Reprogramming RNA processing: an emerging therapeutic landscape
Authors: Christopher R. Neil, Michael W. Seiler, Dominic J. Reynolds, Jesse J. Smith, Frédéric H. Vaillancourt, Peter G. Smith, Anant A. Agrawal
Journal: Trends in Pharmacological Sciences (2022)

mRNA is the result of tightly regulated RNA-processing mechanisms and has a key role in defining proteomic expression, diversity, and cellular fitness.Small-molecule and antisense oligonucleotide-based therapeutics have shown promise in influencing the RNA processing of selective targets, with US Food and Drug Administration-approved drugs for both modalities and new experimental agents in preclinical and clinical development.Targeting RNA processing provides the opportunity to use nucleic acid sequence specificity to approach targets that are challenging to drug at the protein level.Advances in RNA-based platforms and technologies in the space of next-generation sequencing, bioinformatics, structural biology, screening methodologies, and chemistry are converging to inform the rational design of novel therapeutics. The production of a mature mRNA requires coordination of multiple processing steps, which ultimately control its content, localization, and stability. These steps include some of the largest macromolecular machines in the cell, which were, until recently, considered undruggable due to their biological complexity. Building from an expanded understanding of the underlying mechanisms that drive these processes, a new wave of therapeutics is seeking to target RNA processing. With a focus on impacting gene regulation at the RNA level, such modalities offer potential for sequence-specific resolution in drug design. Here, we review our current understanding of RNA-processing events and their role in gene regulation, with a focus on the therapeutic opportunities that have emerged within this landscape. The production of a mature mRNA requires coordination of multiple processing steps, which ultimately control its content, localization, and stability. These steps include some of the largest macromolecular machines in the cell, which were, until recently, considered undruggable due to their biological complexity. Building from an expanded understanding of the underlying mechanisms that drive these processes, a new wave of therapeutics is seeking to target RNA processing. With a focus on impacting gene regulation at the RNA level, such modalities offer potential for sequence-specific resolution in drug design. Here, we review our current understanding of RNA-processing events and their role in gene regulation, with a focus on the therapeutic opportunities that have emerged within this landscape. Traditionally, mRNA (see Glossary) has been viewed as an intermediary between the genetic information encoded within DNA and its ultimate readout as a protein. However, over the past several decades, our understanding of RNA has advanced beyond this limited role as a messenger molecule to include active roles in fine-tuning gene expression. Much of this utility stems from the tremendous amount of information that is encoded within the RNA transcript itself, which allows for the coordinated regulation of a host of downstream processing events in response to dynamic cellular cues [1.Manning K.S. Cooper T.A. The roles of RNA processing in translating genotype to phenotype.Nat. Rev. Mol. Cell Biol. 2017; 18: 102-114Google Scholar]. As a eukaryotic gene is transcribed, a pre-mRNA is generated, which is immediately subject to a series of chemical modifications. Following the initial incorporation of nucleotides into the nascent transcript, 5′ end capping takes place, followed by the removal of large intervening sections of noncoding RNA, termed introns, and subsequent ligation of protein-coding exons through a process referred to as RNA splicing [2.Ramanathan A. et al.mRNA capping: biological functions and applications.Nucleic Acids Res. 2016; 44: 7511-7526Google Scholar,3.Herzel L. et al.Splicing and transcription touch base: co-transcriptional spliceosome assembly and function.Nat. Rev. Mol. Cell Biol. 2017; 18: 637-650Google Scholar]. In coordination with transcriptional termination, the 3′ end undergoes additional processing events that include cleavage and the addition of a polyadenosine (polyA) tail [4.Kumar A. et al.Mechanistic insights into mRNA 3′-end processing.Curr. Opin. Struct. Biol. 2019; 59: 143-150Google Scholar] (Figure 1A ). These processing events are dependent on interactions between cis-acting sequence elements and trans-acting protein and RNA factors, which coordinate the dynamic assembly and function of large ribonucleoprotein (RNP) complexes [4.Kumar A. et al.Mechanistic insights into mRNA 3′-end processing.Curr. Opin. Struct. Biol. 2019; 59: 143-150Google Scholar,5.Shi Y. Mechanistic insights into precursor messenger RNA splicing by the spliceosome.Nat. Rev. Mol. Cell Biol. 2017; 18: 655-670Google Scholar]. These alterations not only define the length and composition of an mRNA, but also provide opportunities for regulation of RNA stability, localization, and translation (Figure 1B). Beyond basal processing, most human genes are subject to alternative splicing and polyadenylation events, which can produce multiple mRNA transcripts, each with potentially distinct coding and regulatory sequences [6.Pan Q. et al.Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing.Nat. Genet. 2008; 40: 1413-1415Google Scholar, 7.Wang E.T. et al.Alternative isoform regulation in human tissue transcriptomes.Nature. 2008; 456: 470-476Google Scholar, 8.Kwan T. et al.Genome-wide analysis of transcript isoform variation in humans.Nat. Genet. 2008; 40: 225-231Google Scholar, 9.Djebali S. et al.Landscape of transcription in human cells.Nature. 2012; 489: 101-108Google Scholar, 10.Braunschweig U. et al.Widespread intron retention in mammals functionally tunes transcriptomes.Genome Res. 2014; 24: 1774-1786Google Scholar]. While these processing events largely occur co-transcriptionally in the nucleus [11.Moteki S. Price D. Functional coupling of capping and transcription of mRNA.Mol. Cell. 2002; 10: 599-609Google Scholar, 12.Fusby B. et al.Coordination of RNA polymerase II pausing and 3′ end processing factor Recruitment with alternative polyadenylation.Mol. Cell. Biol. 2016; 36: 295-303Google Scholar, 13.Drexler H.L. et al.Splicing kinetics and coordination revealed by direct nascent RNA sequencing through nanopores.Mol. Cell. 2020; 77: 985-998Google Scholar, 14.Bedi K. et al.Cotranscriptional splicing efficiencies differ within genes and between cell types.RNA. 2021; 27: 829-840Google Scholar], the continued association of RNA-binding proteins (RBPs) that function throughout RNA metabolism [15.Nostrand E.L.V. et al.A large-scale binding and functional map of human RNA-binding proteins.Nature. 2020; 583: 711-719Google Scholar] ultimately controls transcript expression in the cytoplasm by supporting interactions with either translational machinery or components of the mRNA surveillance and decay pathways [16.Gehring N.H. Roignant J.-Y. Anything but ordinary – emerging splicing mechanisms in eukaryotic gene regulation.Trends Genet. 2020; 37: 355-372Google Scholar,17.Passmore L.A. Coller J. Roles of mRNA poly(A) tails in regulation of eukaryotic gene expression.Nat. Rev. Mol. Cell Biol. 2021; 23: 93-106Google Scholar]. Emerging research suggests that RNA-processing events also signal back to change patterns of host gene transcription itself [18.Xiao R. et al.Pervasive chromatin-RNA binding protein interactions enable RNA-based regulation of transcription.Cell. 2019; 178: 107-121Google Scholar, 19.Fiszbein A. et al.Exon-mediated activation of transcription starts.Cell. 2019; 179: 1551-1565Google Scholar, 20.Fiszbein A. et al.Widespread occurrence of hybrid internal-terminal exons in human transcriptomes.Sci. Adv. 2022; 8: eabk1752Google Scholar]. In this way, mechanisms of RNA processing have the capacity to significantly increase the diversity of transcripts and functions encoded by the genome, with defects in such processing events representing a prevalent source of both rare monogenic disorders and more common chronic diseases with complex patterns of inheritance [21.Scotti M.M. Swanson M.S. RNA mis-splicing in disease.Nat. Rev. Genet. 2016; 17: 19-32Google Scholar,22.Gruber A.J. Zavolan M. Alternative cleavage and polyadenylation in health and disease.Nat. Rev. Genet. 2019; 20: 599-614Google Scholar] (Figure 2).Figure 2Shifts in RNA processing define and contribute to disease states.Show full caption(A) Cis-acting sequence elements coordinate assembly of the spliceosome to drive RNA splicing. (i) Mutations within core splice sites [including the 5′ (green) and 3′ (gold) splice sites, as well as the branch point sequence (red) and polypyrimidine tract (blue)] and (ii) supporting auxiliary sequences (enhancer and silencer sites) frequently shift splicing outcomes within mature mRNAs. Aberrant splicing events that lead to (iii) shifts in frame or (iv) the inclusion of poison exons similarly disrupt gene expression and contribute to an array of splicing-related disorders (examples listed by category). (B) Widespread shifts in alternative splicing patterns (e.g., alternative splice site usage, cassette exon usage, and intron retention) are common in malignancies with underlying mutations in core spliceosomal components (with each of the depicted trans-acting proteins showing high incidence of mutation in patients). (C) Shifts in alternative polyadenylation (APA) to both (i) polyadenylation sites (pink ticks) housed within the gene body (I) or (ii) distal (D) versus proximal (P) sites within a common 3′ untranslated region (UTR) can alter the regulation of an mRNA transcript, contributing to changes in individual gene expression. APA is subject to endogenous regulation through several of the highlighted mechanisms, as well as disruption as a result of mutations (black asterisk) in core regulatory and processing components. Such aberrant APA can contribute to a variety of diseases (examples listed).View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Cis-acting sequence elements coordinate assembly of the spliceosome to drive RNA splicing. (i) Mutations within core splice sites [including the 5′ (green) and 3′ (gold) splice sites, as well as the branch point sequence (red) and polypyrimidine tract (blue)] and (ii) supporting auxiliary sequences (enhancer and silencer sites) frequently shift splicing outcomes within mature mRNAs. Aberrant splicing events that lead to (iii) shifts in frame or (iv) the inclusion of poison exons similarly disrupt gene expression and contribute to an array of splicing-related disorders (examples listed by category). (B) Widespread shifts in alternative splicing patterns (e.g., alternative splice site usage, cassette exon usage, and intron retention) are common in malignancies with underlying mutations in core spliceosomal components (with each of the depicted trans-acting proteins showing high incidence of mutation in patients). (C) Shifts in alternative polyadenylation (APA) to both (i) polyadenylation sites (pink ticks) housed within the gene body (I) or (ii) distal (D) versus proximal (P) sites within a common 3′ untranslated region (UTR) can alter the regulation of an mRNA transcript, contributing to changes in individual gene expression. APA is subject to endogenous regulation through several of the highlighted mechanisms, as well as disruption as a result of mutations (black asterisk) in core regulatory and processing components. Such aberrant APA can contribute to a variety of diseases (examples listed). Building from an increased understanding of the sequence and structural features that underlie RNA-processing events [4.Kumar A. et al.Mechanistic insights into mRNA 3′-end processing.Curr. Opin. Struct. Biol. 2019; 59: 143-150Google Scholar,23.Plaschka C. et al.Structural basis of nuclear pre-mRNA splicing: lessons from yeast.Cold Spring Harb. Perspect. Biol. 2019; 11a032391Google Scholar, 24.Yan C. et al.Molecular mechanisms of pre-mRNA splicing through structural biology of the spliceosome.Cold Spring Harb. Perspect. Biol. 2019; 11a032409Google Scholar, 25.Kastner B. et al.Structural insights into nuclear pre-mRNA splicing in higher eukaryotes.Cold Spring Harb. Perspect. Biol. 2019; 11a032417Google Scholar] and how mutations in these elements drive disease progression [26.Xiong H.Y. et al.The human splicing code reveals new insights into the genetic determinants of disease.Science. 2015; 347: 1254806Google Scholar,27.Soemedi R. et al.Pathogenic variants that alter protein code often disrupt splicing.Nat. Genet. 2017; 49: 848-855Google Scholar], a new wave of therapeutics is emerging with the goal of achieving gene-specific regulation through direct targeting of RNA and RNA–protein complexes. In this review, we explore how the complexity of post-transcriptional gene regulation offers unique opportunities for therapeutic intervention, with a focus on targets previously considered undruggable. We focus on mechanisms of pre-mRNA splicing and 3′ end processing, highlighting emerging therapeutics that are targeting these processes to control gene expression (detailed in Table 1).Table 1Therapeutic modulators of RNA processing in clinical developmentaAbbreviations: AML, acute myeloid leukemia; CMML, chronic myelomonocytic leukemia; MDS, myelodysplastic syndromesTherapeuticTargetDiseaseMechanism of actionDevelopment stage and active clinical trialRefsRNA splicingSplicing eventEteplirsenDMDDMDASO promotes exon 51 skipping in DMD; restores reading frameCompleted Phase III (NCT02255552);FDA approved[35.Charleston J.S. et al.Eteplirsen treatment for Duchenne muscular dystrophy.Neurology. 2018; 90: e2146-e2154Google Scholar,39.Mendell J.R. et al.Eteplirsen for the treatment of Duchenne muscular dystrophy.Ann. Neurol. 2013; 74: 637-647Google Scholar,40.Mendell J.R. et al.Longitudinal effect of eteplirsen versus historical control on ambulation in Duchenne muscular dystrophy.Ann. Neurol. 2016; 79: 257-271Google Scholar]GolodirsenDMDDMDASO promotes exon 53 skipping in DMD; restores reading framePhase III (NCT03532542); Phase IV (NCT04708314);FDA approved[38.Aartsma-Rus A. et al.Development of exon skipping therapies for Duchenne muscular dystrophy: a critical review and a perspective on the outstanding issues.Nucleic Acid Ther. 2017; 27: 251-259Google Scholar]CasimersenDMDDMDASO promotes exon 45 skipping in DMD; restores reading framePhase III (NCT03532542);FDA approved[38.Aartsma-Rus A. et al.Development of exon skipping therapies for Duchenne muscular dystrophy: a critical review and a perspective on the outstanding issues.Nucleic Acid Ther. 2017; 27: 251-259Google Scholar]ViltolarsenDMDDMDASO promotes exon 53 skipping in DMD; restores reading framePhase III (NCT04060199); Phase III (NCT04768062);FDA approved[38.Aartsma-Rus A. et al.Development of exon skipping therapies for Duchenne muscular dystrophy: a critical review and a perspective on the outstanding issues.Nucleic Acid Ther. 2017; 27: 251-259Google Scholar]NusinersenSMN2SMAASO enhances exon 7 inclusion in SMN2; gene compensation for SMN1 deficiencyCompleted Phase III (NCT02292537);FDA approved[36.Michelson D. et al.Evidence in focus: nusinersen use in spinal muscular atrophy: Report of the Guideline Development, Dissemination, and Implementation Subcommittee of the American Academy of Neurology.Neurology. 2018; 91: 923-933Google Scholar,43.Hua Y. et al.Antisense correction of SMN2 splicing in the CNS rescues necrosis in a type III SMA mouse model.Gene Dev. 2010; 24: 1634-1644Google Scholar,44.Passini M.A. et al.Antisense oligonucleotides delivered to the mouse CNS ameliorate symptoms of severe spinal muscular atrophy.Sci. Transl. Med. 2011; 3: 72ra18Google Scholar,46.Hua Y. et al.Peripheral SMN restoration is essential for long-term rescue of a severe spinal muscular atrophy mouse model.Nature. 2011; 478: 123-126Google Scholar,47.Finkel R.S. et al.Nusinersen versus sham control in infantile-onset spinal muscular atrophy.N. Engl. J. Med. 2017; 377: 1723-1732Google Scholar]STK-001SCN1ADravet syndromeASO promotes exon 20N skipping in SCN1A; restores reading framePhase II (NCT04740476)[34.Han Z. et al.Antisense oligonucleotides increase Scn1a expression and reduce seizures and SUDEP incidence in a mouse model of Dravet syndrome.Sci. Transl. Med. 2020; 12: eaaz6100Google Scholar]MilasenCLN7Batten's diseaseASO restricts inclusion of i6 intronic segment in CLN7'N-of-1' clinical trial[48.Kim J. et al.Patient-customized oligonucleotide therapy for a rare genetic disease.N. Engl. J. Med. 2019; 381: 1644-1652Google Scholar]RisdiplamSMN2SMAEnhances exon 7 inclusion in SMN2; gene compensation for SMN1 deficiencyFDA approved[53.Ratni H. et al.Discovery of risdiplam, a selective survival of motor neuron-2 (SMN2) gene splicing modifier for the treatment of spinal muscular atrophy (SMA).J. Med. Chem. 2018; 61: 6501-6517Google Scholar,58.FDA FDA Approves Oral Treatment for Spinal Muscular Atrophy. FDA, 2020Google Scholar]BranaplamSMN2SMAEnhances exon 7 inclusion in SMN2; gene compensation for SMN1 deficiencyPhase I/II (NCT02268552)[54.Cheung A.K. et al.Discovery of small molecule splicing modulators of survival motor neuron-2 (SMN2) for the treatment of spinal muscular atrophy (SMA).J. Med. Chem. 2018; 61: 11021-11036Google Scholar]HTTHDReduces expression of mutant HTT proteinPhase II (NCT05111249)[59.National Library of Medicine A dose range finding study with open-label extension to evaluate the safety of oral LMI070/branaplam in early manifest Huntington's Disease (VIBRANT-HD).https://clinicaltrials.gov/ct2/show/NCT05111249Date: 2021Date accessed: March 1, 2022Google Scholar]PTC-518HTTHDPromotes inclusion of PTC containing pseudoexon from intron 49 of HTTPhase I[60.Bhattacharyya A. et al.Small molecule splicing modifiers with systemic HTT-lowering activity.Nat. Commun. 2021; 12: 7299Google Scholar]Splicing factorE7107SF3bAdvanced solid tumorsSplicing modulationCompleted Phase I (NCT00499499)[73.Eskens F.A.L.M. et al.Phase I pharmacokinetic and pharmacodynamic study of the first-in-class spliceosome inhibitor E7107 in patients with advanced solid tumors.Clin. Cancer Res. 2013; 19: 6296-6304Google Scholar]H3B-8800SF3bAML, CMML, MDSSplicing modulation/synthetic lethalityPhase I (NCT02841540)[74.Seiler M. et al.H3B-8800, an orally available small-molecule splicing modulator, induces lethality in spliceosome-mutant cancers.Nat. Med. 2018; 24: 497-504Google Scholar,75.Steensma D.P. et al.Phase I first-in-human dose escalation study of the oral SF3B1 modulator H3B-8800 in myeloid neoplasms.Leukemia. 2021; D35: 3542-3550Google Scholar]E7820RBM39/CAPERαSolid tumors, AML, MDSUbiquitin-mediated degradation of splicing factorCompleted Phase II (NCT00309179); Phase II (NCT05024994)[78.Uehara T. et al.Selective degradation of splicing factor CAPERα by anticancer sulfonamides.Nat. Chem. Biol. 2017; 13: 675-680Google Scholar,79.Faust T.B. et al.Structural complementarity facilitates E7820-mediated degradation of RBM39 by DCAF15.Nat. Chem. Biol. 2020; 16: 7-14Google Scholar]IndisulamRBM39/ CAPERαSolid tumors, AML, MDSUbiquitin-mediated degradation of splicing factorCompleted Phase II (NCT00165867); completed Phase II (NCT00165880)[77.Han T. et al.Anticancer sulfonamides target splicing by inducing RBM39 degradation via recruitment to DCAF15.Science. 2017; 356: eaal3755Google Scholar,78.Uehara T. et al.Selective degradation of splicing factor CAPERα by anticancer sulfonamides.Nat. Chem. Biol. 2017; 13: 675-680Google Scholar,80.Assi R. et al.Final results of a phase 2, open-label study of indisulam, idarubicin, and cytarabine in patients with relapsed or refractory acute myeloid leukemia and high-risk myelodysplastic syndrome.Cancer. 2018; 124: 2758-2765Google Scholar]7,8-Dimethoxy perphenazineU2AFN/ADisrupts interactions between U2AF homology and ligand motifsPreclinical[81.Jagtap P.K.A. et al.Identification of phenothiazine derivatives as UHM-binding inhibitors of early spliceosome assembly.Nat. Commun. 2020; 11: 5621Google Scholar]NSC 194308U2AFLeukemia cells expressing MDS-associated S34F mutantStalls pre-mRNA splicing by promoting an early-stage U2AF2-RNA complexPreclinical[82.Chatrikhi R. et al.A synthetic small molecule stalls pre-mRNA splicing by promoting an early-stage U2AF2-RNA complex.Cell Chem. Biol. 2021; 28: 1145-1157Google Scholar]Splicing factor modificationSRPIN340SRPK1Age-related macular degeneration; cutaneous melanoma; uveal melanomaVEGF isoform switchPreclinical[84.Amin E.M. et al.WT1 mutants reveal SRPK1 to be a downstream angiogenesis target by altering VEGF splicing.Cancer Cell. 2011; 20: 768-780Google Scholar,85.Gammons M.V. et al.Targeting SRPK1 to control VEGF-mediated tumour angiogenesis in metastatic melanoma.Brit. J. Cancer. 2014; 111: 477-485Google Scholar]SPHINXSRPK1Solid tumors, AMLVEGF isoform switchPreclinical[86.Mavrou A. et al.Serine arginine protein kinase-1 (SRPK1) inhibition as a potential novel targeted therapeutic strategy in prostate cancer.Oncogene. 2015; 34: 4311-4319Google Scholar,87.Tzelepis K. et al.SRPK1 maintains acute myeloid leukemia through effects on isoform usage of epigenetic regulators including BRD4.Nat. Commun. 2018; 9: 5378Google Scholar]SRPKIN-1SRPK1/2Age-related macular degenerationVEGF isoform switchPreclinical[88.Hatcher J.M. et al.SRPKIN-1: a covalent SRPK1/2 inhibitor that potently converts VEGF from pro-angiogenic to anti-angiogenic isoform.Cell Chem. Biol. 2018; 25: 460-470Google Scholar,89.Batson J. et al.Development of potent, selective SRPK1 inhibitors as potential topical therapeutics for neovascular eye disease.ACS Chem. Biol. 2017; 12: 825-832Google Scholar]T-025CLK1/2MYC-driven cancers (breast)Reduced CLK-dependent phosphorylation, splicing alterationPreclinical[94.Iwai K. et al.Anti-tumor efficacy of a novel CLK inhibitor via targeting RNA splicing and MYC-dependent vulnerability.EMBO Mol. Med. 2018; 10e8289Google Scholar]SM08502CLK2/3Solid tumors; colorectal cancerBlocks activity of CLK2/3, disruption of WNT pathwayPhase I (NCT03355066)[95.Tam B.Y. et al.The CLK inhibitor SM08502 induces anti-tumor activity and reduces Wnt pathway gene expression in gastrointestinal cancer models.Cancer Lett. 2020; 473: 186-197Google Scholar]GPS167CLKscolorectal canerBlocks activity of CLKs on SRSF10Preclinical[96.Sohail M. et al.A novel class of inhibitors that target SRSF10 and promote p53-mediated cytotoxicity on human colorectal cancer cells.Nar Cancer. 2021; 3: zcab019Google Scholar]TG693DMD (via CLK1)DMDPromotes exon 31 skipping of DMDPreclinical[97.Sako Y. et al.Development of an orally available inhibitor of CLK1 for skipping a mutated dystrophin exon in Duchenne muscular dystrophy.Sci. Rep. 2017; 7: 46126Google Scholar]KinetinIKBKAP (via CLKs)Familial dysautonomiaPromotes exon 20 inclusion in IKBKAP/ELP1Completed Phase I (NCT02274051)[98.Slaugenhaupt S.A. et al.Rescue of a human mRNA splicing defect by the plant cytokinin kinetin.Hum. Mol. Genet. 2004; 13: 429-436Google Scholar,99.Axelrod F.B. et al.Kinetin improves IKBKAP mRNA splicing in patients with familial dysautonomia.Pediatr. Res. 2011; 70: 480-483Google Scholar]RECTASIKBKAP (via CLKs)Familial dysautonomiaEnhances SRSF6 phosphorylation to enhance inclusion of IKBKAP exon 20Preclinical[100.Yoshida M. et al.Rectifier of aberrant mRNA splicing recovers tRNA modification in familial dysautonomia.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: 2764-2769Google Scholar,102.Ajiro M. et al.Therapeutic manipulation of IKBKAP mis-splicing with a small molecule to cure familial dysautonomia.Nat. Commun. 2021; 12: 4507Google Scholar]BPN-15477IKBKAP (via CLKs)Familial dysautonomiaPromotes exon 20 inclusion in IKBKAPPreclinical[104.Gao D. et al.A deep learning approach to identify gene targets of a therapeutic for human splicing disorders.Nat. Commun. 2021; 12: 3332Google Scholar]EPZ015666 (GSK3235025)PRMT5Multiple myeloma; medulloblastoma; mantle cell lymphomaReduced PRMT5-dependent methylation; splicing disruptionPreclinical[110.Chan-Penebre E. et al.A selective inhibitor of PRMT5 with in vivo and in vitro potency in MCL models.Nat. Chem. Biol. 2015; 11: 432-437Google Scholar,111.Duncan K.W. et al.Structure and property guided design in the identification of PRMT5 tool compound EPZ015666.ACS Med. Chem. Lett. 2016; 7: 162-166Google Scholar]GSK3326595PRMT5Solid tumors, non-Hodgkin lymphoma; breast cancer; AML, MDSReduced PRMT5-dependent methylation; splicing disruption; MDM4 isoform switchPhase I (NCT02783300); Phase II (NCT04676516); Phase I/II (NCT03614728)[112.Gerhart S.V. et al.Activation of the p53-MDM4 regulatory axis defines the anti-tumour response to PRMT5 inhibition through its role in regulating cellular splicing.Sci. Rep. 2018; 8: 9711Google Scholar,113.AbuHammad S. et al.Regulation of PRMT5–MDM4 axis is critical in the response to CDK4/6 inhibitors in melanoma.Proc. Natl. Acad. Sci. U. S. A. 2019; 116: 17990-18000Google Scholar]PF-06939999PRMT5Metastatic solid tumorsReduced PRMT5-dependent methylation; inhibition of co-factor binding; splicing disruptionPhase I (NCT03854227)[114.Jensen-Pergakes K. et al.SAM competitive PRMT5 inhibitor PF-06939999 demonstrates antitumor activity in splicing dysregulated NSCLC with decreased liability of drug resistance.Mol. Cancer Ther. 2021; 21: 3-15Google Scholar]3′ end processingT4 and T5PABPN1nonePromote distal-to-proximal APA usagePreclinical: tool molecule[132.Araki S. et al.Decoding transcriptome dynamics of genome-encoded polyadenylation and autoregulation with small-molecule modulators of alternative polyadenylation.Cell Chem. Biol. 2018; 25: 1470-1484Google Scholar]JTE-607CPSF3AML and Ewing's sarcomaBlocks 3′ end cleavage leading to transcription read-through and RNA instabilityPreclinical[133.Uesato N. et al.JTE-607, a multiple cytokine production inhibitor, ameliorates disease in a SCID mouse xenograft acute myeloid leukemia model.Exp. Hematol. 2006; 34: 1385-1392Google Scholar, 134.Tajima N. et al.JTE-607, a multiple cytokine production inhibitor, induces apoptosis accompanied by an increase in p21waf1/cip1 in acute myelogenous leukemia cells.Cancer Sci. 2010; 101: 774-781Google Scholar, 135.Ross N.T. et al.CPSF3-dependent pre-mRNA processing as a druggable node in AML and Ewing's sarcoma.Nat. Chem. Biol. 2020; 16: 50-59Google Scholar]a Abbreviations: AML, acute myeloid leukemia; CMML, chronic myelomonocytic leukemia; MDS, myelodysplastic syndromes Open table in a new tab RNA splicing is the process by which the removal of noncoding intronic sequences from a pre-mRNA is paired with the ligation of coding exons to generate a mature mRNA transcript [3.Herzel L. et al.Splicing and transcription touch base: co-transcriptional spliceosome assembly and function.Nat. Rev. Mol. Cell Biol. 2017; 18: 637-650Google Scholar] (Box 1). Transcriptome-wide analyses have demonstrated that ≥90% of genes incorporate different combinations of exons or display selective retention of introns as a result of dynamically regulated alternative splicing events [6.Pan Q. et al.Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing.Nat. Genet. 2008; 40: 1413-1415Google Scholar, 7.Wang E.T. et al.Alternative isoform regulation in human tissue transcriptomes.Nature. 2008; 456: 470-476Google Scholar, 8.Kwan T. et al.Genome-wide analysis of transcript isoform variation in humans.Nat. Genet. 2008; 40: 225-231Google Scholar,10.Braunschweig U. et al.Widespread intron retention in mammals functionally tunes transcriptomes.Genome Res. 2014; 24: 1774-1786Google Scholar]. In addition to expanding the coding potential of the genome, alternative splicing patterns broadly impact gene expression and confer specialized regulatory potential for cells. Exons encoding specific functional domains can be incorporated to control protein localization, catalytic or binding activity, and overall stability. Highly conserved premature termination codon-containing exons, termed poison exons, have established roles in the autoregulation of serine/arginine-rich (SR) protein expression and are now understood to be essential for cell growth [29.Leclair N.K. et al.Poison exon splicing regulates a coordinated network of SR protein expression during differentiation and tumorigenesis.Mol. Cell. 2020; 80: 648-665Google Scholar,30.Thomas J.D. et al.RNA isoform screens uncover the essentiality and tumor-suppressor activity of ultraconserved poison exons.Nat. Genet. 2020; 52: 84-94Google Scholar]. Additionally, the coordinated retention of introns is thought to restrict transcript expression to fit tissue-specific needs, with functional ties to cellular differentiation [10.Braunschweig U. et al.Widespread intron retention in mammals functionally tunes transcriptomes.Genome Res. 2014; 24: 1774-1786Google Scholar]. For this reason, RNA mis-splicing is causal in an array of human diseases, with mutations in RNA cis-regulatory elements, core spliceosome components, and trans-acting regulatory factors observed in both monogenic and complex disorders [21.Scotti M.M. Swanson M.S. RNA mis-splicing in disease.Nat. Rev. Genet. 2016; 17: 19-32Google Scholar]. The emergence of high-throughput sequencing of large disease cohorts has provided further insights into the extent to which dysregulation of RNA splicing contributes to disease etiology.Box 1Pre-mRNA processing: mechanisms of RNA splicingThe splicing reaction is catalyzed by a massive RNP complex, termed the spliceosome, which is de novo assembled onto each intron [5.Shi Y. Mechanistic insights into precursor messenger RNA splicing by the spliceosome.Nat. Rev. Mol. Cell Biol. 2017; 18: 655-670Google Scholar,23.Plaschka C. et al.Structural basis of nuclear pre-mRNA splicing: lessons from yeast.Cold Spring