Trans-acting Splicing Factors Research Articles

Long-read RNA-seq demarcates cis- and trans-directed alternative RNA splicing.

Genetic regulation of alternative splicing constitutes an important link between genetic variation and disease. Nonetheless, RNA splicing is regulated by both cis-acting elements and trans-acting splicing factors. Determining splicing events that are directed primarily by the cis- or trans-acting mechanisms will greatly inform our understanding of the genetic basis of disease. Here, we show that long-read RNA-seq, combined with our new method isoLASER, enables a clear segregation of cis- and trans-directed splicing events for individual samples. The genetic linkage of splicing is largely individual-specific, in stark contrast to the tissue-specific pattern of splicing profiles. Analysis of long-read RNA-seq data from human and mouse revealed thousands of cis-directed splicing events susceptible to genetic regulation. We highlight such events in the HLA genes whose analysis was challenging with short-read data. We also highlight novel cis-directed splicing events in Alzheimer's disease-relevant genes such as MAPT and BIN1. Together, the clear demarcation of cis- and trans-directed splicing paves ways for future studies of the genetic basis of disease.

H3.3-G34W in giant cell tumor of bone functionally aligns with the exon choice repressor hnRNPA1L2.

RNA processing is an essential post-transcriptional phenomenon that provides the necessary complexity of transcript diversity prior to translation. Aberrations in this process could contribute to tumourigenesis, and we have previously reported increased splicing alterations in giant cell tumor of bone (GCTB), which carries mutations in the histone variant H3.3 encoding glycine 34 substituted for tryptophan (H3.3-G34W). G34W interacts with several splicing factors, most notably the trans-acting splicing factor hnRNPA1L2. To gain a deeper understanding of RNA processing in GCTB and isogenic HeLa cells with H3.3-G34W, we generated RNA-immunoprecipitation sequencing data from hnRNPA1L2 and H3.3-G34W associated RNAs, which showed that 80% overlapped across genic regions and were frequently annotated as E2F transcription factor binding sites. Splicing aberrations in both GCTB and HeLa cells with H3.3-G34W were significantly enriched for known hnRNPA1L2 binding motifs (p value < 0.01). This splicing aberration differed from hnRNPA1L2 knockouts, which showed alterations independent of H3.3-G34W. Of functional significance, hnRNPA1L2 was redistributed to closely match the H3.3 pattern, likely driven by G34W, and to loci not occupied in normal parental cells. Taken together, our data reveal a functional overlap between hnRNPA1L2 and H3.3-G34W with likely significant consequences for RNA processing during GCTB pathogenesis. This provides novel opportunities for therapeutic intervention in future modus operandi.

Abstract 1873: SpliceCore® a novel ML tool for identifying disease-specific alternative splicing uncovered a promising therapeutic target for triple negative breast cancer

Abstract Accumulating evidence highlights aberrant splicing as a disease-driving event in cancer, affecting tumor progression, metastasis, and therapy resistance. Multiple studies have described the tumor-promoting activity of specific aberrant cis-splicing variants and misregulation of trans-acting splicing factors in patients. Hence, the identification of aberrant splicing patterns has the potential to translate into actionable biomarkers and novel therapeutic targets, particularly for treatment insensitive cancers such as Triple Negative Breast Cancer (TNBC). Envisagenics’ SpliceCore is an innovative cloud-based platform that integrates machine learning (ML) algorithms with high performance computing to analyze large RNA-seq datasets to predict biologically relevant, novel, and highly prevalent tumor-specific alternative splicing (AS) changes. Using SpliceCore, we have analyzed &amp;gt;2500 RNA-seq samples from different breast cancer subtypes as well as normal breast tissue and identified several AS derived therapeutic targets with the potential to translate into therapeutic candidates for TNBC. Here, we report a novel, alternatively spliced isoform that is present in 60.5% of TNBC patients and correlates with poor overall survival. This cytoplasmic protein was previously described as a regulator of the TGFβ pathway; therefore, we have validated the isoform switching action in a TGFβ dependent tumor progression model and demonstrated the activity of Splice Switching Oligos (SSO) to modulate its effect. We observed that this AS was correlated to a differential response on subcellular localization for phSmad2/3, Smad2/3 and TGFβRI. Pretreatment of the TNBC cells with SSO before TGFβ pathway activation modulates its proliferation response by affecting gene expression of p21, c-Myc and Smad7, leading to a decrease of cells in the G2 mitotic phase of the cell cycle and a loss of cell viability after the SSO treatment. More importantly, migratory response induced by TGFβ in TNBC cells was significantly inhibited by SSOs. Pre-treatment with SSO-0205 before TGFβ activation downregulated gene expression of Angiopoietin-like 4, Integrins α5 and β3, TGFβ associated migration markers, which was followed by a 55% decrease in cell migration. Our state-of-the-art ML technology, SpliceCore, has proven its ability to uncover novel disease-specific AS targets and to design splice correcting oligonucleotides for subsequent therapeutic development. Our data provides experimental proof of concept that has uncovered a novel therapeutic target for TNBC, whose aberrant splicing contributes to TNBC pathogenesis by misregulation of the TGFβ pathway. Moreover, SSOs predicted by SpliceCore were able to correct the aberrant splicing downstream effect, thereby uncovering a novel alternative splicing targeted approach for treating TNBC patients. Citation Format: Miguel A. Manzanares, Alyssa Casill, Kendall Anderson, Priyanka Dhingra, Vanessa Frederick, Adam Geier, Martin Akerman, Gayatri Arun. SpliceCore® a novel ML tool for identifying disease-specific alternative splicing uncovered a promising therapeutic target for triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1873.

Roles of Physicochemical and Structural Properties of RNA-Binding Proteins in Predicting the Activities of Trans-Acting Splicing Factors with Machine Learning.

Trans-acting splicing factors play a pivotal role in modulating alternative splicing by specifically binding to cis-elements in pre-mRNAs. There are approximately 1500 RNA-binding proteins (RBPs) in the human genome, but the activities of these RBPs in alternative splicing are unknown. Since determining RBP activities through experimental methods is expensive and time consuming, the development of an efficient computational method for predicting the activities of RBPs in alternative splicing from their sequences is of great practical importance. Recently, a machine learning model for predicting the activities of splicing factors was built based on features of single and dual amino acid compositions. Here, we explored the role of physicochemical and structural properties in predicting their activities in alternative splicing using machine learning approaches and found that the prediction performance is significantly improved by including these properties. By combining the minimum redundancy–maximum relevance (mRMR) method and forward feature searching strategy, a promising feature subset with 24 features was obtained to predict the activities of RBPs. The feature subset consists of 16 dual amino acid compositions, 5 physicochemical features, and 3 structural features. The physicochemical and structural properties were as important as the sequence composition features for an accurate prediction of the activities of splicing factors. The hydrophobicity and distribution of coil are suggested to be the key physicochemical and structural features, respectively.

Abstract P5-17-11: Novel therapeutic target for triple negative breast cancer uncovered by SpliceCore® an innovative platform that identifies disease-specific alternative splicing

Abstract Aberrant splicing is a major hallmark of cancer, affecting tumor progression, metastasis, and therapy resistance. The oncogenic activity of specific cis splicing errors and trans-acting splicing factor misregulation in patient tumors have been demonstrated in multiple studies. As such, cancer-associated splicing dysregulation is a novel source of clinically actionable biomarkers and therapeutic targets, particularly for the treatment of insensitive cancers such as Triple Negative Breast Cancer (TNBC). Envisagenics’ SpliceCore technology is an innovative cloud-based software platform that integrates machine learning (ML) algorithms with high performance computing to analyze large RNA-seq datasets to predict biologically relevant, novel, and highly prevalent tumor specific alternative splicing (AS) changes. Using SpliceCore, we have analyzed &amp;gt;2500 RNAseq samples from different breast cancer subtypes as well as normal breast tissue and identified several AS targets with the potential to translate into therapeutic candidates for TNBC. Interestingly, one of our leading AS targets is an exon skipping isoform that is present in 60.5% of TNBC patients and correlates with poor overall survival, without showing differences in gene expression between all the breast cancer subtypes and healthy patients studied. In addition, SpliceCore was used to predict and design a set of splice switching oligos (SSO) that can efficiently switch the skipping isoform to an inclusion isoform in TNBC cells. The skipping isoform plays a critical role in tumor progression via a TGFβ-dependent mechanism as demonstrated by detailed isoform switching studies using SSO-0205. Pretreatment of the TNBC cells with SSO-0205 24 hours before TGFβ pathway activation modulated TGFβ pathway related protein levels and cellular localization and reversed the cell proliferative response associated with it. This resulted in a strong inhibition of p21 gene expression, accompanied by a 50% decrease on the number of cells in G2, the mitotic phase of the cell cycle, and 40% decrease on cell viability. Additionally, migratory response induced by TGFβ in TNBC cells was also significantly inhibited by SSO-0205 pretreatment, which downregulated ANGPTL4 gene expression followed by a 55% decrease in cell migration. In summary, we were able to uncover a novel therapeutic target for TNBC, whose aberrant splicing contributes to TNBC pathogenesis by promoting an overactivation of the TGFβ pathway. Our results provide experimental proof of concept that demonstrate SpliceCore’s ability to discover novel disease specific AS targets and design splice correcting oligonucleotides for subsequent therapeutic development. Reversal of this aberrant TNBC specific splicing using SSOs represent a new and promising therapeutic approach that will have a significant impact on TNBC treatment and clinical care. Moreover, SpliceCore can be applied to multiple other indications opening a new avenue for therapeutic development in cancer. Citation Format: Miguel A Manzanares, Priyanka Dhingra, Kendall Anderson, Vanessa Frederick, Adam Geier, Alyssa Casill, Martin Akerman, Gayatri Arun. Novel therapeutic target for triple negative breast cancer uncovered by SpliceCore® an innovative platform that identifies disease-specific alternative splicing [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-17-11.

Alternative Splicing in Human Biology and Disease.

Alternative pre-mRNA splicing allows for the production of multiple mRNAs from an individual gene, which not only expands the protein-coding potential of the genome but also enables complex mechanisms for the post-transcriptional control of gene expression. Regulation of alternative splicing entails a combinatorial interplay between an abundance of trans-acting splicing factors, cis-acting regulatory sequence elements and their concerted effects on the core splicing machinery. Given the extent and biological significance of alternative splicing in humans, it is not surprising that aberrant splicing patterns can cause or contribute to a wide range of diseases. In this introductory chapter, we outline the mechanisms that govern alternative pre-mRNA splicing and its regulation and discuss how dysregulated splicing contributes to human diseases affecting the motor system and the brain.

Abstract P170: SpliceCore® a platform for identifying aberrant alternative splicing in triple negative breast cancer for novel therapeutic development

Abstract Splicing dysregulation is a major hallmark of cancer, affecting tumor progression, metastasis, and therapy resistance. Multiple studies have demonstrated the oncogenic activity of specific cis splicing errors and trans-acting splicing factor misregulation in patient tumors. As such, cancer-associated splicing dysregulation is a novel source of clinically actionable biomarkers and therapeutic targets, particularly for treatment insensitive cancers such as TNBC. Envisagenics has developed SpliceCore, an innovative cloud-based software platform that integrates machine learning (ML) algorithms with high performance computing to analyze large RNA-seq datasets to predict biologically relevant, novel, and highly prevalent tumor specific alternative splicing (AS) changes. Using SpliceCore, we have analyzed &amp;gt;2500 RNAseq samples from different breast cancer subtypes as well as normal breast tissue and identified several AS targets with a potential to translate into therapeutic candidates for TNBC. Here, we will discuss one of our most promising AS targets; it is present in 60.5% of TNBC patients and correlates with poor overall survival. Using SpliceCore, we predicted and designed an optimal set of splice switching oligos (SSO) that can efficiently switch the skipping isoform to an inclusion isoform in TNBC cells. Detailed mechanism of action studies of isoform switching by SSO-0205 have demonstrated the critical role of the isoform in a TGFβ dependent tumor progression mechanism. Pretreatment of the TNBC cells with SSO-0205 24 hours before TGFβ pathway activation reversed the cell proliferative response associated with it. This resulted in a strong inhibition of p21 gene expression, accompanied by a 50% decrease on the number of cells in G2, the mitotic phase of the cell cycle, and 40% decrease on cell viability. Additionally, migratory response induced by TGFβ in TNBC cells was also significantly inhibited by SSO-0205 pretreatment, which downregulated ANGPTL4 gene expression followed by a 55% decrease in cell migration. Together, we provide experimental proof of concept to demonstrate SpliceCore’s ability to discover novel disease specific AS targets and design splice correcting oligonucleotides for subsequent correction and therapeutic development. Using this approach, we were able to uncover a novel therapeutic target for TNBC, whose aberrant splicing contributes to TNBC pathogenesis by promoting an overactivation of the TGFβ pathway. Reversal of this aberrant TNBC specific splicing using SSOs represent a new and promising therapeutic approach that will have a significant impact on TNBC treatment and clinical care. Citation Format: Miguel A. Manzanares, Dhingra Priyanka, Kendall Anderson, Vanessa Frederick, Adam Geier, Alyssa Casill, Martin Akerman, Gayatri Arun. SpliceCore® a platform for identifying aberrant alternative splicing in triple negative breast cancer for novel therapeutic development [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P170.

A Reporter Based Cellular Assay for Monitoring Splicing Efficiency.

During gene expression, the vital step of pre-mRNA splicing involves accurate recognition of splice sites and efficient assembly of spliceosomal complexes to join exons and remove introns prior to cytoplasmic export of the mature mRNA. Splicing efficiency can be altered by the presence of mutations at splice sites, the influence of trans-acting splicing factors, or the activity of therapeutics. Here, we describe the protocol for a cellular assay that can be applied for monitoring the splicing efficiency of any given exon. The assay uses an adaptable plasmid encoded 3-exon/2-intron minigene reporter, which can be expressed in mammalian cells by transient transfection. Post-transfection, total cellular RNA is isolated, and the efficiency of exon splicing in the reporter mRNA is determined by either primer extension or semi-quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). We describe how the impact of disease associated 5' splice-site mutations can be determined by introducing them in the reporter; and how the suppression of these mutations can be achieved by co-transfection with U1 small nuclear RNA (snRNA) construct carrying compensatory mutations in its 5' region that basepairs with the 5'-splice sites at exon-intron junctions in pre-mRNAs. Thus, the reporter can be used for the design of therapeutic U1 particles to improve recognition of mutant 5' splice-sites. Insertion of cis-actingregulatory sites, such as splicing enhancer or silencer sequences, into the reporter can also be used to examine the role of U1 snRNP in regulation mediated by a specific alternative splicing factor. Finally, reporter expressing cells can be incubated with small molecules to determine the effect of potential therapeutics on constitutive pre-mRNA splicing or on exons carrying mutant 5' splice sites. Overall, the reporter assay can be applied to monitor splicing efficiency in a variety of conditions to study fundamental splicing mechanisms and splicing-associated diseases.

Revealing Epigenetic Factors of circRNA Expression by Machine Learning in Various Cellular Contexts.

SummaryCircular RNAs (circRNAs) have been identified as naturally occurring RNAs that are highly represented in the eukaryotic transcriptome. Although a large number of circRNAs have been reported, the underlying regulatory mechanism of circRNAs biogenesis remains largely unknown. Here, we integrated in-depth multi-omics data including epigenome, transcriptome, and non-coding RNA and identified candidate circRNAs in six cellular contexts. Next, circRNAs were divided into two classes (high versus low) with different expression levels. Machine learning models were constructed that predicted circRNA expression levels based on 11 different histone modifications and host gene expression. We found that the models achieve great accuracy in predicting high versus low expressed circRNAs. Furthermore, the expression levels of host genes of circRNAs, H3k36me3, H3k79me2, and H4k20me1 contributed greatly to the classification models in six cellular contexts. In summary, all these results suggest that epigenetic modifications, particularly histone modifications, can effectively predict expression levels of circRNAs.

Hallmarks of Splicing Defects in Cancer: Clinical Applications in the Era of Personalized Medicine.

Alternative splicing promotes proteome diversity by using limited number of genes, a key control point of gene expression. Splicing is carried out by large macromolecular machineries, called spliceosome, composed of small RNAs and proteins. Alternative splicing is regulated by splicing regulatory cis-elements in RNA and trans-acting splicing factors that are often tightly regulated in a tissue-specific and developmental stage-specific manner. The biogenesis of ribonucleoprotein (RNP) complexes is strictly regulated to ensure that correct complements of RNA and proteins are coordinated in the right cell at the right time to support physiological functions. Any perturbations that impair formation of functional spliceosomes by disrupting the cis-elements, or by compromising RNA-binding or function of trans-factors can be deleterious to cells and result in pathological consequences. The recent discovery of oncogenic mutations in splicing factors, and growing evidence of the perturbed splicing in multiple types of cancer, underscores RNA processing defects as a critical driver of oncogenesis. These findings have resulted in a growing interest in targeting RNA splicing as a therapeutic approach for cancer treatment. This review summarizes our current understanding of splicing alterations in cancer, recent therapeutic efforts targeting splicing defects in cancer, and future potentials to develop novel cancer therapies.

Histone lysine demethylase KDM4B regulates the alternative splicing of the androgen receptor in response to androgen deprivation.

Alternative splicing is emerging as an oncogenic mechanism. In prostate cancer, generation of constitutively active forms of androgen receptor (AR) variants including AR-V7 plays an important role in progression of castration-resistant prostate cancer (CRPC). AR-V7 is generated by alternative splicing that results in inclusion of cryptic exon CE3 and translation of truncated AR protein that lacks the ligand binding domain. Whether AR-V7 can be a driver for CRPC remains controversial as the oncogenic mechanism of AR-V7 activation remains elusive. Here, we found that KDM4B promotes AR-V7 and identified a novel regulatory mechanism. KDM4B is phosphorylated by protein kinase A under conditions that promote castration-resistance, eliciting its binding to the splicing factor SF3B3. KDM4B binds RNA specifically near the 5′-CE3, upregulates the chromatin accessibility, and couples the spliceosome to the chromatin. Our data suggest that KDM4B can function as a signal responsive trans-acting splicing factor and scaffold that recruits and stabilizes the spliceosome near the alternative exon, thus promoting its inclusion. Genome-wide profiling of KDM4B-regulated genes also identified additional alternative splicing events implicated in tumorigenesis. Our study defines KDM4B-regulated alternative splicing as a pivotal mechanism for generating AR-V7 and a contributing factor for CRPC, providing insight for mechanistic targeting of CRPC.

Alternativesplicing in Alzheimer's disease.

Alzheimer's disease (AD) is the most frequent neurodegenerative disorder in the elderly, occurring in approximately 20% of people older than 80. The molecular causes of AD are still poorly understood. However, recent studies have shown that Alternative Splicing (AS) is involved in the gene expression reprogramming associated with the functional changes observed in AD patients. In particular, mutations in cis-acting regulatory sequences as well as alterations in the activity and sub-cellular localization of trans-acting splicing factors and components of the spliceosome machinery are associated with splicing abnormalities in AD tissues, which may influence the onset and progression of the disease. In this review, we discuss the current molecular understanding of how alterations in the AS process contribute to AD pathogenesis. Finally, recent therapeutic approaches targeting aberrant AS regulation in AD are also reviewed.

Exon size and sequence conservation improves identification of splice-altering nucleotides.

Pre-mRNA splicing is regulated through multiple trans-acting splicing factors. These regulators interact with the pre-mRNA at intronic and exonic positions. Given that most exons are protein coding, the evolution of exons must be modulated by a combination of selective coding and splicing pressures. It has previously been demonstrated that selective splicing pressures are more easily deconvoluted when phylogenetic comparisons are made for exons of identical size, suggesting that exon size–filtered sequence alignments may improve identification of nucleotides evolved to mediate efficient exon ligation. To test this hypothesis, an exon size database was created, filtering 76 vertebrate sequence alignments based on exon size conservation. In addition to other genomic parameters, such as splice-site strength, gene position, or flanking intron length, this database permits the identification of exons that are size- and/or sequence-conserved. Highly size-conserved exons are always sequence-conserved. However, sequence conservation does not necessitate exon size conservation. Our analysis identified evolutionarily young exons and demonstrated that length conservation is a strong predictor of alternative splicing. A published data set of approximately 5000 exonic SNPs associated with disease was analyzed to test the hypothesis that exon size–filtered sequence comparisons increase detection of splice-altering nucleotides. Improved splice predictions could be achieved when mutations occur at the third codon position, especially when a mutation decreases exon inclusion efficiency. The results demonstrate that coding pressures dominate nucleotide composition at invariable codon positions and that exon size–filtered sequence alignments permit identification of splice-altering nucleotides at wobble positions.

Abstract PL02-02: Spliceomics: Alternative RNA splicing as a source of ancestry-related molecular targets in precision oncology and cancer disparities

Abstract PL02-02: Spliceomics: Alternative RNA splicing as a source of ancestry-related molecular targets in precision oncology and cancer disparities

Abstract 750: Ancestry-related differentially spliced and expressed genes in prostate cancer

Abstract Prostate cancer (PCa) affects disproportionally men from different population groups. The Surveillance, Epidemiology, and End Results Program’s (SEER’s) 2018 report reveals that PCa incidence and mortality rates are ~2 times higher among African American (AA) men in comparison with white men. In addition to differences in social, lifestyle and structural determinants of health, there is accumulating evidence for a biological contribution to racial disparity in PCa. To date, most work focused on understanding further the molecular mechanisms underlying racial disparity in PCa has analyzed differential aggregate gene expression and mutation among PCa from patients of different population groups. Our recently published work reported alternative RNA splicing (ARS) as a mechanism promoting tumor aggressiveness and drug resistance in PCa from AA patients. Here, we analyzed The Cancer Genome Atlas (TCGA) data using the Genomic Data Commons to analyze differential aggregate gene expression (2-fold mean change, p &amp;lt; 0.001, Wilcoxon rank sum test) and TCGASpliceSeq to analyze ARS (20% median change, percent spliced in) between PCa from AA and white patients. From our analysis of the 307 PCa specimens from white patients and 49 PCa specimens from AA patients, we identified 71 differentially expressed genes (DEGs) and 73 differential RNA splicing events (DRSEs) between PCa from AA and white patients. 51 of the DEGs (~72%) exhibit increased expression levels in PCa from AA patients compared with white patients. Among the DRSEs, the majority involve exon skipping (35 events, ~48%). Notably, the genes that exhibit differential aggregate gene expression and the genes that undergo differential ARS do not overlap, indicating that ancestry-related differences in aggregate gene expression and ARS can be independent events. However, a significant number of the Gene Ontology terms corresponding to the genes exhibiting ancestry-related differential aggregate gene expression or differential ARS do overlap, indicating that, despite these two distinct mechanisms of regulation (transcription and RNA splicing), both differentially regulate common pathways in PCa between AA and white patients. In addition, we identified 10 trans-acting splicing factors (SFs), whose aggregate gene expression significantly differed between PCa from AA and white patients, suggesting a potential mechanistic relationship between these SFs and the identified DRSEs. These findings increase understanding of molecular mechanisms underlying racial disparity in PCa. Upon further study, such DEGs and DRSEs have the potential to be candidates for novel precision medicine interventions. Citation Format: Muthana Al Abo, Daniel J. George, Jennifer A. Freedman, Steven R. Patierno. Ancestry-related differentially spliced and expressed genes in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 750.

Alteration of splicing factors' expression during liver disease progression: impact on hepatocellular carcinoma outcome.

Trans-acting splicing factors (SF) shape the eukaryotic transcriptome by regulating alternative splicing (AS). This process is recurrently modulated in liver cancer suggesting its direct contribution to the course of liver disease. The aim of our study was to investigate the relationship between the regulation of SFs expression and liver damage. The expression profile of 10 liver-specific SF and the AS events of 7 genes associated with liver disorders was assessed by western-blotting in 6 murine models representing different stages of liver damage, from inflammation to hepatocellular carcinoma (HCC). Relevant SFs (PSF, SRSF3, and SRSF6) and target genes (INSR, SRSF3, and SLK) modulated in mice were investigated in a cohort of 179 HCC patients. Each murine model of liver disease was characterized by a unique SF expression profile. Changes in the SF profile did not affect AS events of the selected genes despite the presence of corresponding splicing sites. In human HCC expression of SFs, including the tumor-suppressor SRSF3, and AS regulation of genes studied were frequently upregulated in tumor versus non-tumor tissues. Risk of tumor recurrence positively correlated with AS isoform of the INSR gene. In contrast, increased levels of SFs expression correlated with an extended overall survival of patients. Dysregulation of SF expression is an early event occurring during liver injury and not just at the stage of HCC. Besides impacting on AS regulation, overexpression of SF may contribute to preserving hepatocyte homeostasis during liver pathogenesis.

Differential NOVA2-Mediated Splicing in Excitatory and Inhibitory Neurons Regulates Cortical Development and Cerebellar Function

RNA-binding proteins (RBPs) regulate genetic diversity, but the degree to which they do so in individual cell types invivo is unknown. We developed NOVA2 cTag-crosslinking and immunoprecipitation (CLIP) to generate functional RBP-RNA maps from different neuronal populations in the mouse brain. Combining cell type datasets from Nova2-cTag and Nova2 conditional knockout mice revealed differential NOVA2 regulatory actions on alternative splicing (AS) on the same transcripts expressed in different neurons. This includes functional differences in transcripts expressed in cortical and cerebellar excitatory versus inhibitory neurons, where we find NOVA2 is required for, respectively, development of laminar structure, motor coordination, and synapse formation. We also find that NOVA2-regulated AS is coupled to NOVA2 regulation of intron retention in hundreds of transcripts, which can sequester the trans-acting splicing factor PTBP2. In summary, cTag-CLIP complements single-cell RNA sequencing (RNA-seq) studies by providing a means for understanding RNA regulation of functional cell diversity.

Modeling and Predicting the Activities of Trans-Acting Splicing Factors with Machine Learning

Alternative splicing (AS) is generally regulated by trans-splicing factors that specifically bind to cis-elements in pre-mRNAs. The human genome encodes ∼1,500 RNA binding proteins (RBPs) that potentially regulate AS, yet their functions remain largely unknown. To explore their potential activities, we fused the putative functional domains of RBPs to a sequence-specific RNA-binding domain and systemically analyzed how these engineered factors affect splicing. We discovered that ∼80% of low-complexity domains in endogenous RBPs displayed distinct context-dependent activities in regulating splicing, indicating that AS is under more extensive regulation than previously expected. We developed a machine learning approach to classify and predict the activities of RBPs based on their sequence compositions and further validated this model using endogenous RBPs and synthetic polypeptides. These results represent a systematic inspection, modeling, prediction, and validation of how RBP sequences affect their activities in controlling splicing, paving the way for de novo engineering of artificial splicing factors.

RBM-5 modulates U2AF large subunit-dependent alternative splicing in C. elegans

ABSTRACTA key step in pre-mRNA splicing is the recognition of 3ʹ splicing sites by the U2AF large and small subunits, a process regulated by numerous trans-acting splicing factors. How these trans-acting factors interact with U2AF in vivo is unclear. From a screen for suppressors of the temperature-sensitive (ts) lethality of the C. elegans U2AF large subunit gene uaf-1(n4588) mutants, we identified mutations in the RNA binding motif gene rbm-5, a homolog of the tumor suppressor gene RBM5. rbm-5 mutations can suppress uaf-1(n4588) ts-lethality by loss of function and neuronal expression of rbm-5 was sufficient to rescue the suppression. Transcriptome analyses indicate that uaf-1(n4588) affected the expression of numerous genes and rbm-5 mutations can partially reverse the abnormal gene expression to levels similar to that of wild type. Though rbm-5 mutations did not obviously affect alternative splicing per se, they can suppress or enhance, in a gene-specific manner, the altered splicing of genes in uaf-1(n4588) mutants. Specifically, the recognition of a weak 3ʹ splice site was more susceptible to the effect of rbm-5. Our findings provide novel in vivo evidence that RBM-5 can modulate UAF-1-dependent RNA splicing and suggest that RBM5 might interact with U2AF large subunit to affect tumor formation.

Abstract A016: KDM4B promotes castration-resistant prostate cancer by regulating alternative splicing of the androgen receptor

Abstract Emergence of constitutively active androgen receptor variant AR-V7 is associated with poor survival of castration-resistant prostate cancer (CRPC). AR-V7 can be generated by the inclusion of cryptic exon CE3 via alternative splicing, but the mechanism of AR-V7 activation remains elusive. Here, we show that histone lysine demethylase KDM4B promotes CRPC via generation of AR-V7. Genetic inactivation of KDM4B in CRPC cells suppressed xenograft growth and abolished tumor AR-V7 expression. Pharmacologic inhibition of KDM4B inhibited tumor growth and sensitized CRPC cells to current antiandrogen therapy. Mechanistically, KDM4B promotes AR-V7 as a trans-acting splicing factor and couples the spliceosome to the chromatin. Our study defines KDM4B-regulated alternative splicing as a pivotal mechanism for generating AR-V7 and a contributing factor for CRPC, providing insight for mechanistic targeting of CRPC. Citation Format: Zhi-Ping Liu. KDM4B promotes castration-resistant prostate cancer by regulating alternative splicing of the androgen receptor [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A016.