. I will employ colocalization single molecule spectroscopy (CoSMoS) to examine the binding and release dynamics of Prp3 and U4 snRNA molecules in individual splicing reactions. I have integrated the Mango-III or iMango RNA aptamer into the 3’ stem loop region of the U4 snRNA to enable fluorophore labeling with cyanine- dye (Cy5) derivatives of the Mango ligand, TO1. Budding yeast strains (S. cerevisiae) expressing Mango-III or iMango U4 snRNA as the sole source of U4 snRNA are viable and showed no significant growth defects. Current work is focused on studying the biochemical and fluorescence properties of splicing extracts made from Mango-U4 yeast strains in the presence of TO1-Cy5. This study will reveal the temporal pathway of U4 snRNP disassembly during spliceosome activation as well as provide a general methodology for labeling endogenous, small RNAs with bright fluorophores for single molecule studies.

Presenting author email [email protected]

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Splicing Mechanism 476 Development of a Yeast-Based High Throughput Screen for Splicing Inhibitor Discovery

Sierra Love, Joshua Paulson, Aaron Hoskins University of Wisconsin-Madison, Madison, WI, USA

Abstract

RNA splicing by the spliceosome is paramount to the regulation of gene expression and transcriptome diversity in eukaryotes. Recently, spliceosomes have been targeted by small molecule inhibitors as potential treatments for a range of human diseases. However, only limited options exist for inhibiting the splicing machinery and many of the most effective drugs inhibit the same binding pocket on the U2 snRNP protein SF3b1. I have developed a high throughput screen (HTS) that employs humanized yeast strains for drug discovery. This HTS is based on measurement of growth inhibition of the humanized strains in the presence of small molecules versus inhibition of wild type strain growth in a parallel, counter-screen. Utilizing the Selleck FDA approved drug library, I have validated the screen platform and have assayed approximately 1200 compounds against multiple humanized yeast strains growing in 3 different types of media. Preliminary results have identified hit compounds which exhibit selective growth inhibition of the humanized yeast strains over wild type and vice versa. Current work is focused on validating these hits using biological and biochemical assays to confirm splicing inhibition as well as expansion of the screen to include ~105 potential drug candidates. My ultimate goal for this project to identify new strategies for therapeutically inhibiting splicing as well as understanding the biochemical mechanisms of inhibition.

Presenting author email

[email protected]

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Splicing Mechanism 534 In vitro Characterization of Maturase-assisted E.r. Group IIC Intron Foward Splicing

Tianshuo Liu1, Anna Pyle1,2 1Yale University, New Haven, CT, USA. 2Howard Hughes Medical Institute, Chevy Chase, MD, USA

Abstract

Group II introns are ribozymes that catalyze the self-splicing reaction through either hydrolytic or branching pathway. Meanwhile, most group II introns encode multi-functional protein cofactors (intron-encoded proteins, IEPs; also known as maturases) that aid in intron forward splicing, reverse splicing, and retro-transposition. Although intron self-splicing biochemistry has been studied extensively, characterization of maturase-assisted intron splicing is still rare and therefore precludes a detailed mechanistic understanding of the functional role of maturase proteins. Our lab has recently discovered the Eubacterium rectale (E.r.) group IIC intron and its encoded maturase (also known as MarathonRT) as a model system to characterize the biochemistry of its self- splicing and maturase-assisted splicing in vitro. I find that E.r. intron self-splicing preferentially undergoes hydrolytic pathway, while adding Marathon to the system completely reverses the trend towards branching. I have examined multiple factors that affect the maturase-assisted E.r. intron splicing outcome. Concurrently, mutagenesis study of the maturase protein has revealed key residues involved in promoting branching splicing. In all, this research has highlighted the functional role of maturase protein in group II intron forward splicing and will provide a more holistic understanding of the group II intron ribonucleoprotein splicing machinery.

Presenting author email [email protected]

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Splicing Mechanism 538 Unwinding spliceosome mysteries using fluorescence microscopy and spectroscopy.

Susan Beaver University of Oregon, Eugene, OR, USA

Abstract

The spliceosome is a complex RNA-protein (RNP) machine responsible for pre-messenger RNA (pre-mRNA) splicing, and its mechanisms are not fully understood. We focus on the action of Prp22, a helicase important to later stages of splicing. Prp22 acts in part by remodeling RNA-RNA and RNA-protein interactions. To investigate this activity on well-controlled substrates, we are using model RNA constructs containing Cy3 fluorophores near the junction between single-stranded and double-stranded domains. Bulk spectroscopic techniques have given us preliminary information about the unwinding behavior of Prp22, suggesting that it remodels RNA structure at junctions between single-stranded and double-stranded domains. We intend to confirm and clarify these results using single-molecule Förster Resonance Energy Transfer (smFRET) microscopy and ultrafast fluorescence upconversion spectroscopy. Our home-built smFRET instrument was built using a total internal reflection geometry to minimize background light, and utilizes an optical fluorescence path to separate donor and acceptor fluorescence spatially for simultaneous imaging. Using fluorescently labeled Prp22 protein and UBC4 pre-mRNA, we will be able to view FRET states for individual complexes, and thus collect equilibrium constants and reaction rates for the binding of the two. This will allow decomposition of bulk spectra into bound and unbound contributions. Meanwhile, fluorescence upconversion spectroscopy will enable measurements of the fluorescence lifetime and spectrum of Cy3 on ultrafast timescales, providing information about how its local environment and solvation properties are altered by the presence of Prp22. This will furnish new insight into spliceosome action, and lay the groundwork for further investigation of these complex RNP machines

Presenting author email [email protected]

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Splicing Mechanism 603 GC-AG introns features in long non-coding and protein-coding genes suggest their role in gene expression regulation

Monah Abou Alezz, Ludovica Celli, Giulia Belotti, Lorenzo Salviati, Arianna Nicastro, Roberta Alfieri, Antonella Lisa, Silvia Bione Institute of Molecular Genetics L.L. Cavalli-Sforza – National Research Council, Pavia, Lombardy, Italy

Abstract

Long non-coding RNAs (lncRNAs) are recognized as a new class of regulatory molecules despite very little is known about their functions in the cellular processes. Due to their overall low expression level and tissue- specificity, the identification and annotation of lncRNA genes still remains challenging. Despite lncRNAs show a low level of sequence conservation, an evolutionary constraint on lncRNA sequences is localized at splicing regulatory elements, suggesting that the recognition of the intron boundaries and splicing of introns is a crucial step required for their function. We exploited recent annotations by the GENCODE compendium to characterize the splicing features of long non-coding genes, in comparison to protein-coding ones, in the human and mouse genome by using bioinformatics approaches. A significant difference in the splice sites usage was observed between the two gene classes. While the frequency of non-canonical GC-AG splice junctions represents about 0.8% of total splice sites in protein-coding genes, we identified a remarkable enrichment of the GC-AG splice sites in long non-coding genes, both in human (3.0%) and mouse (1.9%). In addition, we identified peculiar characteristics of the GC-AG introns in terms of their intron length, a positional bias in the first intron, their donor and acceptor splice sites strength, poly-pyrimidine tract, and alternative polyadenylation signalling. Genes containing GC-AG introns were found conserved in many species across large evolutionary distances and a functional analysis pointed toward their enrichment in specific biological processes such as DNA repair. Moreover, GC-AG introns appeared more prone to alternative splicing and enriched in a special alternative splicing mechanism termed wobble-splicing. Taken together, our data suggests that GC-AG introns represent new regulatory elements mainly associated with lncRNAs, which could contribute to the evolution of complexity, adding a new layer in gene expression regulation.

Presenting author email [email protected]

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Splicing Mechanism 624 Role of RNA helicases in generation of SMN circular RNAs

Joonbae Seo, Eric Ottesen, Diou Luo, Natalia Singh, Ravindra Singh Iowa State University, Ames, IA, USA

Abstract

Humans have two nearly identical copies of Survival Motor Neuron (SMN) gene, SMN1 and SMN2. Low SMN levels due to deletion and/or mutation of SMN1 lead to spinal muscular atrophy (SMA), a major genetic disease associated with infant mortality. SMN2fails to compensate for the loss of SMN1due to skipping of exon 7. Since the full-length mRNAs of both genes code for identical proteins, correction of SMN2 exon 7 splicing provides one of the best therapeutic options. We have recently reported several backsplicing events, which result into generation of a huge repertoire of SMN circular RNAs (circRNAs). In this study, we evaluated the role of three RNA helicases (DDX5, DHX9 and DDX17) on forward and backsplicing. Our rationale to use DHX9 was to specifically look into effect of unwinding of the secondary structures formed by Alu elements, which are disproportionately represented in high numbers in SMN gene. Depletion of DHX9 caused substantial increase in skipping of exon 3 as well neighboring exons of SMN, whereas depletion of other two helicases (DDX5 and DDX17) had no appreciable effect on splicing of SMN exons. We observed enhanced rate of backsplicing events, leading to a noticeable increase in the levels of specific SMNcircRNAs in DHX9-depleted samples. In particular, we observed a correlation between the DHX9-induced exon 3 skipping and the levels of exon 3- containing circRNAs. Implications of these findings in uncovering novel functions of SMN genes and SMA pathology will be discussed.

Reference:Ottesen EW, Luo D, Seo J, Singh NN, Singh RN (2019). Human Survival Motor Neuron genes generate a vast repertoire of circular RNAs. Nucleic Acids Res. 6;46(20):10983-11001. doi: 10.1093/nar/gky770.

Presenting author email [email protected]

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Splicing Mechanism 926 Inhibition of ribosome biogenesis rescues U2 snRNA mutations.

Michelle Seiwald, Jason Talkish, Haller Igel, Manuel Ares Jr. Center for Molecular Biology of RNA, University of California Santa Cruz, Santa Cruz, CA, USA

Abstract

During RNA splicing, the U2 snRNP binds the intron branchpoint (BP) in an ATP-dependent manner. Nucleotides in the loop of the branchpoint-interacting stem-loop (BSL) of U2 snRNA are complementary to the BP in yeast, and cryo-EM models surprisingly revealed an extended helix between the intron and nucleotides in the stem of the BSL. Mutations in the BSL stem predicted to stabilize or destabilize the structure lead to cells that are cold- or temperature-sensitive, implying that the BSL needs to fold and unfold during splicing. This suggests a model where the loop of the folded BSL nucleates contact of U2 snRNA with the BP and unwinding of the BSL stem by the DEAD-box protein Prp5 promotes the extended helix between U2 snRNA and the intron. To explore this, we performed genetic screens in yeast to identify extragenic mutations in factors that suppress growth defects of mutations predicted to stabilize or destabilize the BSL. We found that insertion of a Tyδ element in the 3’ end of NOB1 suppresses the temperature-sensitivity of a U2 snRNA U33 deletion predicted to disrupt the BSL. Nob1 is an endonuclease important for 18S ribosomal RNA formation (rRNA). The Nob1-Tyδ fusion protein removes 27 amino acids from the c-terminus of Nob1 and inhibits 20S pre-rRNA processing. Our lab previously showed that downregulation of intron-rich ribosomal protein gene (RPG) transcription suppress prp11 and prp4 ts- mutations and improves splicing of intron-like sequences not normally efficiently spliced, presumably by reducing the load on the spliceosome. Our model is that disruption of pre-rRNA processing in the nob1-Tyδ strain leads to downregulation of ribosome production in a way that feeds back on transcription of RPGs. In an effort to generalize this observation we have found that mutations in U2 snRNA or prp11 can be suppressed by 1) nob1-Tyδ, 2) downregulation of the RPG transcription factor IFH1, 3) treatment of cells with small molecules that inhibit ribosome biogenesis and 4) deletion of highly expressed introns in non-RPGs. We propose an intricate homeostatic balance exists between ribosome biogenesis (and highly expressed intron-containing r- protein genes) and RNA splicing that is important for cell growth.

Presenting author email [email protected]

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Splicing Mechanism 927 Subsets of Human Introns Using Distinct General Splicing Factors: U2AF- Dependent and SPF45-Dependent Introns as Paradigms

Kazuhiro Fukumura1, Rei Yoshimoto1,2, Tetsuro Hirose3, Kunio Inoue4, Akila Mayeda1 1Institute for Comprehensive Medical Science, Fujita Health University, Toyoake, Aichi, Japan. 2Faculty of Agriculture, Setsunan University, Hirakata, Osaka, Japan. 3Institute for Genetic Medicine, Hokkaido University, Sapporo, Hokkaido, Japan. 4Graduate School of Science, Kobe University, Kobe, Hyogo, Japan

Abstract

Human pre-mRNA introns vary in size from under fifty to over a million nucleotides, so the open question has been how does the spliceosome recognize such diverse substrates. Here we have one of the answers for the short end of the spectrum. We searched for specific splicing factors involved in human short introns by screening siRNAs against 154 human nuclear proteins for activity on a model short 56-nucleotide intron-containing HNRNPH1 pre-mRNA. We identified SPF45 (RBM17) as a novel constitutive splicing factor and whole-transcriptome sequencing of SPF45- deficient cells revealed that SPF45 is essential to splice out the vast majority of short introns with truncated pyrimidine tracts. The hitherto obligate splicing factor U2AF-heterodimer (U2AF65/U2AF35) cannot recognize the truncated pyrimidine tract and SPF45 replaces it for interacting with the U2 snRNP; in which the U2AF- homology motif (UHM) of SPF45 interacts with the UHM-ligand motif (ULM) of the U2 snRNP protein SF3b155 (SF3b1). We propose the existence of two distinct subsets of human short introns; i.e., U2AF-dependent introns and SPF45-dependent introns [bioRxiv doi: https://doi.org/10.1101/784868 version 2]. We previously validated a list of ultra-short introns that includes remarkably atypical G-rich introns with completely inefficient splice sites and branch sites, of which the 49-nt intron in the NDOR1 gene and the 43-nt intron in the ESRP2 gene were analyzed. The involvement of either U2AF or SPF45 is very unlikely because of fully lacking pyrimidine-tract in these G-rich introns. The mechanism of splicing involved in such atypical G- rich introns is enigmatic. We assume the existence of another exotic subset of human ultra-short G-rich introns [Biochem. Biophys. Res. Commun. 423, 289 (2012); Int. J. Mol. Sci. 16, 10376 (2015)].

Presenting author email [email protected]

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Splicing Mechanism 931 Formation of true intron RNA circles by the spliceosome

Haller Igel, Sol Katzman, Manuel Ares, Jr. UC Santa Cruz, Santa Cruz, CA, USA

Abstract

RNA sequencing data from humans, mice, and yeast contain evidence for intron circles in which the 5’ splice site is joined to the 3’ splice site. In addition to molecules with exact 3’ss-5’ss junctions, some have ~3-7 non- templated adenosines or are missing 3’ nucleotides at the junction. We performed targeted RNAseq for 12 yeast introns in wild type and mutant yeast, either before or after treatment with the exonuclease RNaseR or the debranching enzyme Dbr1. The putative circular junctions do arise from circles as judged by their resistance to RNaseR and are joined by a 3’-5’ linkage as judged by their resistance to Dbr1. Intron circles are detected in strains lacking the tRNA ligase gene TRL1, the only other known RNA ligase encoded in the yeast genome besides the spliceosome. Mutation of the spliceosome disassembly factor SPP382/NTR1 greatly increases the number of intron circles, indicating that circularization is in competition with disassembly. A slow acting allele of the disassembly factor Prp43 (Q423N) does not greatly increase accumulation of circles, however shortening of its U6 snRNA substrate enhances accumulation of intron circles, suggesting inhibition at a step prior to binding of Prp43 promotes the reaction. Deletion of TRAMP complex or nuclear exosome subunits alter amounts of circles with non-templated As and missing nucleotides. We propose that after exon release, the 3’ end of the lariat intron leaves its binding site in the P-complex and can bind U5 snRNA as an exon 1 analog in a C-complex-like state. A reaction equivalent to the reverse of the first step of splicing (attack of the tail 3’OH on the branch phosphate) then forms an intron circle, with the branch 2’OH as the leaving group. In rare cases, the 3’ end of the lariat can react with the exosome or TRAMP complex to form circles missing nucleotides and with non-templated adenosines. The biological significance of this set of rare post- splicing reactions is unclear, however it might be relevant to any of several postulated intron transposition mechanisms that invoke reverse splicing by the spliceosome.

Presenting author email [email protected]

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Splicing Mechanism 936 Discard of lariat intermediates between the first and second catalytic steps of pre-mRNA splicing imposes an upper limit 3' splice site distance

Christopher Craddock, Jonathan Staley University of Chicago, Chicago, IL, USA

Abstract

Pre-mRNA splicing is an essential process in eukaryotic gene expression, wherein an intron is excised from precursor mRNA by a dynamic ribonucleoprotein machine known as the spliceosome. The spliceosome assembles de novo onto an intron and excises it in two catalytic steps, known as branching and exon ligation. After the intron is fully excised, the DEAH-box ATPase Prp43 acts to disassemble the spliceosome to allow for recycling of components for another round of splicing. Since the spliceosome contains a single catalytic core, rearrangements are necessary to allow for movement of the branch site, a reactant of the branching catalytic step, out of the catalytic core to allow for docking of the 3' splice site, a reactant for the exon ligation catalytic step. 3' splice site distance is tightly constrained in budding yeast, with a short average effective distance of 25 nucleotides downstream of the branch site. In fission yeast, shortening the distance between the branch site and the 3' splice site promotes exon ligation of TER1 pre-mRNA, a transcript that normally undergoes branching but is released from the spliceosome prior to exon ligation. Interestingly, ATPase-deficient Prp43 alleles also promote TER1 exon ligation, suggesting Prp43 may be a factor that acts to limit 3' splice site distance. Using mathematical modeling, we show that a lariat intermediate discard step between the branching and exon ligation steps is necessary to enforce an upper limit on branch site to 3' splice site distance. Here, we demonstrate the rate of 3' splice site docking slows with increasing distance from the branch site. We also demonstrate that slowing the discard rate increases the allowable distance between the branch site and 3' splice site for mRNA formation. These data suggest a compelling model in which Prp43 enforces an upper limit on 3' splice site distance by kinetically competing with the rate of 3' splice site docking.

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Splicing Mechanism 9 Widespread 3’UTR splicing regulates oncogene expression and tumorigenesis

Jia Jia Chan1, Bin Zhang1, Xavier Roca2, Polly Chen1,3, Yvonne Tay1,3 1Cancer Science Institute, Singapore, Singapore. 2Nanyang Technological University, Singapore, Singapore. 3National University of Singapore, Singapore, Singapore

Abstract

Three prime untranslated regions (3’UTRs) are non-coding segments of messenger RNAs with important regulatory roles that are disrupted in diseases. Although alternative 3’UTRs are widespread in humans and heavily featured in the differential modulation of gene expression and diversification of protein functions, studies have largely focused on 3’UTR lengthening or shortening via alternative polyadenylation and cleavage. Little is known about 3’UTR variants derived from other RNA processing steps. Here, we integrate multiple datasets from different RNA-sequencing platforms to reveal that human 3’UTRs are extensively spliced. Systematic analysis of the spliced 3’UTR characteristics reveals that stop codon proximity, Alu elements and RNA-RNA interactions impact 3’UTR splicing and their potential susceptibility to decay. As proof of concept, we characterize the CTNNB1 3’UTRs and show that the spliced isoform is more abundant in hepatocellular carcinoma and exhibits a stronger oncogenic potential compared to the unspliced. These phenotypes could be facilitated by a combination of factors including evasion of nonsense-mediated decay, enhanced protein stability, translation efficiency and distinct subcellular localization. Overall, our results demonstrate that 3’UTR splicing is a prevalent phenomenon that is upregulated in cancer, suggesting that it may serve as a regulatory mechanism to promote oncogene expression and function to drive tumorigenesis.

Presenting author email [email protected]

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Splicing Regulation & Alternative Splicing 41 Post-translational modification of splicing factors controls the innate immune response to infection

Kelsi West, Haley Scott, Allison Wagner, Robert Watson, Kristin Patrick Texas A&M Health Science Center, Bryan, TX, USA

Abstract

Despite the substantial impact pre-mRNA splicing has on gene expression outcomes, little is known about how the spliceosome itself is regulated during cellular reprogramming. When innate immune cells like macrophages sense a pathogen, they dramatically shift the burden of transcription to genes that encode antimicrobial molecules. While the details of this transcriptional activation are well-characterized, almost nothing is known about pre-mRNA splicing decisions change in response to immune stimuli.

To determine how the spliceosome itself is modified following pathogen sensing, we leveraged a global phosphoproteomics dataset that identified differentially phosphorylated proteins in macrophages infected with the intracellular bacterial pathogen Salmonella enterica serovar Typhimurium. We identified 30+ splicing factors, including SR proteins, hnRNPs, and components of the “core” splicing machinery (SF3b1, CWC22, and SF1), that gain or lose phosphorylation at a key innate immune time-point in Salmonella-infected macrophages. These data support a model whereby splicing factors receive signals in activated macrophages that alter their function (likely via changing protein-protein and protein-RNA interactions) and shape the innate immune gene expression program.

Consistent with these hypotheses, we recently described a role for the splicing factor hnRNP M in repressing expression of important antimicrobial molecules during Salmonella infection and identified a specific serine residue capable of controlling hnRNP M’s ability to repress splicing of pre-mRNAs encoding key inflammatory mediators like IL6 (West et al., 2019). To expand on these results and better understand the nature of the spliceosome in activated macrophages, we generated knockdown RAW 264.7 macrophage cell lines for a number of SR proteins and hnRNPs that were differentially phosphorylated in phospho-dataset (SRSF1, SRSF6, SRSF7, SRSF9, hnRNP F, hnRNP K, hnRNP U, and hnRNP C), infected these cells with Salmonella Typhimurium and performed RNA-seq to determine the contribution of these factors innate immune gene expression and to quantify alternative splicing changes in the absence of these proteins using the MAJIQ algorithm. Our preliminary results suggest a remarkable amount of diversity in pre-mRNAs targeted by each of these splicing regulatory factors, arguing that each of these factors plays a key, albeit underappreciated, role in dictating the timing and abundance of innate immune transcripts.

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Splicing Regulation & Alternative Splicing 43 Control of Adaptive Splicing by DNA Methylation

Ling Liu1, Urmi Das2, Hai Nguyen2, Shervin Pejhan3, Mojgan Rastegar3, Jiuyong Xie1 1Department of Physiology & Pathophysiology, Max Rady College of Medicine, Rady College of Health Sciences, University of Manitoba, Winnipeg, MB, Canada. 21Department of Physiology & Pathophysiology, Max Rady College of Medicine, Rady College of Health Sciences, University of Manitoba, Winnipeg, MB, Canada. 3Department of Biochemistry and Medical Genetics, and Regenerative Medicine Program, Max Rady College of Medicine, Rady College of Health Sciences, University of Manitoba, Winnipeg, MB, Canada

Abstract

Gene expression plays an important role in the long-term changes during cell adaptation to external stimuli, in activities such as hormone secretion, learning & memory or drug addiction. However, little is known about how alternative splicing changes in this process.

To determine if there is transcriptome-wide adaptive splicing, we carried out RNA-Seq analysis of rat pituitary GH3 cells treated with depolarizing concentrations of KCl (50mM) for 6h every day for up to 6 times. Most exons kept a homeostatic level without significant changes. Interestingly, 1,880 exons are differentially spliced upon the 1st or 6th treatment. RT-PCR result of a group of exons validated the different as well as cell- dependent responses to depolarization upon the 1st or repeated treatments. By screening signaling inhibitors, we found that 5-AzaC, an inhibitor of DNA methyl-transferases, significantly induced or changed adaptive splicing in a dose-dependent way in GH3 cells. Bisulfite sequencing of the genomic DNA and RNA-Seq of the corresponding RNA samples indicated that reduced methylation level of exonic DNA is generally correlated with higher fold changes of depolarization-induced splicing in GH3 cells. Importantly, a similar relationship is also observed in the hippocampi of the mCpG-binding MeCP2-null mice stimulated by kainic acid. The relationship is further supported by exonic DNA methylation-dependent splicing changes by the depolarization- activated CaMKIV in in vitro-methylated splicing reporters or their CG to GC mutants. Accompanying the adaptive splicing and methylation changes, there was a specific reduction of the prolactin but not growth hormone transcripts. Moreover, the adaptively spliced exons of synapse-related genes were aberrantly spliced in the hippocampi of Rett syndrome patients. Thus, there is transcriptome-wide adaptive splicing in response to repeated cellular excitation during hormone production or the pathogenesis of Rett syndrome. This change is coupled with the status of exonic DNA methylation. The results suggest that the splicing machinery remodel adaptively with the epigenetic status of DNA methylation during cell adaptation to external stimuli.

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Splicing Regulation & Alternative Splicing 81 Regulation of HNRNPH1 via alternative splicing is disrupted by non-coding mutations in mantle cell lymphoma

Krysta Coyle1, Quratulain Qureshi1, Prasath Pararajalingam1, Nicole Thomas1, Timothy Audas`1, Ryan Morin1,2 1Simon Fraser University, Burnaby, BC, Canada. 2Canada's Michael Smith Genome Sciences Centre, BC Cancer, Vancouver, BC, Canada

Abstract

Non-Hodgkin lymphomas (NHL) are a collection of cancers with each malignancy having distinct clinical management and prognosis. Mantle cell lymphoma (MCL) is particularly genetically heterogeneous and is considered incurable. Through a combination of exome, genome, and targeted sequencing of MCL tumors, we identified recurrent mutations in HNRNPH1 (heterogeneous nuclear ribonucleoprotein H1). These mutations are largely intronic or silent and are associated with a putative cis regulatory region involving a single exon. In RNA-seq data from matched cases, we identified variable representation of two distinct HNRNPH1 isoforms. Based on the reading frame of the affected exons, canonical splicing is predicted to produce a functional protein, while alternative splicing introduces a premature termination codon leading to nonsense-mediated decay. We observed a significantly higher proportion of canonical transcripts in MCL tumors bearing HNRNPH1 mutations, leading us to conclude that mutations in HNRNPH1 significantly alter its splicing. Furthermore, increased canonical splicing results in higher HNRNPH1 protein abundance as determined by immunohistochemical analysis of an MCL tissue microarray. HNRNPH1 mutation status and splicing ratio are associated with shorter survival of MCL patients. We developed an in vitro reporter minigene and introduced three specific mutations corresponding to the patient-identified mutations in HNRNPH1. These mutations lead to an increase in canonical splicing and translation of the HNRNPH1 minigene-derived peptide. Additionally, when HNRNPH1 is overexpressed, we observe a decrease of this peptide, implicating HNRNPH1 in the regulation of its own splicing. This is supported by preliminary data indicating that HNRNPH1 binds its own RNA. Beyond its own splicing, HNRNPH1 is likely also involved in the splicing of additional splicing factors. Overexpression of HNRNPH1 affects the splicing of SRSF3 and HNRNPDL, and many other targets are likely to be identified. This work elucidates a functional role for recurrent non-coding HNRNPH1 mutations and implicates HNRNPH1 expression and splicing of its downstream targets in lymphomagenesis. We continue to explore trans regulatory targets of HNRNPH1 using in vitro and cell-based models. While splicing is a growing field of interest in lymphoma biology, the unique pattern and consequences of these largely silent mutations specifically implicates alternative splicing as an oncogenic mechanism in MCL.

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Splicing Regulation & Alternative Splicing 95 RBFOX2 modulates a metastatic signature of alternative splicing in pancreatic cancer

Amina Jbara1, Chani Stossel2, Kuan-Ting Lin3, Maria Raitses-Gurevich2, Talia Golan2, Adrian R Krainer3, Rotem Karni1 1Department of Biochemistry and Molecular Biology, Institute for Medical Research Israel-Canada, Hebrew University-Hadassah Medical School, Jerusalem, Israel. 2Division of Oncology, Sheba Medical Center Tel Hashomer, Ramat-Gan, Israel. 3Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA

Abstract

The overall survival of metastatic cancer patients has not improved significantly in the past decades due to the fact that most cancer treatments focus on inhibition of cancer growth, with limited emphasis on metastasis. This is especially true for late diagnosis cancers, like pancreatic cancer, where the majority of patients initially present with advanced metastatic disease, with limited response to current treatments, resulting in poor prognosis. Splicing factors expression is frequently altered in cancers, is associated with aberrant alternative splicing programs and have a significant impact on cancer cell development, progression and resistance to therapeutic treatments. Here we present insights for the involvement of splicing factor RBFOX2 in metastatic pancreatic cancer progression and the splicing mechanism it uses to regulate this process. We found that RBFOX2 is downregulated in metastatic pancreatic tumors compared to primary tumors. Overexpression of RBFOX2 in patient-derived metastatic cells inhibited growth, induced cell death and inhibited cell migration in vitro and metastasis in vivo while suppressing RBFOX2 expression promoted the tumorigenesis, migration, and metastasis of primary pancreatic cancer cells. Taken together, these findings suggest that RBFOX2 is a potent metastatic suppressor in metastatic pancreatic cancer. RNA-seq and splicing analysis of RBFOX2 targets revealed a substantial change in pathways that have a direct contribution to metastatic development. To investigate the functional roles of RBFOX2 splicing targets, we developed a CRISPR/Cas9 splicing disruption system as a platform to modulate RNA splicing of RBFOX2 targets. Identification of these aberrant splicing events, which functionally contribute to the metastatic pathogenesis in pancreatic cells, shed light on the mechanisms by which RBFOX2 modulate the metastatic process and have a promising potential to become novel approaches for treatment of metastatic disease.

Presenting author email [email protected]

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Splicing Regulation & Alternative Splicing 118 POINTing out the impact of splicing kinetics on RNA transcription in mammalian cells

Rui Sousa-Luis1, Takayuki Nojima2, Maria Carmo-Fonseca1, Nicholas Proudfoot2 1Instituto de Medicina Molecular João Lobo Antunes, Universidade de Lisboa, Lisboa, Portugal. 2Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom

Abstract

Most splicing reactions take place co-transcriptionally. However, the exact timing of splicing in chromatin and how it is coordinated with transcription remains largely unknown. To address these questions, we have extended our mammalian NET-seq methodology (Nojima, Gomes et al., Cell 2015), to analyse the intact nascent RNA associated with the active site of RNA polymerase II (Pol II). We have named this approach “POlymerase Intact Nascent Transcript-sequencing (POINT-seq)”. This involves immunoprecipitation of Pol II associated full length nascent RNA and its sequencing by both illumina and nanopore long read sequencing platforms. Applying this technique to mammalian cells, we detect a substantial fraction of RNA molecules that have the introns excised while Pol II is transcribing the downstream exons, which supports an intron definition model. Surprisingly, we also detect fully unspliced RNAs that display termination defects and that we predict are a long-lived unspliced RNA category. Moreover, our analysis suggests a sequential splicing pattern that follows the gene order of introns. When splicing is blocked by targeting SF3B1 protein with Pladienolide B, we detect elongation defects with premature termination, especially for long genes. Taken together, our new technology is able to detect nascent intact transcripts associated with Pol II, and reveals new kinetic features of how splicing is coupled to and cross regulated by transcription.

Presenting author email [email protected]

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Splicing Regulation & Alternative Splicing 147 Splicing modulation through inhibition of snRNP biogenesis?

Andrea Pawellek1, David Gray2, Angus I. Lamond1 1Centre for Gene Regulation & Expression, School of Life Sciences, University of Dundee, Dundee, United Kingdom. 2Drug Discovery Unit, School of Life Sciences, University of Dundee, Dundee, United Kingdom

Abstract

An important goal in the splicing field is to identify how both the biogenesis and function of components of the splicing machinery can alter the spectrum of splicing events in cellulo that generate the transcriptome. One obstacle in studying the splicing process is the lack of a diverse set of tool compounds that either block or alter different stages of the splicing process. Our aim over recent years has been to identify and characterize new compounds that target the splicing machinery. We have identified a number of different small molecules and natural products that can inhibit splicing of model pre-mRNAs in vitro and/or modulate alternative splicing in cellulo across a variety of different cancer cell lines. These new splicing modulators induce significantly different phenotypes in cells when compared to the largest group of previously characterized splicing inhibitors, i.e. the SF3B1 inhibitors. Among the new splicing inhibitors we have identified is the small molecule DDD40800, which alters the alternative splicing of endogenous MCL1 transcripts in cells within 30 min after treatment. As shown by RNAseq experiments, treatment of either HeLa, or HEK293 cells with DDD40800 for 24h leads to ~3,000 altered splicing events. A hallmark of splicing modulators is that they cause alterations in either the size, morphology and/or composition of subnuclear bodies. For example, both the SF3B1 inhibitors and hinokiflavone (Pawellek et al., eLife 2017) alter splicing in cells and promote the formation of enlarged splicing speckles in the nucleus. However, immunofluorescence and FISH staining of DDD40800 treated cells does not show formation of mega speckles, but instead causes disruption of Cajal bodies and changes the structure of nucleoli. Related phenotypes are seen after disruption of snRNP biogenesis. Further evidence that DDD40800 might interfere with snRNP biogenesis comes from thermal proteome profiling (TPP), which identified STRAP, a component of the SMN complex, as a potential cellular target of DDD40800. Additional experiments are in progress to characterize in more detail how DDD40800 alters splicing and nuclear structure and to test the hypothesis that it interferes with snRNP biogenesis.

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Splicing Regulation & Alternative Splicing 162 Split-iCRAC combined with NMR structures reveal the motif bound by Npl3 in vivo and an unexpected function of the protein in splicing

Antoine Cléry1, Ahmed Moursy1, Stefan Gerhardy2, Katharina Hembach2, Sanjana Rao2, Mark D. Robinson2, Vikram Panse2, Frédéric H-T. Allain1 1ETH, Zurich, Switzerland. 2University, Zurich, Switzerland

Abstract

Serine-arginine rich proteins (SR proteins) belong to an RNA binding protein family that is involved in multiple steps of RNA metabolism including the regulation of alternative splicing events. In budding yeast, Npl3 is the only SR-like protein that promotes the splicing of pre-mRNAs. In addition, Npl3 is involved in other functions such as linking splicing to chromatin remodeling and nuclear export of mRNA and pre-60S ribosomal subunit. Npl3 is composed of two consecutive RRMs towards the N-terminus followed by a C-terminal RS/RGG domain. The first RRM is canonical and the second is a pseudo-RRM. Although Npl3 binding to RNA is important for its functions, its mode of RNA recognition has still not been characterized. In this study, we performed in yeast a combined split-CRAC and iCLIP approach that we called split-iCRAC and determined the consensus sequence bound by Npl3 in vivo. We found that the RRMs and not the RS/RGG domain of the protein were responsible for the specific interaction of Npl3 with RNA. By determining the NMR structures of the RRMs bound to RNA, we could show that Npl3 RRM2 recognizes a 5´-GNGG-3´ motif, whereas RRM1 binds preferentially upstream of this sequence to a CC dinucleotide. Using these structures, we engineered mutations that decrease the binding of Npl3 RRM1 or RRM2 to RNA and investigated their effect on Npl3 function in yeast. Strikingly, each RRM of Npl3 seems to be involved in a different function of the protein. The binding of RRM2 to RNA is important for the splicing function of Npl3, while RRM1 is important for splicing as well as chromatin remodeling. Finally, these data also show that Npl3 involvement in splicing could come at least in part from its direct and specific interaction with the U2 snRNA. Indeed, we found that the protein can unwind the stem I of the snRNA, which would facilitate the formation of the U2-U6 duplex in the Bact spliceosomal complex.

Presenting author email [email protected]

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Splicing Regulation & Alternative Splicing 176 Identification of tissue-specific alternative splicing regulatory elements in vivo using a random library approach in C. elegans

Sanjana Bhatnagar, John Calarco University of Toronto, Toronto, Ontario, Canada

Abstract

Alternative splicing is a crucial layer of gene expression that is frequently regulated in a tissue-specific manner. The cis-elements involved in alternative splicing regulation are recognized by trans-factors that affect spliceosome recruitment and thus, result in diverse splicing patterns. Hence, the identification and characterization of cis-elements dictating tissue-specific splicing patterns in vivo will contribute to the eventual goal of accurate prediction of splicing patterns for any gene. In our lab, we have implemented a randomized reporter library approach to identify cis-elements that modulate alternative splicing in C. elegans. A library of splicing minigene reporters containing random intronic decamers as potential cis-elements was introduced into C. elegans to study neuron-specific splicing patterns. We collected RNA from these reporter-expressing animals, and prepared cDNA libraries for deep sequencing. Interestingly, data generated from this pilot experiment led to identification of several elements acting as activators and repressors of exon inclusion in neurons. Our analysis has also revealed cis-elements that create strong cryptic 5ʹ splice sites. We now plan on expanding the size of our random library, and to extend this approach to monitor splicing patterns across multiple tissues. Future goals would be to study the sequence features of the potential cis-elements and to further elucidate the major interacting trans-factors. This would give a great insight into tissue-specific alternative splicing regulation. As the size of our datasets increase, we hope to develop more informed computational models to understand the regulation of alternative splicing.

Presenting author email [email protected]

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Splicing Regulation & Alternative Splicing 177 A genome-wide analysis of alternative splicing in tissues and distinct neuronal subtypes in C. elegans

Bina Koterniak University of Toronto, Toronto, Ontario, Canada

Abstract

Alternative splicing plays a major role in establishing tissue-specific transcriptomes. Among the broad tissue types present in metazoans, the central nervous system contains the highest levels of alternative splicing, thought to be necessitated by the heterogeneity of neuronal subtypes. While many documented examples of splicing differences between broad tissue-types exist, there remains much to be understood about the splicing factors and the cis-sequence elements involved in establishing tissue and neuron subtype-specific splicing patterns. Moreover, it is important to understand the functional relevance of specific splicing differences between neuronal subtypes. Using the Translating Ribosome Affinity Purification coupled with deep-sequencing (TRAP-seq) method in C. elegans, we have obtained high coverage transcriptome snapshots for three broad tissue classes (nervous system, muscle, and intestine) and two neuronal subtypes (dopaminergic and serotonergic neurons). We have identified hundreds of isoforms that exhibit distinct splicing patterns across broad tissue types, and also between neuronal subtypes and the rest of the nervous system. Analysis of these alternative splicing patterns has yielded several interesting observations. First, tissue- and neuronal subtype- regulated alternative exons are more likely to be frame-preserving and are enriched in specific cis-regulatory motifs when compared with constitutively spliced exons. Second, regulated exons are more often shorter than constitutive exons, but are flanked by longer intron sequences. Intriguingly, our analysis has also identified examples of highly conserved alternatively spliced micro-exons less than 27 nucleotides in length. Finally, alternatively spliced exons also overlap less frequently with conserved protein domains than constitutively spliced exons but overlap more frequently with intrinsically disordered regions. Collectively, our results indicate an important and rich layer of tissue-specific gene regulation at the level of alternative splicing in C. elegans. Our results also echo observations from comparative analyses of splicing regulation in vertebrates, suggesting that similar evolutionary forces and constraints shape splicing patterns and their influence on protein function across metazoa.

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Splicing Regulation & Alternative Splicing 178 Investigating Regulatory Factors and Evolutionary Conservation of Tissue- Specific Alternative Splicing in Caenorhabditis nematodes

Pallavi Pilaka, John Calarco University of Toronto, Toronto, Ontario, Canada

Abstract

Alternative splicing is a tightly regulated mechanism that can generate multiple mRNA transcripts from the same precursor transcript. The complex interplay between cis-regulatory elements and trans-acting proteins is known to determine tissue-specific splicing patterns. Moreover, there remains much to be understood about how alternative exons and regulated splicing emerge through evolution. Our lab has recently employed Translating Ribosome Affinity Purification coupled with RNA sequencing (TRAP-seq) to assess tissue-specific splicing patterns in C. elegans neuronal, muscle, and intestinal tissue. We identify hundreds of tissue-enriched splice variants in our analysis of these datasets. Four splicing events that were confirmed to demonstrate a drastic change in splicing patterns between two tissue types were selected for analysis (muscle vs. neuronal splicing: ampd-1 exon 9, hlb-1 exon 9, zoo-1 exon 10, and muscle vs. intestine: fhod-1 exon 8). Conserved intronic regions and elements were identified by multi-sequence alignments of homologous gene sequences (C. elegans, C. brenneri, C. briggsae and C. remanei). Interestingly, point mutations in these conserved elements altered splicing patterns and, in some cases, generated cryptic variants. Sequence alignments identified conserved UNC-75/CELF consensus binding sites overlapping with the 5ʹ splice site, adjacent to the alternative exon of zoo-1. unc-75 loss of function mutants and mutagenesis of these binding elements suggests that UNC-75 acts as a repressor of splicing of this alternative exon. Our comparative genomic analysis has thus enriched for active cis-elements playing a key role in defining tissue-specific splicing patterns. We are currently expanding our comparative genomic analysis to include all identified tissue- regulated exons from our transcriptome analysis, and analysis of more than 30 publicly available sequenced Caenorhabditis genomes. Initial observations suggest that different classes of exons (constitutive vs. regulated) showed varying degrees of exon and cis-element conservation throughout the phylogeny, suggesting diverse patterns of evolutionary constraint across splicing events. Taken together, this research will help illuminate the mechanisms involved in species-specific and evolutionarily conserved alternative splicing.

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Splicing Regulation & Alternative Splicing 218 Regulation of intron retention in normal biology and cancer

Ulf Schmitz1, Jaynish S. Shah1, Bijay Dhungel1, Geoffray Monteuuis1, Phuc-Loi Luu2, Veronika Petrova1, Charles G. Bailey1, Ali G. Turhan3, Deborah L. White4, Susan Branford4, Susan Clark2, Timothy P. Hughes5, Justin J-L. Wong1, John E.J. Rasko1 1Centenary Institute, Sydney, NSW, Australia. 2Garvan Institute, Sydney, NSW, Australia. 3Inserm, Paris, France. 4University of Adelaide, Adelaide, SA, Australia. 5University of Adelaide, Adelaide, NSW, Australia

Abstract

Intron retention (IR) is a widespread and conserved form of alternative splicing that enhances transcriptomic complexity in mammalian species. IR is known to modulate gene expression and cell differentiation. Moreover, aberrant IR patterns were associated with various cancers. However, the role of IR in disease and mechanisms regulating IR are not well explored. Using a multi-omics approach we have identified intrinsic features and trans-regulators of IR in haematological cell lineages. We discovered that higher GC content, weak splice sites, and a short length are recurring features of retained introns. We also found significantly reduced DNA methylation levels, increased presence of specific histone marks, and increased nucleosome occupancy around the DNA encoding retained introns. We studied the role and regulation of IR in chronic myeloid leukaemia (CML) and discovered increased IR levels at diagnosis and remission, which is in contrast to the normalisation of gene expression and DNA methylation patterns at remission. IR inhibits the expression of cell cycle regulators in CML, which could be caused by reduced RNA binding protein expression, histone modifications, and reduced DNA methylation.

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Splicing Regulation & Alternative Splicing 223 The multidimensional regulatory network of Alternative Splicing involves a layer of spliceosome cross-regulation.

Malgorzata Rogalska1,2, Estefania Mancini1,2, Xavier Hernandez Alias1,2, Luis Serrano3,2, Juan Valcárcel3,4 1Centre for Genomic Regulation (CRG), Barcelona, Spain. 2Universitat Pompeu Fabra (UPF), Barcelona, Spain. 3(CRG) Centre for Genomic Regulation, Barcelona, Spain. 4Institució Catalana de Recerca i Estudis Avançats (ICREA), Barcelona, Spain

Abstract

Regulation of pre-mRNA splicing greatly contributes to eukaryotic gene regulation. The spliceosome 5 snRNPs and over 200 associated proteins carry out intron removal and can display distinct regulatory effects. To systematically investigate the functions of core and auxiliary splicing factors, we carried out RNA-seq analyses upon the individual knockdown of 305 splicing-related factors. An initial insight arising from these analyses is the complex self-regulatory network by which splicing-related factors regulate each other. By comparing the cross-regulatory network with the network derived from the similarity in downstream targets, we identified a subset of functional connections between core splicing factors that draws the architecture of the regulatory map. Two proteins impacting the highest number of other splicing components are SF3B1, which is a target of anti-tumor drugs and is frequently mutated in cancer, and -unexpectedly- CWC22, a Bactcomplex factor with a late role in spliceosome assembly. Furthermore, by exploring publicly available iClip data, we can model the direct and indirect dependencies between regulators and individual splicing events and thus provide essential information to understand splicing regulation. This is well illustrated by the case of NUMB exon 9, whose inclusion levels are affected by a relatively large number of splicing factors of which only a handful have been shown to directly bind to this region of the pre-mRNA. The cross-regulatory network allows us to rationalize up to 75% of the effects on this exon inclusion event, of which only 18% correspond to factors that directly bind the pre-mRNA. Interestingly, Analysis of The Cancer Genome Atlas (TCGA) data argues that a number of cross- regulatory circuits also operate in various cancer types. Our data imply that analysis and interpretation of the effects of genetic alterations or other perturbations of the splicing machinery require understanding the complexity of the splicing self-regulatory network.

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Splicing Regulation & Alternative Splicing 236 Mechanism and functionality of species-specific CaMKIIβ alternative splicing

Andreas Franz1,2, Nicole Dimos2, Tarek Hilal3, Markus Wahl2,4, Florian Heyd1 1Freie Universität Berlin, Laboratory for RNA Biochemistry, Berlin, Germany. 2Freie Universität Berlin, Laboratory for Structural Biochemistry, Berlin, Germany. 3Freie Universität Berlin, Core Facility BioSupraMol, Berlin, Germany. 4Helmholtz-Zentrum Berlin für Materialien und Energie, Macromolecular Crystallpgraphy, Berlin, Germany

Abstract

Calcium/calmodulin-dependent protein kinase II (CaMKII) is a key player in learning and memory in vertebrates and highly conserved across evolution. CaMKII monomers consist of a conserved kinase domain, followed by a regulatory segment that is connected to a hub domain via a linker. In mammals, CaMKII is encoded by four homologous genes and alternative splicing leads to multiple protein isoforms with variable linker lengths, which have been shown to respond differently to the frequencies and amplitudes of calcium/calmodulin spikes. Isoform-specific responses are achieved by fine-tuning of the equilibria between various active and inactive oligomerized states, but the molecular details of this coupling are presently unclear. Here, we report the discovery of human-specific CaMKIIβ splice isoforms, which are generated through exclusion of exon 15, leading to a reduced linker length. We show that these isoforms reach higher oligomerization states and observe higher maximal activity of the human-specific isoforms. Minigene splicing assays reveal an intronic regulatory sequence responsible for human-specific skipping of exon 15. This regulation is independent of the trans-acting environment, as human-specific exon skipping is also observed in mouse cell lines. Building on this observation, we used Crispr/Cas9 and introduced the human intronic regulatory sequence into the mouse genome, which indeed results in a human-like CaMKIIβ splicing pattern in the brain of mutant mice. Initial analyses of mice with humanized CaMKIIβ splicing suggest altered long-term potentiation (LTP) in CA1 neurons and behavioural phenotypes, which we are currently exploring in more detail. As we have not altered exonic coding regions but only intronic splicing regulatory sequences, any phenotype in these mice is the result of the humanized CaMKIIβ splicing pattern and will thus directly connect species-specific alternative splicing with cognitive abilities and behaviour. By studying this species-specific splicing event on a molecular, cellular and organismal level, our work contributes to the understanding of the role of alternative splicing in generating diverse, functional proteomes and in establishing species identity.

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Splicing Regulation & Alternative Splicing 247 The RNA helicase DDX39B activates FOXP3 RNA splicing to control T regulatory cell fate

Minato Hirano1, Gaddiel Galarza-Muñoz1, Samuel Fagg1, Geraldine Schott1, Liuyang Wang2, Alejandro Antonia2, Jain Vaibhav2, Jason Gibson2, Xiaxing Yu1, Steve Widen1, Farren Briggs2, Dennis Ko2, Simon Gregory2, Shelton Bradrick1, Mariano Garcia-Blanco1 1University of Texas Medical Branch at Galveston, Galveston, TX, USA. 2Duke University, Durham, NC, USA

Abstract

Autoimmune disease is caused by environmental and genetic factors. Genetic factors associated with increased susceptibility to multiple sclerosis (MS), an autoimmune disease of the central nervous system, have been identified, but their mechanisms of action are incompletely understood. Previously, we showed that the pre-mRNA splicing of Interleukin-7 receptor (IL7R), an important receptor for T-cell development and function, is regulated by the RNA helicase DEAD Box Polypeptide 39B (DDX39B) and genetic epistasis between DDX39B and IL7R genes enhances MS risk. In this study, we expand our understanding of the role of DDX39B and found that DDX39B impacted the expression of many genes that play roles in autoimmunity.

We showed that DDX39B controls expression of Forkhead Box P3 (FOXP3), a master regulator of the development, maintenance and function of CD4+/CD25+ T regulatory cells (T regs), a repressor of autoimmunity. Splicing of FOXP3 introns, most of them belong to a new subclass of introns with C-rich polypyrimidine tracts, was sensitive to DDX39B levels, making FOXP3 expression highly sensitive to the levels of this RNA helicase. Rescue assay of DDX39B with/without mutations showed that ATPase activity was required for the splicing of the FOXP3 introns. This aligned with the fact that the ATPase activity of DDX39B requires early steps in spliceosome assembly with U2AF2, and suggesting that DDX39B is important in the commitment step of the splicing. Low DDX39B levels in primary human T regs lead to loss of immune regulatory gene expression and cytokine signatures and gain of effector ones. Given the importance of FOXP3 in autoimmunity, this work cements DDX39B as an important guardian of immune tolerance that can reduce autoimmune disease risk by regulating IL7R splicing and upregulating FOXP3.

Presenting author email [email protected]

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Splicing Regulation & Alternative Splicing 250 Towards a functional splice site strength

Lisa Müller1, Johannes Ptok1, Stephan Theiss2, Heiner Schaal1 1Institute of Virology, Düsseldorf, Germany. 2Institute of Clinical Neuroscience and Medical Psychology, Düsseldorf, Germany

Abstract

The splicing process is an important feature of gene regulation that plays a pivotal role in human gene expression. Major players of splicing regulation are the U1 snRNP and U2 snRNP spliceosomal subunits that initiate the splicing process upon recognition of the splice sites. Additional regulators are splicing regulatory elements (SREs) posing binding sites for regulatory proteins that act on splice site selection in a position dependent manner. A bioinformatics approach towards the evaluation of SREs is the HEXplorer tool. It allows the analysis of sequences by using a fluid approach of overlapping hexamers to make predictions about whether certain regions have enhancing or silencing properties on splice site selection. Furthermore, the HEXplorer algorithm allows the analysis of the consequences of mutations in SREs and splice sites. The bioinformatic identification of changes in splice site use based on mutations is crucial for human diagnostics and might aid in the clinical treatment of patients. Analysing the intricate crosstalk between SREs and the intrinsic splice site strength, the aim is to establish the concept of ‘functional splice site strength’ that also includes the evaluation of variants of uncertain significance (VUS). Therefore, various subgenomic splicing reporters have been employed. Using the HEXplorer algorithm, synthetic SREs were designed that allow to systematically test and quantify the interaction between both sequence elements. The results suggest that the splicing outcome is defined by a function of intrinsic splice site strength that can be calculated by scoring algorithms (e.g. MaxEnt score or H-bond score) together with neighboring splicing regulatory elements. Even splice sites that are highly degenerated from the consensus motif can be rendered into a functional splice site by the supporting activity of SREs in proximity, while strong splice sites can be repressed by neighboring SREs, which becomes especially important in a situation where two competing splice sites are present. It was also found that this crosstalk is quantifiable and can be projected on genome wide association studies. This adds to the accuracy of splice site choice predictions, contributing to the understanding of the fine nuances of splice site choice regulation.

Presenting author email

[email protected]

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Splicing Regulation & Alternative Splicing 271 Engineering Synthetic RNA Binding Proteins to Probe the Mechanisms of Myotonic Dystrophy and Development of Potential New Therapeutics.

Carl Shotwell1,2, Melissa Hale3, Ryan Day1, Andy Berglund2,1 1University of Florida, Gainesville, FL, USA. 2University at Albany, Albany, NY, USA. 3Virginia Commonwealth University, Richmond, VA, USA

Abstract

The Muscleblind-like (MBNL) proteins are master regulators of RNA processing. MBNL sequestration by expanded repeats is a major disease mechanism in several microsatellite expansion disorders, including Myotonic Dystrophy type 1 (DM1) and type 2 (DM2). The expanded CUG (DM1) and CCUG (DM2) repeats present in these diseases cause nuclear sequestration of MBNL proteins through its YGCY binding motif disrupting MBNLs normal cellular function. Multiple disease symptoms correlated with the resulting mis- splicing from MBNL's sequestration. While the MBNL proteins have been well studied, there remains much to be understood about how the protein functions. The proteins themselves are made up of two tandem sets of zinc fingers (ZF1-2 and ZF3-4), an unstructured linker region between the ZF domains, and a C-terminal domain. While previous work shows that certain regions of the linker are important for the splicing regulation of a few targets, it still remains to be understood what about these regions are important and if other regions are important. The overall goal of this project is to design and identify synthetic proteins that displace endogenous MBNL and thereby rescue splicing without off-target effects. The first component of this study is to understand the linker region that is present between ZF2 and ZF3 in order to determine a minimal MBNL1 protein that is active in alternative splicing. To study this region, we have engineered several synthetic proteins that have systematic deletions of the linker region and/or insertions of completely synthetic linkers. Understanding the role of the linker in MBNL’s function will help identify a minimal MBNL protein active in alternative splicing which will aid in the design of future therapeutic proteins. The second component of this study is the design and testing of synthetic MBNL proteins with enhanced binding for CUG/CCUG repeats. The goal is to identify synthetic proteins that only displace the endogenous MBNL proteins from toxic CUG/CCUG repeats leading to splicing rescue without causing off-target effects. Taken together data from both of these components will aid in the development of synthetic MBNL proteins that will serve as the basis for future therapeutic strategies.

Presenting author email [email protected]

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Splicing Regulation & Alternative Splicing 280 Metal tunable catalytic flexibility of the lariat-debranching enzyme, Dbr1

Aiswarya Krishnamohan, Daoming Qin, Jonathan Staley University of Chicago, Chicago, Illinois, USA

Abstract

During the process of pre-mRNA splicing, the intron forms a lariat structure through an unusual 2'-5' linkage. The highly conserved lariat-debranching enzyme, Dbr1, resolves the 2'-5' linkage and generates a 5'-p, allowing degradation by 5'-3' exonucleases – a process critical for RNA decay and biogenesis of miRNAs and snoRNAs. Mis-regulation of Dbr1 has been associated with human diseases such as cancer and amyotrophic lateral sclerosis. Dbr1 activity has also been reported to be involved in susceptibility to viral infection. However, the role played by Dbr1 in these processes remains poorly understood. Nonetheless, these observations underscore the importance of establishing a complete biochemical and biological understanding of this enzyme. Here, we show that the active site of yeast Dbr1 is capable of catalyzing the cleavage of phosphodiester bonds in a variety of contexts beyond the 2'-5' branch linkage, demonstrating a previously unreported expansion of substrate specificity. Our data suggest that the specificity of Dbr1 toward branched RNA substrates depends on the identity of divalent metal ions in the active site. Further, we provide new insight into the evolutionary rationale for the strict conservation of a cysteine residue in the Dbr1 active site, a deviation from other members of the metallo-phosphoesterase family of enzymes. This unexpected catalytic flexibility of Dbr1 reveals the exciting possibility of metal-tunable Dbr1 activity and further, suggests that Dbr1 may have an alternate uncharacterized function in vivo.

Presenting author email [email protected]

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Splicing Regulation & Alternative Splicing 284 A polymorphism in oncolytic reovirus’ μ2 protein dictates its ability to modulate cellular alternative splicing during infection

Simon Boudreault1, Guy Lemay2, Martin Bisaillon1 1Université de Sherbrooke, Sherbrooke, Québec, Canada. 2Université de Montréal, Montréal, Québec, Canada

Abstract

Reovirus is a promising oncolytic virus possessing 10 double-stranded RNA segments that replicates in the cytoplasm of infected cells. REOLYSIN®, a wild-type T3D reovirus strain, is currently in stage 1 to 3 of clinical trials against various cancers. Alternative splicing (AS) is a mRNA maturation step allowing enhanced variability in mature mRNAs originating from the same gene, a process which is dysregulated in cancer cells. Recent evidence shows numerous cases of viruses altering cellular AS during infection, with a limited understanding of the role of these modulations in virus-host interactions. We have previously demonstrated that reovirus infection of L929 murine cells induces changes in the AS pattern of 240 AS events. We sought to determine how reovirus causes these changes in cellular AS, since its replication is cytoplasmic. First, cultivating L929 cells in the presence of infected cells does not lead to a change in AS, highlighting the necessity of reovirus-encoded determinant to trigger these changes. Comparison between two different laboratory T3D strains (T3DS vs T3DK) revealed a strain-dependent modulation in cellular AS, T3DS being a more potent modulator than T3DK. Using reassortant viruses with either T3DS or T3DK M1 segment introduced in the other’s virus genetic background by reverse genetics, we were able to map this strain-dependent phenotype to the M1 segment, which encodes for the μ2 protein. The μ2 protein differs only by two amino acids between T3DS or T3DK; proline (P) or serine (S) at position 208 and glutamine (G) or arginine (R) at position 342, respectively. We introduced separately P208S and Q342R mutations in the T3DS background, and reciprocal mutations in the T3DK background. Following assessment of AS upon infection with these viruses, the P208S substitution in T3DS completely abrogated its impact on AS, and vice versa, S208P mutation in T3DK allowed a strong modulation of cellular AS. Therefore, the amino acid at position 208 governs the ability of reovirus μ2 protein to modulate cellular AS. Investigations are ongoing to explain how μ2 modulates AS and thus improve our knowledge about the role of AS in host-pathogen interactions and oncolytic potential.

Presenting author email [email protected]

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Splicing Regulation & Alternative Splicing 338 Prp18p suppresses cryptic splicing genome-wide

Kevin Roy1,2, Jason Gabunilas2, Indya Weathers2, Joyce Samson2, Guillaume Chanfreau2 1Stanford University, Stanford, CA, USA. 2UCLA, Los Angeles, CA, USA

Abstract

The conserved splicing factor Prp18p plays an important role in promoting the second step of splicing, but it is unknown how it influences splice site selection genome-wide. To understand the role of Prp18p in splicing fidelity, we performed RNA-seq on cells lacking Prp18p. To enable accurate detection of unannotated splicing junctions that could arise from Prp18p inactivation, we developed an approach for high-confidence calling of annotated, unannotated, and non-canonical splice junctions in RNA-sequencing data termed COMPASS (Comparison Of Multiple alignment Programs for Alternative Splice Site detection). We show that COMPASS integration of alignments from STAR, HISAT2, and BBMap provides substantially superior precision and recall compared to each individual aligner.

Using COMPASS, we discovered that loss of Prp18 results in activation of a large number (563) of alternative 3′SS sites, including more than half of the annotated yeast introns (166/280), and with most events previously unreported. Targeted RT-PCR analyses confirmed the identity and relative 3′SS usage predicted by COMPASS. Most of the alternative 3′SS sites diverged from the YAG consensus, with 29% (166) RAG, 28% (156) ending in BG (B=non A), and 20% (115) ending in non-G sequences (including AU), demonstrating a critical role for Prp18p in promoting fidelity of 3′SS sequence selection at the genome-wide level. While the majority of the annotated splicing events decreased in the absence of Prp18p, we also identified an array of unannotated introns not normally spliced by the spliceosome, but for which inactivation of Prp18 triggered splicing, including a previously unreported intron in a Ty1 LTR. Overall we found that 3′SS activated in the absence of Prp18p were characterized by short branchpoint-3′SS distances, a lack of local RNA secondary structures, poly(U) content upstream, and adenosines flanking the intron.

In conclusion, we show that the COMPASS approach is particularly valuable for studying splicing in mutant contexts that decrease splicing fidelity, which allowed us to identify a global role for Prp18p in promoting 3’- splice site fidelity and preventing cryptic splicing in RNAs that are normally not targeted by the spliceosome.

Presenting author email [email protected]

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Splicing Regulation & Alternative Splicing 352 Effect of spacing between inverted repeat elements and the splice junctions on the effectiveness of circRNA splicing

Rita Meganck1,2, Jiacheng Liu1, Andrew Hale1, Katherine Simon2, Marco Fanous2, Jeremy Wilusz3, Nathaniel Moorman1, William Marzluff1, Aravind Asokan2 1UNC Chapel Hill, Chapel Hill, North Carolina, USA. 2Duke University, Durham, North Carolina, USA. 3University of Pennsylvania, Philadelphia, Pennsylvania, USA

Abstract

Circular RNAs (circRNAs) are a class of mammalian RNAs whose prevalence has recently been recognized. Most circRNAs are generated by exonic backsplicing, in which a splice donor site is spliced to an upstream acceptor site. Endogenously circularized exons often contain inverted repeats (e.g. Alu elements) in the flanking introns, which promote backsplicing by bringing the splice junctions in close proximity. However, in different tissues, circularization of the same RNA occurs to different extents, suggesting tissue-specific regulation of circularization. In the current study, we investigated the requirement for spacing of intronic repeats relative to the splice junction, and the impact different IRES elements had on circularization and expression. We used minigene constructs based on three intron pairs (HIPK3, Laccase2, and ZKSCAN1) to produce a circular GFP RNA from a split GFP exon. We discovered an asymmetric spacing requirement for efficient backsplicing. Inserting only a 100 nt sequence into the upstream intron between the acceptor site and the repeat element dramatically reduced circRNA formation. In contrast, insertions into the downstream intron increasing spacing by over 1.5 kb had no effect on circularization. This allowed us to insert additional RNA elements in the downstream intron. We created a construct with optimal expression (5x higher) by deleting regions from both introns, moving the repeat as close to the splice sites as possible. When tested in vivo, circRNA expression from this construct also increased by similar amounts in heart and liver tissue. We tested multiple viral IRES elements to drive translation of the circGFP RNA. Surprisingly, changing the IRES affected not only protein expression, but RNA expression as well. CircRNA levels varied by ~5-fold, and did not correlate with exon size. Increasing exon size by adding a second ORF did not change the amount of circRNA produced. Interestingly, changing the IRES also dramatically affected the types of RNAs produced. Depending on the IRES, a variety of additional linear and circular species were produced. We characterized one smaller circRNA as the product of an additional linear splice within the circRNA in both Poliovirus and KSHV IRESes. These results highlight the importance of exonic elements on backsplicing.

Presenting author email [email protected]

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Splicing Regulation & Alternative Splicing 353 Mass spectrometry exploration of neural exon-regulated protein interactions reveals new modes of transcriptomic regulation

Jonathan Roth1, Ulrich Braunschweig1, Robert Weatheritt1,2, Anne-Claude Gingras1, Benjamin Blencowe1 1University of Toronto, Toronto, Canada. 2Garvan Institute of Medical Research, Darlinghurst, Australia

Abstract

Alternative splicing is a key process contributing to neuronal differentiation and development. In humans ~2500 exons show differential inclusion in the nervous system, approximately half of which are predicted to form protein isoforms. This suggests a widespread role for neural-regulated exons in the control of protein function or stability. We previously employed LUMIER, a high-throughput luminescent-based co- immunoprecipitation assay to assess the function of neural exons in the control of protein-protein interactions (PPIs) (1). One third of the analyzed exons affected PPIs and these data provided new insight into neural exon functions. Subsequent work from the Vidal laboratory using the yeast two-hybrid assay revealed extensive human splice isoform-dependent PPIs (2). A limitation of these assays is that they were performed in non- neural contexts, and LUMIER requires prior knowledge of interactors to be assayed. Accordingly, we have developed an affinity purification coupled to mass spectrometry (AP-MS) strategy in Neuro2a cells to detect exon-dependent PPI changes in an unbiased manner. Of 18 analyzed regulatory factors with neural differential exons, six displayed prominent exon-dependent PPIs. Interestingly, a neural exon in the Sin3A-associated protein 30 binding protein (Sap30bp) was found to affect interactions with multiple exon junction complex components. RNA-Seq analysis of Sap30bp knockdown cells re-expressing each isoform revealed a role for its neural exon in the regulation of a specific subclass of short (<100nt) introns, the function of which are under investigation. Additionally, a neural exon in Chromatin Target of Prmt1 (Chtop), which has been implicated in transcription and mRNA export, affected its interaction with Prmt1. Interestingly, a recent report (3) has shown that Chtop preferentially binds terminal exons and regulates alternative polyadenylation. Initial results from isoform rescue experiments coupled to RNA-Seq indicates that the Chtop neural exon controls alternative polyadenylation of transcripts linked to neurogenesis. Collectively, the results illustrate the utility of AP-MS in the discovery of neural isoform dependent protein interactions that impact different facets of transcriptomic regulation and that are linked to nervous system development. 1. Ellis et al. (2012), Molecular Cell, 46(6):884-92 2. Yang et al. (2016), Cell, 164(4):805-17 3. Viphakone et al. (2019), Molecular Cell, 75(2):310-323

Presenting author email [email protected]

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Splicing Regulation & Alternative Splicing 363 Temporal control of ESRP2 licenses fetal-to-adult switch in alternative splicing required for hepatocyte maturation

Sushant Bangru1,2, Jackie Chen1, Waqar Arif1, Frances Alencastro3, Russ P Carstens4, Andrew Duncan3, Auinash Kalsotra1,2 1Dept. of Biochemistry, University of Illinois, Urbana-Champaign, Urbana, Illinois, USA. 2Cancer center@University of Illinois, Urbana-Champaign, Urbana, Illinois, USA. 3Dept. of Pathology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA. 4Perelman school of medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA

Abstract

The postnatal period of development is critical for mammalian tissues and is coordinated through precise activation of genetic circuits that govern differentiation, growth, and maturation of individual cell lineages. Here, we describe a cell type- and developmental stage-specific program of alternative splicing that drives the transition of fetal-to-adult protein isoforms in the liver. In-depth transcriptome and phenotypic analyses of fetal, postnatal day 14, and adult mouse hepatocytes revealed an essential role for alternative splicing in controlling cell size, cell proliferation, polyploidy, and hepatic metabolism. We find that the postnatal switch in splicing is a prerequisite for driving terminal differentiation and functional maturation of hepatocytes and that ESRP2 is a major regulator for these developmental splicing decisions. Targeted deletion of Esrp2 in the mouse liver demonstrated its requirement in generating and maintaining the adult splicing patterns of nearly one/fifth of hepatocyte mRNAs. To further determine if premature expression of ESRP2 was sufficient to activate the adult splicing program in neonatal livers, we engineered a Tet-inducible, hepatocyte-specific ESRP2 transgenic mouse model. Because Esrp2 gene is silent in fetal livers and the protein is only expressed two weeks after birth, we induced the transgenic neonates—via mother’s breastfeed—and harvested their liver tissues at postnatal day six as a midpoint between birth and two weeks. Remarkably, earlier-than-normal expression of ESRP2 was sufficient to turn on the adult splicing program, evoke premature cell-cycle exit, early polyploidization, and induce early maturation of hepatocytes. Additionally, we also employed the transgenic mouse model to define the first transcriptome-wide binding landscape of ESRP2 using eCLIP in adult and fetal hepatocytes revealing in vivo binding specificities and functionality. Collectively, these findings uncover that ESRP2 acts in a strict temporal window to stimulate the expression of adult protein isoforms, which dictate postnatal maturation and functional competence of hepatocytes.

Presenting author email [email protected]

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Splicing Regulation & Alternative Splicing 378 Chromatin variation is a major determinant of variation in isoform usage

Benjamin Fair, Stephanie Lozano, Yi Zeng, Jonathan Staley, Yang Li University of Chicago, Chicago, IL, USA

Abstract

The processing of pre-mRNA into mature messenger RNA (mRNA) occurs predominantly during transcription, allowing close interactions between chromatin regulators, RNA polymerase, and splice factors. Associations between chromatin and RNA processing have been documented in the past. However, to what extent variation in chromatin affects RNA processing remains unknown. Here we measure RNA splicing genome wide in chromatin and nucleoplasm subcellular fractions, along with histone modifications (H3K9me3, H3K36me2, and H3K36me3) and PolII occupancy in lymphoblastoid cell lines. Importantly, profiling the RNA in the nucleoplasm allows readout of splicing independent of cytoplasmic decay processes which perturb total cellular RNA, while profiling of nascent RNA associated with chromatin allows insight into the cotranscriptional splicing dynamics. Utilizing the natural occurring genetic variation present across cell lines derived from 68 individuals, we identify the quantitative trait loci (QTL) that associate chromatin, polymerase occupancy, and cotranscriptional splicing phenotypes, to genetic variation. Our detailed accounting of how genetic variants affect both chromatin and splicing phenotypes may elucidate the relative contribution of chromatin variation to splicing, and also detail the molecular mechanisms of genetic variants which contribute to complex phenotypes and disease.

Presenting author email [email protected]

Topic category

Splicing Regulation & Alternative Splicing 392 Regulation of splice site selection, the proximity rule revisited

Francisco Carranza, Hossein Shenasa, Klemens Hertel University of California Irvine, Irvine, CA, USA

Abstract

Splice site selection is a critical step for pre-mRNA splicing. It is known that splice site selection is influenced by a combination of features, including splice site strength, the length of exons and the length of their flanking introns. Depending on the gene architecture, the spliceosome recognizes splice sites using two different mechanisms. When exons are flanked by long introns their splice sites are defined across the exon. When exons are flanked by short introns their splice sites are defined across the intron. When two splice sites compete for selection it is unclear whether the strength of the splice site or the intron-exon architecture dictates splice site selection. Classical experiments have described a proximity rule in which the proximity between the paired 5' and 3' splice site directs splice patterns. We have revisited the proximity rule through the lens of the exon/intron definition modes of splice site recognition. A computational analysis of alternative splice site selection complemented with mini-gene analysis evaluated how the intron definition and exon definition mode of spliceosome assembly influence alternative splice site selection. For intron definition, the 5’ splice site most proximal to the downstream 3’ splice site is selected, as predicted by classical in vitro experiments. However, in the context of exon definition the 5’ splice site most proximal to the upstream 3’ splice site of the same exon is preferentially selected. Interestingly, the selection biases triggered by architectural features of the pre-mRNA can only be overridden by significant differences in the strength of the competing 5’ splice sites. Our results demonstrate that the proximity rule plays a significant role in 5’ splice site selection, however, the splice site strength of the two competing 5’ splice site can compensate and overrule proximity effects.

Presenting author email [email protected]

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Splicing Regulation & Alternative Splicing 446 Molecular mechanisms and functional impact of aberrant splicing in diffuse intrinsic pontine gliomas

Ammar Naqvi1,2, Brian Ennis1,2, Krutika Gaonkar1,2, Mateusz Koptyra1,2, Jessica Foster1,2, Michael Koldobskiy3, Yuankun Zhu1,2, Miguel Brown1,2, Bo Zhang1,2, Phillip Storm1,2, Adam Resnick1,2, Jo Lynne Rokita1,2 1Children's Hospital of Philadelphia, Philadelphia, PA, USA. 2The Center for Data Driven Discovery in Biomedicine, Philadelphia, PA, USA. 3Center for Epigenetics and Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MA, USA

Abstract

Fewer than 1% of children diagnosed with diffuse intrinsic pontine glioma (DIPG) survive for more than 5 years because no effective therapies exist for these patients. Here, we sought to identify and characterize mechanisms of aberrant splicing in primary DIPG tumors (n=47). We observe transcriptome-wide aberrant splicing in DIPG compared to normal brainstem, including changes that corresponded to exons mapping to known protein domains and functional sites. We found 9,805 differential splicing variations corresponding to 4,734 genes, including known tumor suppressors and oncogenes and hypothesize that aberrant splicing of cancer driver genes plays a role in DIPG tumor formation. To investigate this, we assessed splicing factor dysregulation and whether this impacted known cancer transcripts. We discovered several splicing factors to be mis-spliced. For example, aberrant splicing in SRSF3 created mRNA transcripts with poison exons, canonical targets for nonsense-mediated decay (NMD) and a subset of other splicing factors, such as CELF4, that were down-regulated in DIPG. We next performed differential methylation analysis and identified regions of local hypermethylation over certain exons, demonstrating that splicing factor dysregulation could be explained by increased non-coding exon inclusion in SRSF3 or differential methylation of CELF4. Next, we focused on recurrent splicing changes (n>=10) and observed significant exon inclusion in tumor suppressor SMARCA4, an integral member of the SWI/SNF family of proteins involved in chromatin remodeling. We identified significant aberrant splicing events in 16 of the 27 members of the SWI/SNF complex in DIPG, including significantly higher incidence of exon 7 skipping with a percent-spliced-in difference of ~70% in DPF2, representing a complete mRNA transcript switch. Finally, we observed universal down-regulation of the splicing factor SRRM4, a known regulator for neural-specific microexons and identified 245 known microexons lost or gained in DIPG tumors. Moreover, a quarter of these changes were found in known cancer genes, most frequently causing gain of a clathrin-binding site in tumor suppressor BIN1 and loss of an out-of-frame microexon in the oncogene BAK1. Altogether, our results suggest that aberrant splicing changes may be an alternative mechanism driving DIPG tumorigenesis we can potentially harness clinically.

Presenting author email [email protected]

Topic category

Splicing Regulation & Alternative Splicing 454 Arg-tRNA Synthetase Regulates SRRM2 Nuclear Condensate Trafficking and mRNA Processing

Haissi Cui1, Jolene Diedrich1, Douglas Wu2, Justin Lim3, Ryan Nottingham2, James Moresco1, John R Yates1, Benjamin Blencowe3, Alan Lambowitz2, Paul Schimmel1,4 1The Scripps Research Institute, La Jolla, CA, USA. 2University of Texas at Austin, Austin, TX, USA. 3University of Toronto, Toronto, Ontario, Canada. 4The Scripps Research Institute, Jupiter, FL, USA

Abstract

Aminoacyl-tRNA synthetases (aaRS) are mostly thought of in the context of their primary enzymatic function, the charging of tRNAs with their cognate amino acid as a part of mRNA translation. Recently, new roles of aaRS aside from translation have been discovered, leading to a renewed appreciation of aaRS as signal transducers and regulatory proteins. These functions are often mediated by interaction partners. Using a proteomics approach, we discovered a specific and robust interaction between Arginyl-tRNA synthetase (ArgRS), and Serine/Arginine-repetitive protein-2 (SRRM2), a spliceosomal protein and nuclear speckle component. ArgRS and SRRM2 colocalize in the nucleus in the proximity of distinct SRRM2-rich puncta. Therefore, we measured fluorescent recovery of SRRM2 after photobleaching in live cells and found that reduction of ArgRS increased the mobility of SRRM2. As SRRM2 contributes to RNA processing, we next explored whether processing of different RNA species would be affected by ArgRS. While small RNAs and tRNAs were mostly unchanged, exon usage in mRNAs differed upon ArgRS knock down. We further focused on defining ArgRS-dependent mRNA splice junctions and identified a set of 40 genes that are differentially spliced upon ArgRS knock down, among them growth factor receptors. This finding links a member of the translational apparatus to mRNA processing and and suggests a new role of an aminoacyl-tRNA synthetase in the regulation of alternative splicing.

Presenting author email [email protected]

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Splicing Regulation & Alternative Splicing 455 AMPK affects alternative splicing via SRSF1 phosphorylation

Eri Matsumoto, Yuji Yamamoto, Jun Inoue, Yu Matsumoto, Tsukasa Suzuki Tokyo University of Agriculture, Tokyo, Japan

Abstract

AMP-activated protein kinase (AMPK) is a Serine/Threonine protein kinase that regulates cellular energy homeostasis by inhibiting anabolic processes and activating catabolic processes. Recent studies have demonstrated that metformin, which is an AMPK activator, modifies alternative splicing. However, no direct substrate of AMPK for alternative splicing has been reported. Thus, we purified proteins binding with AMPK using the Tandem Affinity Purification (TAP) assay and identified them by LC-MS/MS to find novel AMPK substrates. We identified Serine/Arginine-rich splicing factor 1 (SRSF1) as a candidate of AMPK substrate. We found that AMPK directly phosphorylated SRSF1 at Ser133 in an RNA recognition motif 2 (RRM2), which is specifically binds to GA-rich ESE sequences. Interestingly, Ser133 phosphorylation suppressed the interaction between SRSF1 and specific RNA sequences. Moreover, AMPK regulated the SRSF1-mediated alternative splicing of the proto-oncogene MST1R (Ron) by suppressing its interaction with Ron pre-mRNA. The findings of this study revealed that the AMPK-dependent phosphorylation of SRSF1 at Ser133 inhibited the ability of SRSF1 to bind RNA and regulated alternative splicing. Therefore, our results are the first to demonstrate that AMPK regulates alternative splicing via SRSF1 phosphorylation.

Presenting author email [email protected]

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Splicing Regulation & Alternative Splicing 463 Srsf10 and the minor spliceosome control tissue-specific and dynamic SR protein expression

Stefan Meinke, Marco Preussner, Florian Heyd FU Berlin, Berlin, Germany

Abstract

Minor and major spliceosomes control splicing of distinct intron types and are thought to act largely independent of one another. SR proteins are essential splicing regulators mostly connected to the major spliceosome. Here, we show that SRSF10 expression is controlled through an autoregulated minor intron, tightly correlating SRSF10 with minor spliceosome abundance across different tissues and differentiation stages. Surprisingly, all other SR proteins also correlate with the minor spliceosome and Srsf10, and abolishing SRSF10 autoregulation induces expression of all SR proteins. Our data thus reveal extensive crosstalk and a global impact of the minor spliceosome on major intron splicing.

Presenting author email [email protected]

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Splicing Regulation & Alternative Splicing 469 Evolutionary conserved kinases as body temperature sensors

Miriam Strauch, Florian Heyd Freie Universitaet, Berlin, Germany

Abstract

Alternative pre-mRNA splicing gives rise to multiple mRNA isoforms thereby increasing the structural and functional space of the proteome. Alternative splicing is a fast process and therefore represents an ideal mechanism for cells to respond to internal and external changes. Temperature is one external clue that influences every aspects of life. We have shown that subtle changes in temperature can globally reprogram alternative splicing and that this is controlled by temperature dependent phosphorylation of SR proteins via Cdc2-like kinases (CLKs). Interestingly, CLKs are conserved throughout eukaryotic evolution and we have shown that CLKs from different organisms react extremely sensitive to temperature changes within the physiologically relevant temperature range of the respective species. In our current work, we have further characterized CLKs from divers organisms including the parasite Plasmodium falciparum, which is responsible for the tropical disease malaria tropica. There is a global interest in reducing new malaria infections and a recent paper suggested PfCLK3 as a potential drug target. Using our established in vitro kinase assays, we show that PfCLK1 and PfCLK3 indeed react to temperature changes with the highest activity betweeb 22 °C and 26 °C, the optimal living temperature of the host Anopheles. Intriguingly, almost no activity could be observed between 36 °C and 40 °C, the physiological relevant temperature range for humans. These data point to a role of temperature-controlled PfCLK activity in regulating gene expression profiles in the parasite when entering the human host. These data also suggest that a PfCLK3 inhibitor would be more efficient when used at temperatures that the parasite experiences when residing in Anopheles, as this is where the kinase is most active. Our data shed light on the functionality of body temperature controlled CLK activity in a model system that is highly relevant for human health.

Presenting author email [email protected]

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Splicing Regulation & Alternative Splicing 500 Rbfox2 regulation of β cell function

Nicole Moss, Lori Sussel University of Colorado, Anschutz Medical Campus, Aurora, Colorado, USA

Abstract

Functional hormone-secreting endocrine cells are vital for regulating blood glucose homeostasis. Maintenance and establishment of a robust endocrine cell population remains a critical challenge for individuals with Type 1 and Type 2 diabetes. Significant research efforts have been directed at understanding the formation and function of endocrine cells to not only assess the pathogenesis of the disease, but also explore potential treatment avenues. Recent studies have shown dysregulation of RNA-binding proteins (RBPs) and aberrant mRNA splicing activity in endocrine cells of diabetic patients compared to healthy controls. Additionally, our lab has shown differential splicing of over 1,000 genes between endocrine cell types. RNA regulation is in part orchestrated by RBPs. RBPs, including Rbfox2, are enriched in endocrine cells of pancreatic islets and can become dysregulated under stress. We found that conditional loss of Rbfox2 from pancreatic progenitors in mice leads to impaired blood glucose regulation without a significant change in β cell mass, suggesting that β cell function may be disrupted. There is mounting evidence, both published and unpublished, that Rbfox2 not only acts as a splicing regulator but also contributes to transcript stability. In my preliminary data I find that the loss of Rbfox2 results in the decreased expression of >1,000 genes that do not undergo Rbfox2 mediated alternative splicing. Further investigation of these differentially expressed genes reveals that many have Rbfox2 putative binding sites in their 3’UTR. Moreover, my own analysis of published Rbfox2 CLIP-Seq datasets in neuronal cell lines reveals a high proportion of binding events in 3’UTRs as opposed to the intron- exon binding commonly observed when Rbfox2 regulates alternative splicing. These data suggest that Rbfox2 could be serving two functions in the β cell: 1) modulating mRNA stability, and 2) coordinating alternative splicing. Our data identifies both direct and indirect mechanism of modulating insulin secretion through RNA regulation in the pancreatic β cell.

Presenting author email [email protected]

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Splicing Regulation & Alternative Splicing 505 mTOR-driven widespread exon skipping renders multifaceted gene regulation and proteome complexity

Sze Cheng1, Naima Ahmed Fahmi2, Meeyeon Park1, Jae-Woong Chang1, Jiao Sun2, Kai Thao1, Hsin-Sung Yeh1, Wei Zhang2, Jeongsik Yong1 1University of Minnesota Twin Cities, Minneapolis, MN, USA. 2University of Central Florida, Orlando, FL, USA

Abstract

Mammalian target of rapamycin (mTOR) pathway is important for cell proliferation, survival, and protein translation. This signaling pathway is often hyperactivated in many diseases, including cancer. mTOR activation has previously been shown to shorten 3’UTR of transcripts involved in the ubiquitin-mediated proteasome degradation pathway. However, the role of mTOR on alternative splicing regulation remains unclear. Here, we investigated transcriptome-wide alternative splicing events and their functional proteomic relevance in mTOR-activated MEFs and human cancer cells. We found that mTOR activation leads to widespread exon skipping events. We further revealed that these exon skipping cases in the 5’UTR can regulate translation efficiency while cases in the coding region can affect protein post-translational modifications sites. Additionally, protein isoforms generated from alternatively spliced transcripts have differences in protein stability. Lastly, we found that the transcriptome-wide exon skipping by mTOR activation is partially mediated by splicing factor srsf3 upregulation. Together, these findings suggest the role of mTOR signaling in alternative splicing and its impact on proteome complexity.

Presenting author email [email protected]

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Splicing Regulation & Alternative Splicing 551 Role for pre-mRNA secondary structure in regulating splicing

Ramya Rangan, Rhiju Das Stanford University, Stanford, CA, USA

Abstract

In the first stages of RNA splicing, sequence-distant 5’ splice sites and branch point sequences must be recognized with high specificity over cryptic alternatives. In some cases, RNA secondary structure has been implicated in this process, for instance by bringing together the correct 5’ splice site and branch site sequences spatially via base pairing in the RPS17b intron of Saccharomyces cerevisiae. Here we ask whether secondary structure properties can act as general mechanisms for promoting efficient splicing. We look across Saccharomyces cerevisiae introns and use a variety of secondary structure prediction methods to generate consensus ensembles for intron and control sequences, finding numerous features in intron sequences more often than expected, including 5' splice site and branch point co-localization, the lack of branch point sequence protection, and the formation of zipper stems between key splicing sequences. We have accumulated chemical mapping data from in vitro constructs and in vivo modification in yeast to assess the formation of these structures, and we are collecting these data on a genome-wide scale for yeast pre-mRNA. Additionally, we are investigating the possibility that the presence of these structural features can distinguish true introns from cryptic alternatives, which appear to not be spliced despite having splice site motifs in expressed regions. By better understanding the role of pre-mRNA secondary structure, we can move closer to a predictive model for splice site selection.

Presenting author email [email protected]

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Splicing Regulation & Alternative Splicing 558 Mechanistic and functional differences in RNA binding and processing activities of the muscle- and non-muscle RBFOX2 isoforms

Ullas Valiya Chembazhi1, Chaitali Misra1, Sarah Matatov1, Sushant Bangru1,2, Auinash Kalsotra1,2 1Department of Biochemistry, University of Illinois, Urbana-Champaign, Illinois, USA. 2Cancer Center@illinois, University of Illinois, Urbana-Champaign, Illinois, USA

Abstract

Myotonic Dystrophy type 1, or DM1, is a multisystemic genetic disorder and the most common form of adult- onset muscular dystrophy. Cardiac dysfunctions occur in ~80% of DM1 patients and are the second leading cause of disease-related deaths. Recently, we demonstrated that the upregulation of a non-muscle splice isoform of RBFOX2 (RBFOX240) in DM1 heart tissue induces cardiac conduction defects in DM1 individuals. Here, we performed eCLIP and high-resolution RNA-sequencing studies on cardiomyocytes isolated from wild Δ43/Δ43 type (expressing the normal muscle-specific RBFOX243 isoform), Rbfox2 (expressing the non-muscle RBFOX240 isoform), and RBFOX240 overexpressing (OE) mice to determine how aberrant expression of the RBFOX240 protein causes missplicing of target genes and triggers cardiac conduction abnormalities. By integrating genome-wide RNA binding and processing activities of the two RBFOX2 isoforms, we identify the non-muscle RBFOX240 isoform-driven missplicing events that may directly contribute to DM1-related cardiac pathology and arrhythmogenesis. Further, through subnuclear fractionation and protein-protein interaction studies, we demonstrate that the higher-order assembly of LASR (large assembly of splicing regulators) complexes formed by the RBFOX240 isoform boost its splicing activity and promote the generation of pathogenic splice variants of voltage-gated ion channels and other components of the cardiac conduction system.

Presenting author email [email protected]

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Splicing Regulation & Alternative Splicing 574 Dynamic Cytoplasmic Regulation of Full-Length Alternative mRNA Isoforms in Human Cells

Jolene Draper, Alison Tang, Chris Vollmers, Jeremy Sanford UCSC, Santa Cruz, CA, USA

Abstract

The vast majority of metazoan genes are not only spliced, but also capable of producing multiple mature mRNA isoforms. Characterization of these isoforms has relied on microarray assays and short-read sequencing combined with computational prediction of overall isoform architecture. New longread platforms like PacBio and Oxford Nanopore Technologies (ONT) provide the ability to look at the bona fide full length isoform structure and abundance of alternative mRNA isoforms. The question remains, which of these mRNA isoforms are biologically relevant. Here, we have utilized a short time course of human neural differentiation to take advantage of the occurrence of myriad evolutionarily-conserved and developmentally-regulated alternative splicing events in early neural development. We use short-read Illumina and long-read ONT sequencing combined with sucrose gradient fractionation of cytoplasm to query polyribosome association of mRNA isoforms. The Rolling Circle Amplification to Concatemeric Consensus (R2C2) greatly increases ONT read identity and detection of alternative transcript ends. These data provide insight into which isoforms are likely translated in each of two cell states, which are likely under specific methods of translational control, and which are likely substrates for translation-dependent decay programs such as nonsense mediated decay.

Presenting author email [email protected]

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Splicing Regulation & Alternative Splicing 616 Factors promoting SRC1 alternative splicing in Saccharomyces cerevisiae

Shravan Kumar Mishra, Balashankar R., Poulami Choudhuri IISER Mohali, Mohali, Punjab, India

Abstract

S. cerevisiae SRC1 intron has two competing and non-canonical 5’ splice sites (referred to as alternative and constitutive 5’ss). Spliceosome promotes SRC1 alternative splicing by using both 5’ss. Usage of the alternative 5’ss is controlled by non-covalent associations of the ubiquitin-like protein Hub1 with splicing factors Snu66 and Prp5. However, more proteins are likely to participate in SRC1 alternative splicing. Here we show that the process requires additional proteins of the spliceosomal core, including the Prp8-101 surface, and subunits of the retention and splicing (RES) complex. Hub1 proximity to selected spliceosomal core proteins supports alternative splicing, whereas RES subunits appear to function differently. The collective list of alternative splicing factors suggests a mechanism – slow splicesomes at SRC1 5’ss promotes splicing using both. As SRC1 alternative splicing via competing 5’ss requires a subset of spliceosomal proteins and regulators, other forms of alternative splicing might similarly require concerted action of distinct molecules.

Presenting author email [email protected]

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Splicing Regulation & Alternative Splicing 623 Circular RNAs of human SMN genes incorporate novels exons derived from the intronic and intergenic sequences

Eric Ottesen, Diou Luo, Joonbae Seo, Natalia Singh, Ravindra Singh Iowa State University, Ames, IA, USA

Abstract

Low levels of Survival Motor Neuron (SMN) protein due to deletions or mutations of SMN1causes spinal muscular atrophy (SMA), a devastating genetic disease of infants and children. SMN2, a nearly identical copy of SMN1, fails to compensate for loss of SMN1due to predominant skipping of exon 7. SMN is universally required to sustain essential cellular functions, including but not limited to transcription, splicing, translation, and RNA trafficking. SMNgenes harbor unusually high levels of primate-specific Alu elements capable of base pairing with each other, making them ideal candidates for the generation of circular RNAs (circRNAs). To confirm the nature and abundance of circRNAs generated by SMN genes, we performed circRNA-specific PCR on samples depleted of linear RNA by RNase R. Our results revealed a huge repertoire of circRNAs universally expressed by both SMNgenes. The most surprising finding was the incorporation of four novel exons from the intergenic region, confirming that SMNgenes are transcribed downstream of the last known exon. Further studies revealed presence of the intergenic exons in the linear transcripts as well. Depletion of DHX9, an RNA and DNA helicase associated with resolving Alu-mediated structures, resulted in a drastic increase in expression of the downstream novel exons. Our results reveal a diverse population of SMN mRNAs harboring different 3' untranslated regions. While substantially expanding the length of SMNgenes, these findings bring a new perspective towards our understanding of SMNgene regulation and function.

Reference:Ottesen EW, Luo D, Seo J, Singh NN, Singh RN(2019). Human Survival Motor Neuron genes generate a vast repertoire of circular RNAs. Nucleic Acids Res. 6;46(20):10983-11001. doi: 10.1093/nar/gky770.

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Splicing Regulation & Alternative Splicing 636 KDM3A regulates alternative splicing of cell-cycle genes following DNA damage

Mai Baker, Mayra Petasny, Mercedes Bentata, Gillian Kay, Eden Engal, Yuval Nevo, Sara Dahan, Maayan Salton Hebrew University, Jerusalim, Israel

Abstract

Changes in the cellular environment result in chromatin structure alteration, which in turn regulates gene expression. To learn about the effect of the cellular environment on the transcriptome, we studied the H3K9 de-methylase KDM3A. Using RNA-seq, we found that KDM3A regulates the transcription and alternative splicing of genes associated with cell cycle and DNA damage. We showed that KDM3A undergoes phosphorylation by PKA at serine 265 following DNA damage and that the phosphorylation is important for a proper cell cycle regulation. We demonstrated that SAT1 alternative splicing regulated by KDM3A plays a role in cell cycle regulation. Furthermore, we found that KDM3A’s demethylase activity is not needed for SAT1 alternative splicing regulation. In addition, we identified KDM3A’s protein partner ARID1A, the SWI/SNF subunit, and SRSF3 as regulators of SAT1 alternative splicing and showed that KDM3A is essential for SRSF3 binding to SAT1 pre-mRNA. These results suggest that KDM3A serves as a sensor of the environment and an adaptor for splicing factor binding. Our work reveals chromatin sensing of the environment in the regulation of alternative splicing.

Presenting author email [email protected]

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Splicing Regulation & Alternative Splicing 643 Cleavage factor Im regulates intron detention of the SAM synthetase MAT2A RNA

Anna Scarborough, Olga Hunter, Kuanqing Liu, Ashwani Kumar, Chao Xing, Benjamin Tu, Nicholas Conrad UT Southwestern Medical Center, Dallas, TX, USA

Abstract

S-adenosylmethionine (SAM) is the essential methyl donor for nearly all cellular methylation events. SAM levels are critical for regulating a variety of cellular functions, but the factors that regulate intracellular SAM concentrations remain incompletely understood. The N6-adenosine methyltransferase METTL16 regulates intron detention of MAT2A, the only SAM synthetase expressed in most cells. Our working model proposes that under SAM-replete conditions, METTL16 binds and methylates a vertebrate-conserved hairpin (hp1) in the MAT2A 3´ UTR. Efficient methylation by METTL16 correlates with detention of the last intron of MAT2A and nuclear degradation of the transcript. Under SAM-starved conditions, METTL16 dwell-time on MAT2A hp1 increases, presumably due to slower enzymatic turnover. The resulting increased occupancy of METTL16 promotes splicing of the detained intron to produce a functional MAT2A mRNA. However, the mechanism of METTL16 induction of MAT2A splicing remains unknown. To identify factors that regulate MAT2A splicing, we generated a reporter gene that fuses GFP with the MAT2A detained intron and 3´ UTR, integrated this into the genome of HCT116 cells, and isolated a clone in which SAM starvation induces GFP. We used this line to perform unbiased genome-wide CRISPR screens to identify factors required for induction of MAT2A upon SAM depletion. We identified the cleavage and polyadenylation complex factor Im (CFIm) component NUDT21 (CFIm25) to be necessary for induction of MAT2A. NUDT21 lacks known splicing domains, but complexes with CPSF6 (CFIm68) and CPSF7 (CFIm59), which contain RS domains. Upon knock-down of NUDT21, or co-depletion of CPSF6 and CPSF7, MAT2A fails to induce splicing under SAM limiting conditions, mimicking the phenotype of METTL16 knock-down. Moreover, mutation of a NUDT21 binding site decreases MAT2A splicing in reporter assays. Importantly, depletion of NUDT21 results in a decrease of intracellular SAM, but not in cells with a genomic deletion in the MAT2A detained intron. This strongly supports the idea that NUDT21 depletion alters SAM levels due to its function in MAT2A splicing, not due to its well described activity in alternative polyadenylation. Our work suggests METTL16’s role in SAM homeostasis requires CFIm and implies a novel role for the CFIm in SAM metabolism.

Presenting author email [email protected]

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Splicing Regulation & Alternative Splicing 672 The splicing factor hnRNP M is a critical regulator of innate immune gene expression in macrophages

Kelsi West, Haley Scott, Sylvia Torres-Odio, A.Phillip West, Kristin Patrick, Robert Watson Texas A&M University Health Science Center, Bryan, TX, USA

Abstract

While transcriptional control mechanisms of innate immune gene expression are well characterized, almost nothing is known about how pre-mRNA splicing decisions influence, or are influenced by, macrophage activation. Here, we demonstrate that the splicing factor hnRNP M can be controlled by pathogen sensing cascades and acts as a critical repressor of innate immune gene expression by influencing pre-mRNA splicing efficiency via post-translational modification of RNA-binding proteins. Through an unbiased RNA-seq approach, we found a unique regulon of genes that are upregulated when hnRNP M is removed, including IL6, a critical pro-inflammatory cytokine. We have found hnRNP M is enriched at the chromatin level, at the IL6 genomic locus, in an RNA-dependent manner. Importantly, this association is relieved when cells are activated, suggesting hnRNP M is released in order to de-repress IL6 expression. We also have identified specific phosphorylation sites that impact hnRNP M’s ability to associate with chromatin and regulate IL6 expression. These are the first data to describe a role for hnRNP phosphorylation in regulating gene expression. To detect specific splicing changes that occur when hnRNP M is removed, we utilized bioinformatics approaches to study alternatively spliced transcripts. Using RNA-Seq reads, we computationally identified differential splicing events (e.g. exon inclusion) that take place more often when hnRNP M is removed, providing evidence that hnRNP M acts at the level of splicing. Additional computational analysis of our RNA-seq data identified a number of transcripts whose splicing is altered during bacterial infection. While some of these events relied on hnRNP M, a number of alternative splicing decisions changed in response to infection alone (S. Typhimurium and Mycobacterium tuberculosis), suggesting that the innate immune transcriptome has a vast amount of unappreciated complexity at the level of alternatively spliced isoforms. Together, our results indicate that hnRNP M is a repressor of splicing and its ability to inhibit IL6 maturation is regulated by phosphorylation events downstream of pathogen sensing. We are currently investigating how immune signaling pathways regulate hnRNP M, how hnRNP M intron recognition regulates IL6 transcript levels, and how protein partners of hnRNP M play a role in this process.

Presenting author email [email protected]

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Splicing Regulation & Alternative Splicing 675 Signal-dependent Regulation of Alternative Splicing in Innate Immunity

Frank Lee1,2, Chelsea Harris1, Kevin Davidson1, Scott Alper1,2 1National Jewish Health, Department of Biomedical Research and The Center for Genes, Environment, and Health, Denver, Colorado, USA. 2University of Colorado Department of Immunology and Microbiology, Aurora, Colorado, USA

Abstract

Innate immunity is the first line of defense in fighting infection. During infection, Toll-like receptors (TLRs) sense pathogens and relay signals through the adaptor protein MyD88 to activate NF-kB and induce inflammation. Alternatively spliced isoforms in the TLR signaling pathway have been shown to play an important regulatory role. Here we investigate the MyD88 adaptor to study the mechanism of alternative splicing regulation in the innate immune system. The MyD88 gene produces two alternatively spliced isoforms, MyD88-L (long) and MyD88-S (short). MyD88-L induces inflammation when TLRs are activated by pathogens. In contrast, MyD88-S, whose expression is induced by TLR activation, is a dominant-negative inhibitor of inflammation. Thus, pathogen-induced production of MyD88-S represents a negative feedback loop that limits inflammation and prevents chronic inflammatory disease. We have established a model to investigate how lipopolysaccharide (LPS) from Gram-negative bacteria induces MyD88-S production. We found that MyD88 alternative splicing is regulated by the TLR4-NF-kB signaling pathway. Using a MyD88 minigene construct that faithfully recapitulates LPS-induced alternative splicing, we determined that MyD88 alternative splicing regulation is not coupled to the MyD88 transcription state. We are currently screening for splicing factors that mediate the effects of LPS on MyD88 alternative splicing. Understanding the mechanism that regulates LPS- induced alternative splicing of MyD88 will be beneficial for designing new therapies to prevent or treat chronic inflammatory diseases.

Presenting author email [email protected]

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Splicing Regulation & Alternative Splicing 710 Ptbp1-mediated splicing regulation of a chromatin modifier to alter gene expression during neuronal development

Mohammad Nazim, Anthony Linares, Kyu-Hyeon Yeom, Douglas Black University of California, Los Angeles, Department of Microbiology, Immunology, and Molecular Genetics, Los Angeles, CA, USA

Abstract

The mammalian SWI/SNF chromatin remodeling complexes, including the Brg/Brm-associated factor (BAF) complex, play crucial roles in regulating the genomic architecture. We discovered that in brain, one BAF complex subunit, Dpf2, is expressed almost entirely as an alternatively spliced variant that potentially targets BAF to new genomic loci as a part of the neuronal gene expression program. Inclusion of exon 7 creates a longer isoform (Dpf2-L), where 14 amino acids are inserted into the C2H2-type zinc finger domain. The zinc finger domain split by exon 7 is suggested to bind promoter regions and alter gene expression through downstream tandem PHD finger domains that recognize acetylated H3 and H4 histone tails. Expression data from days E15 to P60 of mouse cortical development confirm a shift from the canonical Dpf2-S to Dpf2-L isoform during brain development. We found that the splicing regulatory protein Ptbp1 regulates the alternative splicing of Dpf2 exon 7. As Ptbp1 is depleted early in neuronal differentiation, exon 7 becomes derepressed to create Dpf2-L. RNA-seq and iCLIP-seq data from mouse ESCs and primary differentiated neurons indicate that exon 7 inclusion is directly repressed by Ptbp1. Analyses of a reporter minigene spanning Dpf2 exons 6 to 8 identified multiple Ptbp1 binding motifs in both the upstream and downstream introns, as well as a weak 5’ splice site (5’SS), that together repress exon 7 splicing. SiRNA-mediated knockdown of Ptbp1, or disruption of the Ptbp1 binding motifs by site-directed mutagenesis promote inclusion of exon 7. Conversion of the weak 5’SS to an optimal 5’SS results in near exclusive inclusion of exon 7. Applying CRISPR/Cas9- mediated genome editing, we generated mouse ESC lines expressing either one of the two Dpf2 isoforms. ChIP-seq and ATAC-seq in these cells revealed altered binding of the two Dpf2 isoforms in specific genomic loci, particularly in genomic regions with higher chromatin accessibility. In separate proteomic studies, we examined the assembly and subunit composition of BAF complexes containing the two alternatively spliced isoforms of Dpf2. In total, these studies provide new understanding about the interplay between the chromatin and the RNA level regulation during neuronal development.

Presenting author email [email protected]

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Splicing Regulation & Alternative Splicing 743 Multiple competing RNA structures dynamically control alternative splicing in human ATE1 gene

Marina Kalinina1, Dmitry Skvortsov2, Svetlana Kalmykova1, Timofei Ivanov1, Olga Dontsova1,2, Dmitri Pervouchine1 1Skolkovo Institute for Science and Technology, Moscow, Russian Federation. 2Moscow State University, Moscow, Russian Federation

Abstract

The mammalian Ate1 gene encodes the arginyl transferase enzyme which is essential for embryogenesis, male meiosis, and regulation of the cytoskeleton. Reduced levels of Ate1 are associated with malignant transformations and serve as a prognostic indicator of prostate cancer metastasis. The tumor suppressor function of Ate1 depends on the inclusion of one of the two mutually exclusive exons (MXE), named 7a and 7b. Here, we report that the molecular mechanism underlying MXE splicing involves five conserved regulatory intronic elements, termed here R1-R5, of which R1 and R4 compete for base pairing with R3, while R2 and R5 form an ultra-long-range complementary bridge spanning 30 Kb. The competing base pairings between R1, R3, and R4 likely originated from a single ancestor hairpin at the same time when exons 7a and 7b were created by a tandem genomic duplication, while R2 and R5 evolved later to counteract the expansion of the intron downstream of exon 7b. In minigenes, single and double mutations that disrupt base pairings lead to the loss of MXE splicing, while compensatory double and triple mutations that restore the RNA structure also revert splicing to that of the wild type. The splicing changes induced by locked nucleic acid (LNA) oligonucleotide probes complementary to R1-R5 further confirm this mechanism in the endogenous Ate1 pre-mRNA. At that, the 7a/7b isoform ratio depends on RNA Pol II elongation rate, indicating that the two groups of structures, R1- R3-R4 and R2-R5, dynamically interact to maintain MXE splicing. The molecular mechanism presented here opens new possibilities for the development of therapeutic solutions, including antisense correction of splicing

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[email protected]

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Splicing Regulation & Alternative Splicing 744 Conserved long-range RNA structures associated with splicing markup and pre-mRNA processing of human genes

Svetlana Kalmykova1, Timofei Ivanov1, Stepan Denisov1, Roderic Guigo2, Dmitri Pervouchine1 1Skolkovo Institute for Science and Technology, Moscow, Russian Federation. 2Center for Genomic Regulation and UPF, Barcelona, Spain

Abstract

Eukaryotic genes are expressed as single-stranded RNA molecules that fold into complicated secondary and tertiary structures. Remarkably, many of these structures contain long stretches of complementarity nucleotides with the free energy of interaction exceeding that of the folding of large protein domains. In this work, we present an extended catalog of the so-called panhandle RNA structures, e.g., pairs of conserved complementary regions (CCR) in the human protein-coding transcriptome that may interact over long distances. These structures are associated with RNA processing signals and footprints of RNA-binding proteins (RBP), depleted of population polymorphisms, show evidence of compensatory evolution, and are supported by RNA structure probing data. Global trends in the positioning of these structures suggest that, together with RBP binding sites, they constitute a global network of regulatory signals that define gene-level splicing markup, including RNA bridges and double-stranded regions that approximate distant exons or suppress cryptic splice sites. These regulatory RNA structures are highly dynamic and form co-transcriptionally depending on the elongation rate of RNA Pol II. An intriguing observation is that, in addition to previously known association with intron borders, panhandle RNA structures also tend to loop out transcript ends, suggesting their involvement in 5’- and 3’-end processing. We hypothesize that, at least in some genes, RNA structure may serve as an intra-molecular crosslink that controls the dynamic competition between pre-mRNA splicing, cleavage, and polyadenylation.

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[email protected]

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Splicing Regulation & Alternative Splicing 745 Global cross-regulatory network couples the expression and alternative splicing of RNA-binding proteins by nonsense-mediated mRNA decay

Anastasia Danchurova1, Sergei Margasyuk2, Zoya Chervontseva1, Natalia Logvina2, Timofei Zatsepin1,2, Dmitri Pervouchine1 1Skolkovo Institute for Science and Technology, Moscow, Russian Federation. 2Moscow State University, Moscow, Russian Federation

Abstract

Nonsense-mediated decay (NMD) is a eukaryotic mRNA surveillance system that selectively degrades transcripts with premature termination codons (PTC). Many RNA-binding proteins (RBP) regulate their own expression levels by a negative feedback loop, in which an RBP binds its own pre-mRNA and causes alternative splicing to introduce a PTC, thereby leading to degradation. Instances of cross-regulation of RBP by this mechanism have been reported (e.g., PTBP1/PTBP2, RBM10/RBM5, etc). Here, we present an integrative analysis of three data sources generated by the ENCODE consortium: eCLIP assays for a large RBP panel, shRNA inactivation of NMD pathway, and shRNA-depletion of RBPs followed by RNA-seq, to identify novel such auto- and cross-regulatory feedback loops. In combination with co-expression gene networks, protein-protein interaction data, RBP motif predictions, and RBP overexpression data, it allowed identification of a remarkably complex regulatory network. As a proof of principle, we overexpressed SRSF7 in HEK293 cell line to confirm the model of its autoregulation and to validate its cross-regulated targets, SRSF4, SRSF10, and SRSF11. We extended the model of SFPQ autoregulation that was proposed previously, in which SFPQ gene product binds its mRNA and promotes switching to an alternative distal 3'-UTR that is targeted by NMD. We hypothesize that abnormally high expression of SFPQ in cancer, particularly in hepatocellular carcinoma could be due to the loss of this negative feedback. We also suggest specific splicing events that could be implicated in auto and cross- regulatory feedback loops in RBM39, HNRNPM, U2AF1, RPS3, and U2AF2 genes. Taken together, our results uncovered a sophisticated network of alternative splicing coupled with NMD that contains many more feedback and feed-forward connections than it is described in the literature today. Our findings suggest that auto- and cross-regulation through alternative splicing and NMD represent a ubiquitous form of post- transcriptional control of gene expression, and that deregulation of gene expression in disease could well be due to breakdowns in this regulatory mechanism.

Presenting author email

[email protected]

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Splicing Regulation & Alternative Splicing 772 Intron retention is a robust marker of intertumoral heterogeneity in pancreatic ductal adenocarcinoma

Daniel J. Tan1, Mithun Mitra1,2, Alec M. Chiu3, Hilary A. Coller1,2 1Department of Molecular, Cell, and Developmental Biology, UCLA, Los Angeles, CA, USA. 2Department of Biological Chemistry, David Geffen School of Medicine, UCLA, Los Angeles, CA, USA. 3Bioinformatics Interdepartmental Program, UCLA, Los Angeles, CA, USA

Abstract

Pancreatic Ductal Adenocarcinoma (PDAC) is an aggressive cancer with a 5-year survival rate of <8%. Previous studies have found strong associations between alternative splicing and cancer-related phenotypes. Unsupervised clustering of 76 PDAC patients based on five different splicing types: ES (exon skip), IR (intron retention), A5 (alternative 5’ splice site), A3 (alternative 3’ splice site), and MES (multiple exon skip) revealed that analysis of IR generated two clusters that scored high on measures of compaction and separation from each other. Patients in IR cluster 1 (IR-1) had lower overall IR levels and significantly poorer clinical outcomes compared to patients in IR-2. Comparison of these two IR clusters with previously determined gene expression clusters showed no significant concordance, thus indicating IR is a measure of PDAC tumor-to-tumor heterogeneity that is independent of gene expression. The genes corresponding to the set of 262 differentially retained introns between the two IR clusters showed enrichment for splicing factors and oncogenes. Twenty IR events out of 262 were found to be independently predictive of clinical outcome. Out of 259 RNA binding protein (RBP) genes that were differentially expressed between IR-1 and IR-2, the motifs for 30 RBPs were significantly enriched in the 262 differentially retained introns. Additionally, the expression of 25 RBPs was highly correlated (Pearson correlation coefficient≥0.7, adjusted p<0.05) with IR levels of 139 introns out of 262. Network analysis suggested that retention of these introns could be mediated by protein-protein interactions between RBPs. IR-based clustering of 20 other cancer types found IR events to be generally asymmetrically distributed between the clusters for each cancer, like PDAC. IR was also found to be a potentially robust biomarker for intertumoral heterogeneity in Kidney Renal Clear Cell Carcinoma (KIRC) and Prostate Adenocarcinoma (PRAD). Comparison of differentially expressed RBPs between the IR clusters for PDAC, KIRC, and PRAD revealed 194 common RBPs. In sum, our studies suggest IR as a robust parameter contributing to tumor-to-tumor heterogeneity.

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Splicing Regulation & Alternative Splicing 798 Meristem-Defective, a novel splicing factor essential for root meristem development in Arabidopsis thaliana.

Helen Thompson1, Weiran Shen1, Christiane Calixto2, David Dolan1, Medhavi Kakkar1, Sina Mozaffari-Jovin3, Carl Jones1, Sushma Nagaraja Grellscheid1, Keith Lindsey1 1Durham University, Durham, United Kingdom. 2Dundee University, Dundee, United Kingdom. 3Max Planck Institute of Biophysical Chemistry, Gottingen, Germany

Abstract

Around 61% of Arabidopsis intron-containing genes undergo alternative splicing. Splice isoform variants are associated with different cell types, organs, and developmental stages, and involvement in abiotic stress response and circadian clock gene regulation is known. We are investigating the role of a novel splicing factor MDF in the development of the Arabidopsis root meristem. The MDF gene is expressed in areas of increased mitotic activity such as the root and apical meristems and functions independently of auxin regulation. The null T-DNA mutant mdf-1 displays aberrant root meristem development, and essential meristem regulatory genes are down-regulated. MDF is homologous to human SART1, and our modelling predicts it has a similar tertiary structure. SART1 interacts with Prp6 and Prp8 in the human U4/U6 snRNP during B complex formation, and we are investigating whether MDF interacts similarly within the Arabidopsis spliceosome. Our RNA sequencing and HR-RTPCR analysis show that in mdf-1 components of the splicing cycle are alternatively spliced along with a subset of SR genes reported to be active in root tissues. MDF also appears to have a role in the abiotic stress response through isoform switching of two splice variants.

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Splicing Regulation & Alternative Splicing 816 Minor splicing is required for voltage-gated sodium and calcium channels expression in cardiomyocytes

Pablo Montañés Agudo, Simona Aufiero, Simona Casini, Ingeborg van der Made, Yigal M. Pinto, Carol Ann Remme, Esther E. Creemers Amsterdam UMC, location AMC, Amsterdam, Netherlands

Abstract

Background. The vast majority of introns (>99%) are removed by the major spliceosome. However, there is a small subset of introns (~700), that requires a different complex, called the minor spliceosome. The existence of major and minor introns, with different splicing consensus sequences, implies that minor intron-containing genes require not one, but two different spliceosomes. The removal of these minor introns has been postulated to be a bottleneck in RNA processing since it is less efficient than the removal of major introns. Interestingly, important genes for cardiomyocyte biology contain minor introns, including sodium and calcium voltage-gated ion channels, multiple MAP kinases, sarcomeric proteins and calpains. We hypothesize that minor intron splicing plays a major role in cardiac physiology by regulating the expression of minor intron-containing genes at the post-transcriptional levels.

Methods and Results. Using RNA-Seq data, we found that minor introns are significantly less retained than major introns in the mouse and human heart. To test the relevance of minor intron retention, we performed a gapmer-mediated knockdown of the minor spliceosome component U6atac in neonatal rat ventricular cardiomyocytes. A 60% reduction of U6atac resulted in a dramatic increase of minor intron retention in multiple cardiac genes, including the sodium channel Scn5a/Nav1.5 and the L-type calcium channel Cacna1c/Cav1.2. Consequently, we observed a reduction of these ion channels at the protein level and we confirmed a 70% decrease in the sodium current density by patch-clamp.

Conclusion. Minor splicing is an active step of RNA processing in cardiomyocytes. Knockdown of the minor spliceosome component U6atac results in minor intron retention, which reduces the expression of minor intron- containing genes and affects the electrophysiological properties of cardiomyocytes.

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Splicing Regulation & Alternative Splicing 831 A combinatorially regulated RNA splicing signature predicts breast cancer EMT states and patient survival

Yushan Qiu1,2, Jingyi Lyu1, Mikayla Dunlap1, Samuel Harvey3, Chonghui Cheng1 1Baylor College of Medicine, Houston, TX, USA. 2Shenzhen University, Shenzhen, China. 3Northwestern University, Chicago, IL, USA

Abstract

Epithelial-mesenchymal transition (EMT) is a critical developmental process that is often dysregulated in cancer. Post-transcriptional regulatory pathways are increasingly recognized to play a critical role in EMT regulation, especially alternative splicing, which is a major contributor to proteome diversity. In this study, we identified an alternative splicing signature from The Cancer Genome Atlas (TCGA) breast cancer patients that is able to stratify epithelial and mesenchymal tumor types. This alternative splicing signature contains 9 events that showed increased exon inclusion, and 16 events showing increased exon skipping during EMT. Using the 25-gene splicing signature, the EMT status of cell line data from CCLE can be accurately predicted. Using RBP expression from the TCGA, bipartite networks between RBPs and alternative splicing events were constructed, revealing the complexity of RBP regulation during EMT. This analysis not only connected well- studied RBPs and splicing events, but also nominated previously unknown RBPs that may play a role in EMT alternative splicing regulation. The alternative splicing events can separate patient survival based on exon inclusion levels, and a combined signature using four alternative splicing events can accurately predict patient survival, outperforming gene expression methods. This study highlights the global importance of alternative splicing regulation during EMT and provided a possible clinical application for using EMT-associated splicing as a predictor of survival.

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Splicing Regulation & Alternative Splicing 840 A dynamic splicing program insures proper synaptic connections in the developing cerebellum

Donatella Farini1,2, Eleonora Cesari3,4, Robert J. Weatheritt5,6, Gina La Sala7, Chiara Naro3,8, Vittoria Pagliarini3,8, Davide Bonvissuto3, Vanessa Medici1,2, Marika Guerra3,4, Chiara Di Pietro7, Francesca Romana Rizzo9,10, Alessandra Musella10, Valeria Carola2,11, Diego Centonze9,12, Benjamin J. Blencowe5, Daniela Marazziti7, Claudio Sette2,3 1Department of Biomedicine and Prevention, University of Rome Tor Vergata, Rome, Italy. 2Fondazione Santa Lucia, IRCCS, Rome, Italy. 3Department of Neuroscience, Section of Human Anatomy, Catholic University of the Sacred Heart, Rome, Italy. 4Fondazione Policlinico Universitario A. Gemelli, Rome, Italy. 5Donnelly Centre and Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada. 6EMBL Australia, Garvan Institute of Medical Research, Darlinghurst, New South Wales, Australia. 7Institute of Cell Biology and Neurobiology, CNR, Monterotondo, Rome, Italy. 8Fondazione Policlinico Universitario A. Gemelli, IRCCS, Rome, Italy. 9Department of Systems Medicine, University of Rome Tor Vergata, Rome, Italy. 10San Raffaele Pisana and University San Raffaele, IRCCS, Rome, Italy. 11Department of Dynamic and Clinical Psychology, University of Rome Sapienza, Rome, Italy. 12Unit of Neurology, IRCCS Neuromed, Isernia, Italy

Abstract

Tight coordination of gene expression in the developing cerebellum is crucial for the establishment of neuronal circuits governing motor and cognitive functions. However, transcriptional changes alone do not explain all the switches underlying neuronal differentiation. Herein, we have unveiled a widespread and highly dynamic splicing program that affects synaptic genes in cerebellar neurons. Search for motifs enriched in the modulated exons implicated the splicing factor Sam68 as a regulator of this program. Sam68 controls splicing of exons with weak branchpoints, by directly binding near the 3¢ splice site and competing with U2AF recruitment. Ablation of Sam68 disrupts splicing regulation of synaptic genes associated with neurodevelopmental diseases and impairs synaptic connections and firing of Purkinje cells, thus resulting in motor coordination defects, ataxia and abnormal social behaviour. These findings uncover an unexpectedly dynamic splicing regulatory network that shapes the synapse in early life and governs establishment of motor and cognitive circuitry in the developing cerebellum.

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Splicing Regulation & Alternative Splicing 848 VulExMap: Detection of exons which are vulnerable to exonic splicing mutations

Lise Lolle Holm, Katharina Flugt, Thomas Koed Doktor, Michael Hansen, Alexander Grønning, Brage Storstein Andresen University of Southern Denmark, Odense M, Denmark

Abstract

It has for a long time been accepted that exonic mutations may cause aberrant splicing by altering splicing regulatory elements (SREs) located outside the splice sites. Consequently, software for predicting the effect of mutations on individual SREs is now widely used in precision medicine. Unfortunately, it is typically presumed that all exons are equally dependent on SREs and that predicted effects of mutations on SREs will have the same effect in all exons. However, exonic splicing mutations (ESMs) tend to cluster in particular exons of a gene and similar mutations in SREs located in different exons do not always have identical effects. We have defined a new subgroup of constitutive exons that are more likely to be affected by ESMs because they have an inherent suboptimal splicing efficiency and therefore are more dependent on the balance between positive and negative SREs. We coin these exons as vulnerable and have created a tool, VulExMap, which uses large RNA-seq datasets to assign exon vulnerability, based on small but significant levels of constitutive exon skipping. We show that approx. 20% of all constitutive exons are vulnerable, and that vulnerable exons as a group have characteristics of a suboptimal splicing context, such as weaker splice site strengths, lower density of positive SREs and higher density of negative SREs. VulExMap currently uses 3 different datasets to categorize exon vulnerability and allows the user to upload their own annotations of mutations to observe if they are located in a vulnerable exon. We have generated a database of 144 validated ESMs and we show by analysis with VulExMap that ESMs are several fold more frequent in vulnerable exons. Moreover, we demonstrate that ESMs that reside in vulnerable exons generally produce smaller changes to the splicing code, whereas ESMs in more resilient exons result in a more severe change to the splicing code. Our goal is that VulExMap will be used as a tool to determine exon vulnerability as a pre-requisite before assessing the potential effects of mutations on splicing. VulExMap is available at https://vulexmap.compbio.sdu.dk

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Splicing Regulation & Alternative Splicing 864 The patterns of Alu exonisation in human cancer

Mariela Cortés López1, Laura Schulz1, Kathi Zarnack2, Julian König1 1Institute of Molecular Biology, Mainz, Germany. 2Buchmann Institute for Molecular Life Sciences, Frankfurt, Germany

Abstract

Alu elements are primate-specific repetitive sequences and account for more than 10% of the human genome. Previous studies have reported the emergence of novel exons from Alu elements where single point mutations can generate new splice sites. There is evidence that some Alu exons can be translated, contributing to the protein diversity whereas others have been shown to promote the degradation of transcripts via non-sense mediated decay (NMD) as a consequence of the introduction of premature stop codons. The mechanisms and consequences of Alu exonisation are not completely understood. In this study we took advantage of the available cancer data in The Cancer Genome Atlas (TCGA) to investigate the patterns of Alu exonisation upon disease. Several annotated exons overlap with Alu elements, here we characterize the exon inclusion profiles of more than 3000 Alu derived exons within different cancer cohorts. Alternative splicing regulation depends largely of the interaction of the trans-acting factors with the sequence elements of the regulated regions (cis-elements). We identified single nucleotide variants within the Alu elements and neighbouring regions that seem to have an influence over the exonisation patterns on a patient- level. The connection between cis-elements and further identification of trans-acting factors in the regulatory context of Alu exons will allow us to understand some general aspects of alternative splicing and evolution.

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Splicing Regulation & Alternative Splicing 887 Regulation of Alternative Splicing by PARP-1 Extends to Circular RNA Biogenesis

Rebekah Eleazer, Elena Matveeva, Yvonne Fondufe-Mittendorf University of Kentucky, Lexington, KY, USA

Abstract

Poly(ADP-ribose) Polymerase 1 (PARP-1) is an abundant, multifunctional, nuclear enzyme that synthesizes ADP- ribose polymers and catalyzes the addition of polyADP-ribose to target biomolecules (PARylation). PARP-1 is well-known for its role in DNA damage response, where its PARylation of histones at sites of DNA breaks is critical for DNA repair. However, recent studies outline diverse roles for PARP and PARylation in nearly every step of RNA biogenesis. We previously showed that PARP-1 regulates co-transcriptional splicing both through direct interaction with splicing factors and through modulation RNA polymerase II kinetics, two non-mutually exclusive mechanisms. We showed that this role of PARP-1 is context specific. Circular RNAs (circRNAs) produced from protein coding genes are formed through an alternative splicing event called backsplicing, however the factors which facilitate backsplicing over the more efficient linear splicing events are not fully understood. We hypothesized that PARP1 in regulating alternative splicing, could be regulating backsplicing formation. Two factors known to promote backsplicing are the dimerization of splicing factors and increased rates of RNA polymerase II. Since PARP-1 exerts influence on gene expression at promoters and directs alternative splicing through regulation of splicing factors and RNA polymerase II kinetics, we hypothesized that it would also regulate circRNA biogenesis. Here we re-analyze RNA-seq data from PARP-1 knockdown and PARylation inhibited cells to uncover data on the role of PARP-1 on circRNA expression. Direct digital counting was used to validate changes in circRNA expression and to measure changes in the circular:linear ratio. With our very stringent analysis of total RNA-seq data, few circRNA candidates (~30) were able to pass our threshold. CircRNA expression can be regulated at the promoter level, which results in co-expression of its linear mRNA counterpart. We showed in further analysis of circular:linear ratio of these candidates that PARP-1 regulates circRNA biogenesis at both the splicing level and at the level of transcriptional initiation. These results recapitulate previous findings that suggest a context-specific effect of PARP-1 in RNA biogenesis and processing. Future studies will determine the functional impact of these circRNAs as well as how PARP1 influences PARP1-chromatin binding to regulate circRNA biogenesis.

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Splicing Regulation & Alternative Splicing 890 Thermosensitive alternative splicing senses and mediates temperature adaptation in Drosophila

Ane Martin Anduaga1, Naveh Evantal2, Sebastian Kadener1 1Brandeis University, Waltham, MA, USA. 2The Hebrew University of Jerusalem, Jerusalem, Israel

Abstract

We previously described a strong temperature dependent alternative splicing on timeless. In particular, we found that apart from the canonical isoform (tim-L), which is expressed at all temperatures; there are two isoforms (tim-short&cold and tim-cold) that are highly upregulated at cold temperatures while tim-M is highly expressed at 29°C. We developed a battery of genetic tools to assess the role of each of these isoforms. We first generated UAS lines that would knock-down specific isoforms using a short hairpin strategy. We found that the reduction of tim-L and tim-cold levels resulted on high arrhythmicity; while the line targeting tim-L and tim-M isoforms had high arrhythmicity only in the highest temperature. Additionally, we created tim-cold CRISPR mutants that resulted on either locking the isoform (preventing the generation of the canonical tim-L) or its abrogation (eliminating the intron and, thus, resulting on a lack of tim-cold production). We also performed behavioral experiments at different temperatures (18, 25 and 29°C) to test the capabilities of these mutant flies to adapt to colder and warmer conditions.

Presenting author email [email protected]

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Splicing Regulation & Alternative Splicing 910 Antisense targeting of decoy exons can reduce intron retention and increase protein expression in human erythroblasts

Marilyn Parra1, Weiguo Zhang1, Jonathan Vu2, Mark DeWitt2, John Conboy1 1Lawrence Berkeley National Laboratory, Berkeley, CA, USA. 2University of California, Berkeley, Berkeley, CA, USA

Abstract

The decoy exon model has been proposed to regulate a subset of intron retention (IR) events involving predominantly larger introns (>1kb). Experiments with minigene splicing reporters have shown that decoy exons can function in heterologous retained introns, and that decoy splice sites are essential for IR activity. Our working hypothesis is that decoys act by engaging intron-terminal splice sites so as to compete with cross- intron interactions required for intron excision. The decoy model predicts that antisense oligonucleotides should be able to block decoy splice sites in endogenous pre-mRNA, thereby reducing IR and increasing productive gene expression. Indeed, we now demonstrate that a morpholino designed to target a decoy 5′ splice site in intron 4 of the O-GlcNAc transferase (OGT) gene was able to reduce IR from ~80% to ~20% in primary human erythroblasts. Decreased IR was accompanied by increases in spliced OGT RNA and OGT protein expression. The remaining component of OGT IR was refractory to antisense treatment and might be mediated by independent mechanism(s). In contrast, for three other introns tested, retention was strongly dependent on decoy function, since antisense targeting of decoy 5′ splice sites greatly reduced (SNRNP70) or nearly eliminated (SF3B1) IR in two widely expressed splicing factors, and also greatly reduced IR in transcripts encoding the erythroid-specific structural protein, alpha-spectrin (SPTA1). Morpholinos targeting the 3’ splice sites of these decoys were predicted to have lower binding affinity, and not surprisingly they exhibited more modest activity in reducing IR. Together these results show that modulating decoy exon function can dramatically alter IR, and suggest that dynamic regulation of decoy exons could be a mechanism to fine tune gene expression post-transcriptionally in many cell types.

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Splicing Regulation & Alternative Splicing 956 Alternative splicing of Gephyrin settles inhibitory synapse diversity

Raphael Dos Reis1, Etienne Kornobis2, Alyssa Pereira1, Judit Carrasco3, Céline Jahannault-Talignani1, Christian Muchardt2, Andreas Schlosser4, Hans Maric4, Fabrice Ango1, Eric Allemand2,5 1IGF, montpellier, France. 2Institut Pateur, paris, France. 3Max Planck Institute of Immunobiology and epigenetics, freiburg, Germany. 4University of Würzburg, Würzburg, Germany. 5INSERM, Paris, France

Abstract

The diversity of inhibitory synapses is upheld by multiple classes of interneurons with distinct morphologies, connectivity patterns and physiological properties. Yet, the specific molecular determinants of inhibitory synapse diversity remain largely unknown. Here we studied the universal scaffold and master regulator of inhibitory synapses, namely gephyrin (Gphn). We discovered 277 Gphn splice variants expressed during brain development, generating a GPHN proteome with distinct neuronal localization and neurotransmitter receptor clustering properties. Our data reveals how changes in expression of Gphn isoforms control inhibitory synapse formation in the cerebellar cortex, thus demonstrating that isoform complexity of a single postsynaptic scaffolding protein contributes to the molecular diversity of inhibitory synapses in the brain.

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Splicing Regulation & Alternative Splicing 960 A possible role for weak RNA-protein interactions in exon recognition

Larry Chasin Columbia University, New York, NY, USA

Abstract

Using saturation mutagenesis of a model 51 nt exon we recently showed that almost all of 91 human RBPs tested showed a significant correlation between binding affinity for mutated exonic 7mer sequences and splicing (Ke et al., doi: 10.1101/gr.219683.116). Binding affinities in those experiments were measured by RNA Compete and listed in the CISBP-RNA database The relative affinities for each RBP are normalized by setting the mean or the ~16000 7mers to 0 and the SD to +/-1. We have now confirmed this apparent promiscuity using 7mer affinities for 62 RBPs measured by Bind-n-Seq. A deeper analysis of these data revealed that 30% of these significant correlations (FDR< 0.05) occurred even when the relative affinities available were limited to z-scores of < |2|. Similar results were obtained using Bond-n-Seek data. How can relative binding affinities near or below the mean for all possible 7‑mers be functional in splicing? One possible explanation for these numerous observations is that an RBP with a low binding affinity may be a surrogate for a different RBP, one not included in the CISBP-RNA database. The latter RBP would bind with similar sequence specificity but with a much stronger affinity and would be the real driver of the phenotype. A more interesting possibility is that RBPs with low relative affinity can and do influence splicing, even if they may have relatively rapid off rates. For instance, if we conservatively assume a diffusion limited on‑rate of about 106 M-1s-1 for a protein (Schlosshauer and Baker 2004) then an RBP that binds an RNA sequence with an exceedingly weak Kd of 1 mM will have a residence time (half-life) on RNA of about a millisecond. Coupled with the high effective concentrations of abundant RBPs in a crowded nucleoplasm or in a liquid-liquid phase RNA granule this sojourn may be sufficient to compete in a consequential way with tighter binders. The functional test used here may be detecting interactions not seen in most physical assays. Weak binding could be an oft overlooked feature of functional in vivo macromolecular interactions, including transcription factor binding and cell-cell recognition.

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Splicing Regulation & Alternative Splicing 400 Post-transcriptional regulation of poly(A) binding proteins controls protein synthesis and cardiac growth during development and disease

Joe Seimetz1, Bo Zhang1, Chaitali Misra1, Sandip Chorghade1, Stefan Bresson2, Nicholas Conrad2, Auinash Kalsotra1 1University of Illinois Urbana Champaign, Urbana, Illinois, USA. 2University of Texas Southwestern, Dallas, Texas, USA

Abstract

Cellular growth and function depend heavily on protein synthesis. However, it is becoming clear that global protein synthesis rates are not static, but rather vary widely both temporally and between cell types. These differences are necessary for normal development, growth, and homeostasis. Particularly, the rate of protein synthesis in the adult heart is one of the lowest amongst tissues but increases markedly in response to stress and hypertrophic stimuli. The molecular bases for these historical observations, however, remain poorly understood. Post-transcriptional modifications can greatly influence translation rates to achieve regulated protein synthesis. Particularly, the 3’UTR sequence and poly(A) tail length can influence translation through RNA binding proteins (RBPs) and non-coding RNAs (ncRNAs). Nuclear poly(A) binding protein (PABPN1) regulates the 3’UTR of transcripts through alternative polyadenylation (APA) This alters the UTR sequence which influences how downstream regulatory molecules bind to and modulate the target mRNA. PABPN1 is also deterministic of poly(A) tail length. Longer poly(A) tails are associated with enhanced transcript stability and increased translation rates through polysome formation. We discovered that PABPN1 expression is dynamically regulated in the heart. This is correlated with decreased protein synthesis rates, widespread APA, and shorter poly(A) tail lengths. In periods of adult hypertrophy protein synthesis rates are increased and concurrent changes to APA and poly(A) tail length occurs; PABPN1 becomes reexpressed during these periods of hypertrophy. Our data demonstrates a model by which cardiac maturation and hypertrophy are driven by changes in translation mediated by APA and poly(A) tails, regulated, in part, by PABPN1.

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Polyadenylation & 3' end formation 605 Intronic polyadenylation evasion leads to reduced mRNA isoform diversity and neurodegeneration

Geneva LaForce1, Jordan Farr1, Evren Gumus2,3, Cydni Akesson1, Otis Pinkard4, Katherine Johnson1, Thomas Sweet4, Jeff Coller4, Eric Wagner5, Polyxeni Philippidou6, Ashleigh Schaffer1 1Department of Genetics and Genome Sciences, Case Western Reserve University, Cleveland, OH, USA. 2Department of Medical Genetics, Mugla Sitki Kocman University, Mugla, Turkey. 3Department of Medical Genetics, University of Harran, Sanliurfa, Turkey. 4Center for RNA Science and Therapeutics, Case Western Reserve University, Cleveland, OH, USA. 5Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX, USA. 6Department of Neuroscience, Case Western Reserve University, Cleveland, OH, USA

Abstract

Tight regulation of mRNA isoform expression is essential for neuronal development, maintenance, and function; however, the factors governing this process are largely unknown. Here we show the multifunctional RNA kinase, CLP1, promotes usage of mRNA polyadenylation sites within introns to enrich isoform diversity and attenuate long gene expression in neurons, and that dysregulation of this process leads to neurodegeneration. We develop induced pluripotent stem cell derived motor neurons from patients with the pathogenic, bi-allelic, CLP1 p.R140H variant that display progressive neuronal loss and perform transcriptome analysis. We find reduced CLP1 function leads to a distinct pattern of mRNA processing defects in motor neurons characterized by reduced intronic polyadenylation (IPA) and elevated expression of full-length isoforms of long genes harboring IPA sites. Clp1 p.R140H mutant mice recapitulate the human disease and show correlated gene expression changes with the patient-derived motor neurons, validating transcriptional dysregulation in motor neuron disease. Lastly, we mine publicly available mRNA 3’-end sequencing data and find sporadic Parkinson’s Disease brain tissue shows the same pattern of reduced IPA and upregulation of long genes, identifying a previously unrecognized mRNA misprocessing signature in neurodegeneration that suggests a common mechanism of disease.

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Polyadenylation & 3' end formation 613 FttA is a CPSF73 homologue that terminates transcription in Archaea

Travis Sanders1, Breanna Wenck1, Jocelyn Selan2, Mathew Barker1, Stavros Trimmer1, Julie Walker1, Thomas Santangelo1 1Colorado State University, Fort Collins, Colorado, USA. 2Colorado State University, Fort Collins, Coloraod, USA

Abstract

Regulated gene expression is achieved in large part by controlling the activities of essential, multi-subunit RNA polymerase transcription elongation complexes (TECs). The extreme stability required of TECs to processively transcribe large genomic regions necessitates robust mechanisms to terminate transcription. Efficient transcription termination is particularly critical for gene-dense bacterial and archaeal genomes wherein continued transcription would necessarily transcribe immediately adjacent genes, result in conflicts between the transcription and replication apparatuses and the coupling of transcription and translation would permit loading of ribosomes onto aberrant transcripts. Only select sequences or transcription termination factors can disrupt the otherwise extremely stable TEC and we demonstrate that one of the last universally conserved archaeal proteins with unknown biological function is the Factor that terminates transcription in Archaea (FttA). FttA resolves the dichotomy of a prokaryotic gene structure (operons and polarity) and eukaryotic molecular homology (general transcription apparatus) observed in Archaea. This missing-link between prokaryotic and eukaryotic transcription regulation provides the most parsimonious link to the evolution of the processing activities involved in RNA 3’-end formation in Eukarya.

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Polyadenylation & 3' end formation 617 Adding and subtracting poly(A) tails in the nucleus

Kathryn Williams, Richa Singhania, Jonathan Wattis, Cornelia de Moor University of Nottingham, Nottingham, United Kingdom

Abstract

Poly(A) tails affect multiple aspects of gene regulation: they help identify mRNAs for nuclear export, enhance translation efficiency, and are essential to regulating mRNA degradation rate. A poly(A) tail of 200-250 residues is thought to be uniformly added to newly synthesised mRNA and later gradually removed in the cytoplasm, allowing degradation of the mRNA itself. We have shown that poly(A) tail addition is not uniform; soon after serum induction, transiently expressed mRNAs are exported with long poly(A) tails, but towards the end of the transcription pulse the tails for new transcripts are much shorter. In contrast, housekeeping mRNAs consistently receive only 50-70 adenosines both before and throughout the serum response and do not appear to be gradually deadenylated. Our novel 3’ deep sequencing technique, Pat-Quant-Seq can be used to investigate nuclear poly(A) regulation thanks to its low input requirements. Our first set data confirmed that for most mRNAs, poly(A) tail size is already determined in the nucleus. Knockdown of the Ccr4-Not deadenylase subunit, Cnot1 increased nuclear poly(A) tail size for all mRNAs tested, indicating it is involved in nuclear poly(A) tail regulation. Chromatin fractionation revealed differences in the location of mRNA deadenylation between genes. Nascent poly(A) regulation could both conserve energy and enhance the precision with which gene expression is controlled by narrowing the peak in mature mRNA levels, thus limiting the time during which translation can occur and perhaps enhancing translation efficiency.

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Polyadenylation & 3' end formation 648 The C. elegans 3′ UTRome v2 reveals principles of mRNA cleavage and polyadenylation in eukaryotic transcriptomes

Hannah Steber1, Christina Gallante2, Shannon O'Brien2, Po-Lin Chiu3, Marco Mangone1 1Arizona State University, tempe, AZ, USA. 2Arizona State University, Tempe, AZ, USA. 3Arizona State UniversityTempe, Tempe, AZ, USA

Abstract

3′ Untranslated regions (3′ UTRs) of mRNAs emerged as central regulators of cellular function because they contain important but poorly characterized cis-regulatory elements targeted by a multitude of regulatory factors. The model Caenorhabditis elegans is ideal to study these interactions because it possesses a well- defined 3′ UTRome. To improve its annotation, we have used a genome-wide bioinformatics approach to download raw transcriptome data for 1088 transcriptome data sets corresponding to the entire collection of C. elegans trancriptomes from 2015 to 2018 from the Sequence Read Archive at the NCBI. We then extracted and mapped high-quality 3′-UTR data at ultradeep coverage. Here, we describe the updated version of the worm 3′ UTRome (v2). This resource contains high-quality 3′-UTR data mapped at single-base ultra resolution for 23,084 3′-UTR isoform variants corresponding to 14,788 protein-coding genes and is updated to the latest release of WormBase. In addition to the release of the resource, we have also performed a comparative analysis of the C. elegans cleavage and polyadenylation complex (CPC) and used our updated 3’UTRome v2 to perform in vivo studies to probe principles of mRNA cleavage and polyadenylation in this organism. We found that the CPC in C. elegans is highly conserved to its human counterpart, with most of the functional domains and critical amino acids preserved. While most of the 3’UTRs possess a known Polyadenylation signal element (PAS) localized around -19 nt from the cleavage site (AAUAAA), non-canonical PAS 3’UTRs possess a less stringent requirement but preserve the chemical nature of the element which is RRYRRR. The majority of C. elegans 3’UTRs terminate with a terminal Adenosine nucleotide, which we speculate is included by the RNA polymerase II during the transcription step. This Adenosine nucleotide is required for proper cleavage since its removal impacts the location of the cleavage site in vivo. The worm 3′ UTRome v2 represents the most comprehensive and high-resolution 3′-UTR data set available in C. elegans and provides a novel resource to investigate the mRNA cleavage and polyadenylation reaction, 3′- UTR biology, and miRNA targeting in a living organism.

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Polyadenylation & 3' end formation 757 The C2H2-zinc finger protein Sp1 is a novel regulator of eukaryotic alternative polyadenylation

Jingwen Song, Syed Nabeel-Shah, Shuye Pu, Hyunmin Lee, Ulrich Braunschweig, Zuyao Ni, Edyta Marcon, Guoqing Zhong, Debashish Ray, Kevin C.H. Ha, Xinghua Guo, Zhaolei Zhang, Timothy R. Hughes, Benjamin J. Blencowe, Jack F. Greenblatt Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, ON, Canada

Abstract

Most eukaryotic pre-mRNAs are matured by undergoing 3’ end cleavage and polyadenylation (CPA) via the assembly of multiprotein complexes. The majority of human genes have more than one CPA sites and generate different lengths of mRNAs via alternative polyadenylation (APA). C2H2-zinc finger proteins (C2H2-ZNFs) constitute a large class of putative transcription factors. Previously we used ChIP-seq with inducible GFP- tagged constructs of 130 C2H2-ZNFs to show that most human C2H2-ZNFs bind DNA sequences specifically. Interestingly, some of the non-KRAB C2H2-ZNFs bind preferentially to splice and termination sites, as well as 3’ ends, indicating their possible roles in splicing and transcription termination. However, whether and how these DNA-binding proteins play a role in 3’end formation remained largely unknown.

Herein we identified the C2H2 transcription factor Specificity protein 1 (Sp1) as a broad regulator of APA, governing the usage of distal cleavage sites. By applying QAPA (Quantification of APA) method on deep RNA- seq data, we show that depletion of Sp1 induces the 3’UTR lengthening of 2344 mRNAs among total 6526 mapped transcripts. iCLIP-seq reveals that Sp1 binds an AG-rich RNA sequence upstream of the CPA sites at target 3’ UTRs. Additionally, we report that Sp1 prevents the CPA at distal cleavage sites via interacting with multiple CPA subunits and impeding the occupancy of CFIm to RNAs. By applying bioinformatic analysis on TCGA breast cancer data, we observed that the lengths of 3’ UTRs of target genes are generally related to Sp1 expression levels, implying the role of Sp1 in APA regulation in breast cancer. This study contributes to the current understanding of the diverse functions of ZNF transcription factors by revealing a novel role of Sp1 as an APA regulator in tumorigenesis.

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Polyadenylation & 3' end formation 780 Investigating the key mechanisms of alternative polyadenylation

Rachael Turner1, Paul Harrison1, Bernhard Dichtl2, Traude Beilharz1 1Monash University, Melbourne, Victoria, Australia. 2Deakin University, Melbourne, Victoria, Australia

Abstract

The 3’-untranslated region (3’-UTR) marks an important landscape for regulation of mRNA fate. Through the binding of key regulatory elements such as microRNAs and RNA binding proteins, the 3’-UTR can influence the stability, localisation and translatability of mRNA. In eukaryotes, up to 80% of genes regulate 3’-UTR length by encoding multiple locations for cleavage and polyadenylation (CPA) within the nascent transcript, a process known as alternative polyadenylation (APA). A naturally occurring adenosine analogue, cordycepin (3’- deoxyadenosine), is known to cause bulk mRNA shortening through chain termination due to its lack of a reactive hydroxyl group at the 3’ position. Using poly(A)-tail-focused deep sequencing, PAT-seq, we show that, unexpectedly, treatment with cordycepin induces bulk transcript lengthening and increased usage of distal CPA sites in yeast. A similar result was found for yeast strains with defective CPA machinery, implicating CPA complex stoichiometry as a key determinant of CPA site choice. Using yeast nucleosome occupancy data, we also show that genes that are able to undergo alternative polyadenylation have a wider nucleosome depleted region at the 3’-end of the gene suggesting that the higher ordering of the gene itself plays a significant role in CPA site usage.

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Polyadenylation & 3' end formation 799 LIN28-dependency and tissue specificity of TUT4/7-mediated RNA uridylation in tumorigenesis

Ragini Medhi1,2, Jon Price2, Eric Miska1,2 1Department of Genetics, University of Cambridge, Cambridge, United Kingdom. 2Wellcome Trust/Cancer Research UK Gurdon Institute, Cambridge, United Kingdom

Abstract

Terminal uridylation of mRNAs with short poly(A) tails (<25 nucleotides) is catalysed by the terminal uridyl transferases TUT4 and TUT7 (TUT4/7) and results in direction-dependent RNA decay. However, the mechanisms of recruitment of TUT4/7 to these mRNAs is unclear. TUT4/7 is also recruited to another species of RNA called microRNAs (miRNAs) through a physical interaction with the protein LIN28A. LIN28A recognises the stem-loop structure of miRNA precursors (pre-miRNA) and recruits TUT4/7. Consequently, the pre-miRNA is oligo-uridylated and degraded. On the contrary, in absence of LIN28A, TUT4/7 mono-uridylates pre-miRNAs and aids in their biogenesis. Here, we report LIN28A-mediated functions of TUT4/7 in cancer and report differences observed upon loss of uridylation in presence or absence of LIN28A. We observe defects in wound healing and cell migration in TUT4/7-/- mutants of LIN28A-postive ovarian cancer cell line IGROV1. Conversely, these defects are not observed in TUT4/7-/- mutants of LIN28A-negative prostate cancer cell line DU145. Furthermore, LIN28A is endogenously overexpressed in IGROV1 TUT4/7-/- mutants and we observe that upon stable integration of LIN28A in DU145, LIN28A is overexpressed in the TUT4/7-/-mutants. Therefore, this suggests that LIN28A levels are regulated in a TUT4/7-dependent manner. We explore possible mechanisms of TUT4/7 regulation on other select mRNAs. As LIN28A protein recognises RNA motifs within mRNA sequences, we explore mRNA targets regulated by TUT4/7 after potential initial binding by LIN28A. In addition, we report mRNA targets and miRNA targets of TUT4/7 that are LIN28A-dependent and independent of or specific to the tissue type. This study adds to the ever-increasing evidence of the importance LIN28A-TUT4/7 interaction ranging from embryonic development to cancer. In addition, we provide a mechanism of selective target decay in tumorigenesis.

Presenting author email [email protected]

Topic category

Polyadenylation & 3' end formation 933 Regulation of neural mRNA 3’-end landscape by Elav/Hu family RNA binding proteins

Lu Wei, Seungjae Lee, Sonali Majumdar, Binglong Zhang, Piero Sanfilippo, Eric Lai MSKCC, New York, NY, USA

Abstract

Cleavage and polyadenylation (CPA) is an essential processing step during the maturation of metazoan mRNAs. The CPA machinery recognizes polyA signals in pre-mRNA and guides transcript cleavage and addition of polyA tails. Recent studies reveal that the majority of metazoan transcripts undergo alternative polyadenylation (APA), yielding isoforms with distinct 3' UTRs. We and others found that the nervous system of Drosophila and mammals exhibits a very distinct APA landscape, with broad utilization of very long 3’ UTRs. However, the underlying factors and mechanisms that specifies the extended neural 3' UTR landscape, and its associated biological consequences, remain largely elusive. Drosophila Elav, the founding member of the ELAV/Hu RNA binding protein (RBP) family, was reported to be critical for this process. Unexpectedly, we find that elav mutants, long thought to be embryonic lethal, can survive as early larvae, and that elav null central nervous system (CNS) maintains substantial neural 3' UTR extensions. Meanwhile, two Elav orthologs Rbp9 and Fne, which have high structural similarity as Elav, have similar capacities to induce a lengthened 3' UTR landscape in an ectopic setting in non-neural context. These factors promote accumulation of chromatin-associated, 3' UTR-extended, nascent transcripts, through inhibition of proximal polyadenylation site (PAS) usage. Interestingly, we observed Fne, which usually localizes in cytoplasmic compartment in normal CNS, shows partial nuclear localization in elav mutants. Further, genomic analysis of global 3’ UTR of mutant CNS indicated that elav/fne double mutant CNS exhibit strong and broad loss of neural APA. Overall, this constitutes the first genetic setting that can strongly abrogate the neural extended 3' UTR landscape. Moreover, the overlapping activities of Drosophila ELAV/Hu RBPs in this process may inform how the mammalian neural APA is established.

Presenting author email [email protected]

Topic category

Polyadenylation & 3' end formation