Splicing Fidelity Research Articles

Identification of BAY61-3606 Derivatives With Improved Activity in Splicing Modulation That Induces Inclusion of Cassette Exons Similar to the Splicing Factor 3B Subunit 1 Mutation.

Splicing modulation by a small compound offers therapeutic potential for diseases caused by splicing abnormality. However, only a few classes of compounds that can modulate splicing have been identified. We previously identified BAY61-3606, a multiple kinase inhibitor, as a compound that relaxes the splicing fidelity at the 3' splice site recognition. We have also reported the synthesis of derivatives of BAY61-3606. In this study, we tested those compounds for their splicing modulation capabilities and identified two contrasting compounds. These compounds were further investigated for their effects on the whole transcriptome, and analysis of changes in transcription and splicing revealed that the highly active derivative in the splicing reporter assay also showed significantly higher activity in modulating the splicing of endogenously expressed genes. Particularly, cassette exon inclusion was highly upregulated by this compound, and clustering analysis revealed that these effects resembled those in splicing factor 3b subunit 1 (SF3B1) K700E mutant cells but contrasted with those of the splicing inhibitor H3B-8800. Additionally, a group of serine/arginine-rich (SR) protein genes was identified as representatively affected, likely via modulation of poison exon inclusion. This finding could guide further analysis of the mode of action of these compounds on splicing, which could be valuable for developing drugs for diseases associated with splicing abnormalities.

CCAR1 promotes DNA repair via alternative splicing

DNA repair is directly performed by hundreds of core factors and indirectly regulated by thousands of others. We massively expanded a CRISPR inhibition and Cas9-editing screening system to discover factors indirectly modulating homology-directed repair (HDR) in the context of ∼18,000 individual gene knockdowns. We focused on CCAR1, a poorly understood gene that we found the depletion of reduced both HDR and interstrand crosslink repair, phenocopying the loss of the Fanconi anemia pathway. CCAR1 loss abrogated FANCA protein without substantial reduction in the level of its mRNA or that of other FA genes. We instead found that CCAR1 prevents inclusion of a poison exon in FANCA. Transcriptomic analysis revealed that the CCAR1 splicing modulatory activity is not limited to FANCA, and it instead regulates widespread changes in alternative splicing that would damage coding sequences in mouse and human cells. CCAR1 therefore has an unanticipated function as a splicing fidelity factor.

The exon junction complex is required for DMD gene splicing fidelity and myogenic differentiation

Deposition of the exon junction complex (EJC) upstream of exon-exon junctions helps maintain transcriptome integrity by preventing spurious re-splicing events in already spliced mRNAs. Here we investigate the importance of EJC for the correct splicing of the 2.2-megabase-long human DMD pre-mRNA, which encodes dystrophin, an essential protein involved in cytoskeletal organization and cell signaling. Using targeted RNA-seq, we show that knock-down of the eIF4A3 and Y14 core components of EJC in a human muscle cell line causes an accumulation of mis-splicing events clustered towards the 3′ end of the DMD transcript (Dp427m). This deregulation is conserved in the short Dp71 isoform expressed ubiquitously except in adult skeletal muscle and is rescued with wild-type eIF4A3 and Y14 proteins but not with an EJC assembly-defective mutant eIF4A3. MLN51 protein and EJC-associated ASAP/PSAP complexes independently modulate the inclusion of the regulated exons 71 and 78. Our data confirm the protective role of EJC in maintaining splicing fidelity, which in the DMD gene is necessary to preserve the function of the critical C-terminal protein–protein interaction domain of dystrophin present in all tissue-specific isoforms. Given the role of the EJC in maintaining the integrity of dystrophin, we asked whether the EJC could also be involved in the regulation of a mechanism as complex as skeletal muscle differentiation. We found that eIF4A3 knockdown impairs myogenic differentiation by blocking myotube formation. Collectively, our data provide new insights into the functional roles of EJC in human skeletal muscle.

Identification of MYC intron 2 regions that modulate expression.

MYC pre-mRNA is spliced with high fidelity to produce the transcription factor known to regulate cellular differentiation, proliferation, apoptosis, and alternative splicing. The mechanisms underpinning the pre-mRNA splicing of MYC, however, remain mostly unexplored. In this study, we examined the interaction of heterogeneous nuclear ribonucleoprotein C (HNRNPC) with MYC intron 2. Building off published eCLIP studies, we confirmed this interaction with poly(U) regions in intron 2 of MYC and found that full binding is correlated with optimal protein production. The interaction appears to be compensatory, as mutational disruption of all three poly(U) regions was required to reduce both HNRNPC binding capacity and fidelity of either splicing or translation. Poly(U) sequences in MYC intron 2 were relatively conserved across sequences from several different species. Lastly, we identified a short sequence just upstream of an HNRNPC binding region that when removed enhances MYC translation.

MYCN and SNRPD3 cooperate to maintain a balance of alternative splicing events that drives neuroblastoma progression

Many of the pro-tumorigenic functions of the oncogene MYCN are attributed to its regulation of global gene expression programs. Alternative splicing is another important regulator of gene expression and has been implicated in neuroblastoma development, however, the molecular mechanisms remain unknown. We found that MYCN up-regulated the expression of the core spliceosomal protein, SNRPD3, in models of neuroblastoma initiation and progression. High mRNA expression of SNRPD3 in human neuroblastoma tissues was a strong, independent prognostic factor for poor patient outcome. Repression of SNRPD3 expression correlated with loss of colony formation in vitro and reduced tumorigenicity in vivo. The effect of SNRPD3 on cell viability was in part dependent on MYCN as an oncogenic co-factor. RNA-sequencing revealed a global increase in the number of genes being differentially spliced when MYCN was overexpressed. Surprisingly, depletion of SNRPD3 in the presence of overexpressed MYCN further increased differential splicing, particularly of cell cycle regulators, such as BIRC5 and CDK10. MYCN directly bound SNRPD3, and the protein arginine methyltransferase, PRMT5, consequently increasing SNRPD3 methylation. Indeed, the PRMT5 inhibitor, JNJ-64619178, reduced cell viability and SNRPD3 methylation in neuroblastoma cells with high SNRPD3 and MYCN expression. Our findings demonstrate a functional relationship between MYCN and SNRPD3, which maintains the fidelity of MYCN-driven alternative splicing in the narrow range required for neuroblastoma cell growth. SNRPD3 methylation and its protein-protein interface with MYCN represent novel therapeutic targets.Hypothetical model for SNRPD3 as a co-factor for MYCN oncogenesis. SNRPD3 and MYCN participate in a regulatory loop to balance splicing fidelity in neuroblastoma cells. First MYCN transactivates SNRPD3 to lead to high-level expression. Second, SNRPD3 and MYCN form a protein complex involving PRMT5. Third, this leads to balanced alterative splicing (AS) activitiy that is favorable to neuroblastoma. Together this forms as a therapeutic vulnerability where SNRPD3 perturbation or PRMT5 inhibitors are selectively toxic to neuroblastoma by conditionally disturbing splicing activity.

Abstract B018: MTA-cooperative PRMT5 inhibitors selectively modulate RNA splicing in MTAP-deleted cancer cells across histologies

Abstract PRMT5 is a type II arginine methyltransferase that forms an active complex with methylosome protein 50 (MEP50) to catalyze the symmetric dimethylation (SDMA) of arginine residues in proteins that regulate biological roles including modulation of apoptosis, DNA damage response and RNA processing. Some of the best characterized PRMT5 substrates are SNRPB, SNRPD1 and SNRPD3, which are necessary for the formation of the spliceosome and therefore RNA splicing fidelity. The development of MTA-cooperative PRMT5 inhibitors, including the clinical stage inhibitors TNG908 and TNG462, have been shown in preclinical studies to selectively inhibit PRMT5 in MTAP-deleted cancer cells while sparing normal, MTAP-intact cells. Consistent with this selective, on-target mechanism, treatment with TNG908 in preclinical studies results in increased aberrant RNA splicing in MTAP-deleted cells relative to MTAP-intact cells. As MTAP loss occurs in 10-15% of all human cancer, the identification of a signature of alternative splicing events may report pharmacodynamic activity of PRMT5 inhibitors and potentially predict patient response. Similar splicing alterations caused by PRMT5 inhibition were identified in preclinical MTAP-deleted cancer models representing glioblastoma, non-small cell lung cancer, among others, suggesting the events are histology-agnostic. Additionally, titration of exogenous MTA, an endogenous inhibitor of PRMT5, recapitulated the observed splicing events in preclinical MTAP-proficient cells. Similar findings were observed utilizing a pharmacological inhibitor of MTAP suggesting that the identified alternative splicing events are dependent on accumulation of MTA. Collectively, these data suggest that a PRMT5-dependent RNA splicing signature can be developed to identify solid tumors that may best respond to MTA-cooperative PRMT5 inhibitors as well as monitor their pharmacodynamic activity. Citation Format: Matthew R Tonini, Samuel R Meier, Shangtao Liu, Kevin M Cottrell, John P Maxwell, Jannik N Andersen, Alan Huang, Luisa Cimmino, Kimberly J Briggs. MTA-cooperative PRMT5 inhibitors selectively modulate RNA splicing in MTAP-deleted cancer cells across histologies [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B018.

An ATP-independent role for Prp16 in promoting aberrant splicing.

The spliceosome is assembled through a step-wise process of binding and release of its components to and from the pre-mRNA. The remodeling process is facilitated by eight DExD/H-box RNA helicases, some of which have also been implicated in splicing fidelity control. In this study, we unveil a contrasting role for the prototypic splicing proofreader, Prp16, in promoting the utilization of aberrant 5' splice sites and mutated branchpoints. Prp16 is not essential for the branching reaction in wild-type pre-mRNA. However, when a mutation is present at the 5' splice site or if Cwc24 is absent, Prp16 facilitates the reaction and encourages aberrant 5' splice site usage independently of ATP. Prp16 also promotes the utilization of mutated branchpoints while preventing the use of nearby cryptic branch sites. Our study demonstrates that Prp16 can either enhance or impede the utilization of faulty splice sites by stabilizing or destabilizing interactions with other splicing components. Thus, Prp16 exerts dual roles in 5' splice site and branch site selection, via ATP-dependent and ATP-independent activities. Furthermore, we provide evidence that these functions of Prp16 are mediated through the step-one factor Cwc25.

Identification of pararosaniline as a modifier of RNA splicing in Caenorhabditis elegans.

Posttranscriptional splicing of premessenger RNA (mRNA) is an evolutionarily conserved eukaryotic process for producing mature mRNA that is translated into proteins. Accurate splicing is necessary for normal growth and development, and aberrant splicing is increasingly evident in various human pathologies. To study environmental factors that influence RNA splicing, we employed a fluorescent Caenorhabditis elegans in vivo splicing reporter as a biomarker for splicing fidelity to screen against the US EPA ToxCast chemical library. We identified pararosaniline hydrochloride as a strong modifier of RNA splicing. Through gene expression analysis, we found that pararosaniline activates the oxidative stress response and alters the expression of key RNA splicing regulator genes. Physiological assays show that pararosaniline is deleterious to C. elegans development, reproduction, and aging. Through a targeted RNAi screen, we found that inhibiting protein translation can reverse pararosaniline's effect on the splicing reporter and provide significant protection against long-term pararosaniline toxicity. Together, this study reveals a new chemical modifier of RNA splicing and describes translation inhibition as a genetic mechanism to provide resistance.

Conserved and divergent signals in 5' splice site sequences across fungi, metazoa and plants.

In eukaryotic organisms the ensemble of 5' splice site sequences reflects the balance between natural nucleotide variability and minimal molecular constraints necessary to ensure splicing fidelity. This compromise shapes the underlying statistical patterns in the composition of donor splice site sequences. The scope of this study was to mine conserved and divergent signals in the composition of 5' splice site sequences. Because 5' donor sequences are a major cue for proper recognition of splice sites, we reasoned that statistical regularities in their composition could reflect the biological functionality and evolutionary history associated with splicing mechanisms. Results: We considered a regularized maximum entropy modeling framework to mine for non-trivial two-site correlations in donor sequence datasets corresponding to 30 different eukaryotes. For each analyzed species, we identified minimal sets of two-site coupling patterns that were able to replicate, at a given regularization level, the observed one-site and two-site frequencies in donor sequences. By performing a systematic and comparative analysis of 5'splice sites we showed that lineage information could be traced from joint di-nucleotide probabilities. We were able to identify characteristic two-site coupling patterns for plants and animals, and propose that they may echo differences in splicing regulation previously reported between these groups.

Splicing quality control mediated by DHX15 and its G-patch activator SUGP1

Pre-mRNA splicing is surveilled at different stages by quality control (QC) mechanisms. The leukemia-associated DExH-box family helicase hDHX15/scPrp43 is known to disassemble spliceosomes after splicing. Here, using rapid protein depletion and analysis of nascent and mature RNA to enrich for direct effects, we identify a widespread splicing QC function for DHX15 in human cells, consistent with recent invitro studies. We find that suboptimal introns with weak splice sites, multiple branch points, and cryptic introns are repressed by DHX15, suggesting a general role in promoting splicing fidelity. We identify SUGP1 as a G-patch factor that activates DHX15's splicing QC function. This interaction is dependent on both DHX15's ATPase activity and on SUGP1's U2AF ligand motif (ULM) domain. Together, our results support a model in which DHX15 plays a major role in splicing QC when recruited and activated by SUGP1.

Pre-mRNA splicing order is predetermined and maintains splicing fidelity across multi-intronic transcripts.

Combinatorially, intron excision within a given nascent transcript could proceed down any of thousands of paths, each of which would expose different dynamic landscapes of cis-elements and contribute to alternative splicing. In this study, we found that post-transcriptional multi-intron splicing order in human cells is largely predetermined, with most genes spliced in one or a few predominant orders. Strikingly, these orders were conserved across cell types and stages of motor neuron differentiation. Introns flanking alternatively spliced exons were frequently excised last, after their neighboring introns. Perturbations to the spliceosomal U2 snRNA altered the preferred splicing order of many genes, and these alterations were associated with the retention of other introns in the same transcript. In one gene, early removal of specific introns was sufficient to induce delayed excision of three proximal introns, and this delay was caused by two distinct cis-regulatory mechanisms. Together, our results demonstrate that multi-intron splicing order in human cells is predetermined, is influenced by a component of the spliceosome and ensures splicing fidelity across long pre-mRNAs.

MEN1 is a regulator of alternative splicing and prevents R-loop-induced genome instability through suppression of RNA polymerase II elongation.

The fidelity of alternative splicing (AS) patterns is essential for growth development and cell fate determination. However, the scope of the molecular switches that regulate AS remains largely unexplored. Here we show that MEN1 is a previously unknown splicing regulatory factor. MEN1 deletion resulted in reprogramming of AS patterns in mouse lung tissue and human lung cancer cells, suggesting that MEN1 has a general function in regulating alternative precursor mRNA splicing. MEN1 altered exon skipping and the abundance of mRNA splicing isoforms of certain genes with suboptimal splice sites. Chromatin immunoprecipitation and chromosome walking assays revealed that MEN1 favored the accumulation of RNA polymerase II (Pol II) in regions encoding variant exons. Our data suggest that MEN1 regulates AS by slowing the Pol II elongation rate and that defects in these processes trigger R-loop formation, DNA damage accumulation and genome instability. Furthermore, we identified 28 MEN1-regulated exon-skipping events in lung cancer cells that were closely correlated with survival in patients with lung adenocarcinoma, and MEN1 deficiency sensitized lung cancer cells to splicing inhibitors. Collectively, these findings led to the identification of a novel biological role for menin in maintaining AS homeostasis and link this role to the regulation of cancer cell behavior.

Targeted high-throughput mutagenesis of the human spliceosome reveals its in vivo operating principles

The spliceosome is a staggeringly complex machine, comprising, in humans, 5 snRNAs and >150 proteins. We scaled haploid CRISPR-Cas9 base editing to target the entire human spliceosome and investigated the mutants using the U2 snRNP/SF3b inhibitor, pladienolide B. Hypersensitive substitutions define functional sites in the U1/U2-containing A complex but also in components that act as late as the second chemical step after SF3b is dissociated. Viable resistance substitutions map not only to the pladienolide B-binding site but also to the G-patch domain of SUGP1, which lacks orthologs in yeast. We used these mutants and biochemical approaches to identify the spliceosomal disassemblase DHX15/hPrp43 as the ATPase ligand for SUGP1. These and other data support a model in which SUGP1 promotes splicing fidelity by triggering early spliceosome disassembly in response to kinetic blocks. Our approach provides a template for the analysis of essential cellular machines in humans.

A network of DZF proteins controls alternative splicing regulation and fidelity.

Proteins containing DZF (domain associated with zinc fingers) modules play important roles throughout gene expression, from transcription to translation. Derived from nucleotidyltransferases but lacking catalytic residues, DZF domains serve as heterodimerization surfaces between DZF protein pairs. Three DZF proteins are widely expressed in mammalian tissues, ILF2, ILF3and ZFR, which form mutually exclusive ILF2-ILF3 and ILF2-ZFR heterodimers. Using eCLIP-Seq, we find that ZFR binds across broad intronic regions to regulate the alternative splicing of cassette and mutually exclusive exons. ZFR preferentially binds dsRNA in vitro and is enriched on introns containing conserved dsRNA elements in cells. Many splicing events are similarly altered upon depletion of any of the three DZF proteins; however, we also identify independent and opposing roles for ZFR and ILF3 in alternative splicing regulation. Along with widespread involvement in cassette exon splicing, the DZF proteins control the fidelity and regulation of over a dozen highly validated mutually exclusive splicing events. Our findings indicate that the DZF proteins form a complex regulatory network that leverages dsRNA binding by ILF3 and ZFR to modulate splicing regulation and fidelity.

Abstract 1637: MAT2A inhibition in MTAP-/- tumors confers mechanistic vulnerabilities to multiple clinically actionable synthetic lethal drug combinations

Abstract Methylthioadenosine phosphorylase (MTAP) is co-deleted with the tumor suppressor CDKN2A in approximately 15% of solid tumors and can elicit dependency on methionine adenosyltransferase 2A (MAT2A) production of S-adenosyl methionine (SAM), the major methyl donor for cellular methyltransferase reactions. IDE397, a potent small molecule inhibitor of MAT2A, was developed to exploit this synthetic lethal relationship for therapeutic benefit in patients harboring MTAP-/- cancers and demonstrates selective anti-tumor activity in MTAP-/- models. Evaluation of IDE397 efficacy across a panel of 48 MTAP-/- PDX models revealed consistent tumor growth inhibition (TGI>60%) across diverse lineages, with distinct enrichment of tumor regressions in squamous lung cancers. To further enhance the efficacy of IDE397 within and among tumor lineages, we sought to identify rational combination partners. A three-pronged approach was employed that dovetailed molecular profiling of IDE397 drug effects in vivo, chemogenomic evaluation of selective drug sensitivities in MTAP-/- cell lines across the CCLE, and high-throughput in vitro drug combination screens. Unbiased pathway analysis of IDE397-dependent changes in MTAP-/- PDX transcriptomes revealed perturbations in pre-mRNA splicing, DNA damage repair, and mitotic spindle assembly as commonly enriched across diverse models. Evaluation of altered pre-mRNA splicing patterns suggested that many of these changes may be a consequence of selective impairment of PRMT5-dependent support of spliceosome activity. Notably, sensitivities to drugs that inhibit the fidelity of pre-mRNA splicing, genome stability, and microtubule stability were enriched in MTAP-/- cell lines, suggesting that the biological processes selectively altered by IDE397 in tumors are already partially impaired by MTAP deletion. Finally, an IDE397 in vitro drug combination viability screen of over 400 drugs returned taxanes, platins, topoisomerase inhibitors, splicing inhibitors, and anti-folates as selectively synergistic with IDE397 in MTAP-/- models. Synergy between IDE397 and pemetrexed was of particular interest given mechanistic convergence of the methionine salvage and folate cycle pathways on nucleotide synthesis in the context of MTAP-/-. Accordingly, this combination provided enhanced tumor growth inhibition in vivo in MTAP-/- NSCLC and bladder models and is currently under evaluation in phase 1. Collectively, these observations indicate that MAT2A inhibition can generate cell states in MTAP-/- tumor cells that are selectively vulnerable to approved chemotherapies and targeted therapies. These synergistic relationships may provide a predictive biomarker strategy for multiple IDE397 synthetic lethal combination therapies. Citation Format: Kimberline Y. Gerrick, Jenny Laraio, Kelsey Annen, Hoang Tran, Marcus M. Fischer, Yevgeniy Freyman, Geeta Sharma, Isaac Bishof, Stephen Federowicz, Divya Pankajakshan, Claire L. Neilan, Biju Mangatt, Anthony Mazurek, Mark R. Lackner, Michael White, Zineb Mounir. MAT2A inhibition in MTAP-/- tumors confers mechanistic vulnerabilities to multiple clinically actionable synthetic lethal drug combinations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1637.

DNA Repair and Therapeutic Strategies in Cancer Stem Cells.

First-line cancer treatments successfully eradicate the differentiated tumour mass but are comparatively ineffective against cancer stem cells (CSCs), a self-renewing subpopulation thought to be responsible for tumour initiation, metastasis, heterogeneity, and recurrence. CSCs are thus presented as the principal target for elimination during cancer treatment. However, CSCs are challenging to drug target because of numerous intrinsic and extrinsic mechanisms of drug resistance. One such mechanism that remains relatively understudied is the DNA damage response (DDR). CSCs are presumed to possess properties that enable enhanced DNA repair efficiency relative to their highly proliferative bulk progeny, facilitating improved repair of double-strand breaks induced by radiotherapy and most chemotherapeutics. This can occur through multiple mechanisms, including increased expression and splicing fidelity of DNA repair genes, robust activation of cell cycle checkpoints, and elevated homologous recombination-mediated DNA repair. Herein, we summarise the current knowledge concerning improved genome integrity in non-transformed stem cells and CSCs, discuss therapeutic opportunities within the DDR for re-sensitising CSCs to genotoxic stressors, and consider the challenges posed regarding unbiased identification of novel DDR-directed strategies in CSCs. A better understanding of the DDR mediating chemo/radioresistance mechanisms in CSCs could lead to novel therapeutic approaches, thereby enhancing treatment efficacy in cancer patients.

The Role of PRMT5 in Immuno-Oncology.

Immune checkpoint inhibitor (ICI) therapy has caused a paradigm shift in cancer therapeutic strategy. However, this therapy only benefits a subset of patients. The difference in responses to ICIs is believed to be dependent on cancer type and its tumor microenvironment (TME). The TME is favorable for cancer progression and metastasis and can also help cancer cells to evade immune attacks. To improve the response to ICIs, it is crucial to understand the mechanism of how the TME is maintained. Protein arginine methyltransferase 5 (PRMT5) di-methylates arginine residues in its substrates and has essential roles in the epigenetic regulation of gene expression, signal transduction, and the fidelity of mRNA splicing. Through these functions, PRMT5 can support cancer cell immune evasion. PRMT5 is necessary for regulatory T cell (Treg) functions and promotes cancer stemness and the epithelial-mesenchymal transition. Specific factors in the TME can help recruit Tregs, tumor-associated macrophages, and myeloid-derived suppressor cells into tumors. In addition, PRMT5 suppresses antigen presentation and the production of interferon and chemokines, which are necessary to recruit T cells into tumors. Overall, PRMT5 supports an immunosuppressive TME. Therefore, PRMT5 inhibition would help recover the immune cycle and enable the immune system-mediated elimination of cancer cells.

Aberrant splicing in human cancer: An RNA structural code point of view.

Alternative splicing represents an essential process that occurs widely in eukaryotes. In humans, most genes undergo alternative splicing to ensure transcriptome and proteome diversity reflecting their functional complexity. Over the last decade, aberrantly spliced transcripts due to mutations in cis- or trans-acting splicing regulators have been tightly associated with cancer development, largely drawing scientific attention. Although a plethora of single proteins, ribonucleoproteins, complexed RNAs, and short RNA sequences have emerged as nodal contributors to the splicing cascade, the role of RNA secondary structures in warranting splicing fidelity has been underestimated. Recent studies have leveraged the establishment of novel high-throughput methodologies and bioinformatic tools to shed light on an additional layer of splicing regulation in the context of RNA structural elements. This short review focuses on the most recent available data on splicing mechanism regulation on the basis of RNA secondary structure, emphasizing the importance of the complex RNA G-quadruplex structures (rG4s), and other specific RNA motifs identified as splicing silencers or enhancers. Moreover, it intends to provide knowledge on newly established techniques that allow the identification of RNA structural elements and highlight the potential to develop new RNA-oriented therapeutic strategies against cancer.

BAY61-3606 Alters snRNP Composition and Enhances Usage of Suboptimal Splice Acceptor Site.

Intron recognition by the spliceosome mainly depends on conserved intronic sequences such as 5' splice sites, 3' splice sites, and branch sites. Therefore, even substitution of just a single nucleotide in a 5' or 3' splice site abolishes the splicing at the mutated site and leads to cryptic splice site usage. A number of disease-causative mutations have been found in 5' and 3' splice sites, but the genes with these mutations still maintain the correct protein-coding sequence, so recovery of splicing at the mutated splice site may produce a normal protein. Mutations in the spliceosome components have been shown to change the balance between the conformational transition and disassembly of the spliceosome, which affects the decision about whether the reaction of the incorporated substrate will proceed. In addition, the lower disassembly rate caused by such mutations induces splicing of the mutated splice site. We hypothesized that small compounds targeting the spliceosome may include a compound mimicking the effect of those mutations. Thus, we screened a small-compound library and identified a compound, BAY61-3606, that changed the cellular small nuclear ribonucleoprotein composition and also showed activity of enhancing splicing at the mutated 3' splice site of the reporter gene, as well as splicing at the suboptimal 3' splice site of endogenous cassette exons. These results indicate that further analysis of the mechanism of action of BAY61-3606 could enable modulation of the fidelity of splicing.

Decreased spliceosome fidelity and egl-8 intron retention inhibit mTORC1 signaling to promote longevity

Changes in splicing fidelity are associated with loss of homeostasis and aging, yet only a handful of splicing factors have been shown to be causally required to promote longevity, and the underlying mechanisms and downstream targets in these paradigms remain elusive. Surprisingly, we found a hypomorphic mutation within ribonucleoprotein RNP-6/poly(U)-binding factor 60 kDa (PUF60), a spliceosome component promoting weak 3′-splice site recognition, which causes aberrant splicing, elevates stress responses and enhances longevity in Caenorhabditis elegans. Through genetic suppressor screens, we identify a gain-of-function mutation within rbm-39, an RNP-6-interacting splicing factor, which increases nuclear speckle formation, alleviates splicing defects and curtails longevity caused by rnp-6 mutation. By leveraging the splicing changes induced by RNP-6/RBM-39 activities, we uncover intron retention in egl-8/phospholipase C β4 (PLCB4) as a key splicing target prolonging life. Genetic and biochemical evidence show that neuronal RNP-6/EGL-8 downregulates mammalian target of rapamycin complex 1 (mTORC1) signaling to control organismal lifespan. In mammalian cells, PUF60 downregulation also potently and specifically inhibits mTORC1 signaling. Altogether, our results reveal that splicing fidelity modulates lifespan through mTOR signaling.