hnRNPM protects against the dsRNA-mediated interferon response by repressing LINE-associated cryptic splicing.

RNA splicing is pivotal in post-transcriptional gene regulation, yet the exponential expansion of intron length in humans poses a challenge for accurate splicing. Here, we identify hnRNPM as an essential RNA-binding protein that suppresses cryptic splicing through binding to deep introns, maintaining human transcriptome integrity. Long interspersed nuclear elements (LINEs) in introns harbor numerous pseudo splice sites. hnRNPM preferentially binds at intronic LINEs to repress pseudo splice site usage for cryptic splicing. Remarkably, cryptic exons can generate long dsRNAs through base-pairing of inverted ALU transposable elements interspersed among LINEs and consequently trigger an interferon response, a well-known antiviral defense mechanism. Significantly, hnRNPM-deficient tumors show upregulated interferon-associated pathways and elevated immune cell infiltration. These findings unveil hnRNPM as a guardian of transcriptome integrity by repressing cryptic splicing and suggest that targeting hnRNPM in tumors may be used to trigger an inflammatory immune response, thereby boosting cancer surveillance.

Splicing-specific transcriptome-wide association uncovers genetic mechanisms for schizophrenia.

Recent studies have highlighted the essential role of RNA splicing, a key mechanism of alternative RNA processing, in establishing connections between genetic variations and disease. Genetic loci influencing RNA splicing variations show considerable influence on complex traits, possibly surpassing those affecting total gene expression. Dysregulated RNA splicing has emerged as a major potential contributor to neurological and psychiatric disorders, likely due to the exceptionally high prevalence of alternatively spliced genes in the human brain. Nevertheless, establishing direct associations between genetically altered splicing and complex traits has remained an enduring challenge. We introduce Spliced-Transcriptome-Wide Associations (SpliTWAS) to integrate alternative splicing information with genome-wide association studies to pinpoint genes linked to traits through exon splicing events. We applied SpliTWAS to two schizophrenia (SCZ) RNA-sequencing datasets, BrainGVEX and CommonMind, revealing 137 and 88 trait-associated exons (in 84 and 67 genes), respectively. Enriched biological functions in the associated gene sets converged on neuronal function and development, immune cell activation, and cellular transport, which are highly relevant to SCZ. SpliTWAS variants impacted RNA-binding protein binding sites, revealing potential disruption of RNA-protein interactions affecting splicing. We extended the probabilistic fine-mapping method FOCUS to the exon level, identifying 36 genes and 48 exons as putatively causal for SCZ. We highlight VPS45 and APOPT1, where splicing of specific exons was associated with disease risk, eluding detection by conventional gene expression analysis. Collectively, this study supports the substantial role of alternative splicing in shaping the genetic basis of SCZ, providing a valuable approach for future investigations in this area.

Navigating the landscape of epitranscriptomics and host immunity.

RNA modifications, also termed epitranscriptomic marks, encompass chemical alterations to individual nucleotides, including processes such as methylation and editing. These marks contribute to a wide range of biological processes, many of which are related to host immune system defense. The functions of immune-related RNA modifications can be categorized into three main groups: regulation of immunogenic RNAs, control of genes involved in innate immune response, and facilitation of adaptive immunity. Here, we provide an overview of recent research findings that elucidate the contributions of RNA modifications to each of these processes. We also discuss relevant methods for genome-wide identification of RNA modifications and their immunogenic substrates. Finally, we highlight recent advances in cancer immunotherapies that aim to reduce cancer cell viability by targeting the enzymes responsible for RNA modifications. Our presentation of these dynamic research avenues sets the stage for future investigations in this field.

A Multiplexed Assay for Exon Recognition Reveals that an Unappreciated Fraction of Rare Genetic Variants Cause Large-Effect Splicing Disruptions.

Mutations that lead to splicing defects can have severe consequences on gene function and cause disease. Here, we explore how human genetic variation affects exon recognition by developing a multiplexed functional assay of splicing using Sort-seq (MFASS). We assayed 27,733 variants in the Exome Aggregation Consortium (ExAC) within or adjacent to 2,198 human exons in the MFASS minigene reporter and found that 3.8% (1,050) of variants, most of which are extremely rare, led to large-effect splice-disrupting variants (SDVs). Importantly, we find that 83% of SDVs are located outside of canonical splice sites, are distributed evenly across distinct exonic and intronic regions, and are difficult to predict a priori. Our results indicate extant, rare genetic variants can have large functional effects on splicing at appreciable rates, even outside the context of disease, and MFASS enables their empirical assessment at scale.

Allele-specific alternative splicing and its functional genetic variants in human tissues.

Alternative splicing is an RNA processing mechanism that affects most genes in human, contributing to disease mechanisms and phenotypic diversity. The regulation of splicing involves an intricate network of cis-regulatory elements and trans-acting factors. Due to their high sequence specificity, cis-regulation of splicing can be altered by genetic variants, significantly affecting splicing outcomes. Recently, multiple methods have been applied to understanding the regulatory effects of genetic variants on splicing. However, it is still challenging to go beyond apparent association to pinpoint functional variants. To fill in this gap, we utilized large-scale data sets of the Genotype-Tissue Expression (GTEx) project to study genetically modulated alternative splicing (GMAS) via identification of allele-specific splicing events. We demonstrate that GMAS events are shared across tissues and individuals more often than expected by chance, consistent with their genetically driven nature. Moreover, although the allelic bias of GMAS exons varies across samples, the degree of variation is similar across tissues versus individuals. Thus, genetic background drives the GMAS pattern to a similar degree as tissue-specific splicing mechanisms. Leveraging the genetically driven nature of GMAS, we developed a new method to predict functional splicing-altering variants, built upon a genotype-phenotype concordance model across samples. Complemented by experimental validations, this method predicted >1000 functional variants, many of which may alter RNA-protein interactions. Lastly, 72% of GMAS-associated SNPs were in linkage disequilibrium with GWAS-reported SNPs, and such association was enriched in tissues of relevance for specific traits/diseases. Our study enables a comprehensive view of genetically driven splicing variations in human tissues.

dsRID: in silico identification of dsRNA regions using long-read RNA-seq data.

MOTIVATION: Double-stranded RNAs (dsRNAs) are potent triggers of innate immune responses upon recognition by cytosolic dsRNA sensor proteins. Identification of endogenous dsRNAs helps to better understand the dsRNAome and its relevance to innate immunity related to human diseases. RESULTS: Here, we report dsRID (double-stranded RNA identifier), a machine-learning-based method to predict dsRNA regions in silico, leveraging the power of long-read RNA-sequencing (RNA-seq) and molecular traits of dsRNAs. Using models trained with PacBio long-read RNA-seq data derived from Alzheimer's disease (AD) brain, we show that our approach is highly accurate in predicting dsRNA regions in multiple datasets. Applied to an AD cohort sequenced by the ENCODE consortium, we characterize the global dsRNA profile with potentially distinct expression patterns between AD and controls. Together, we show that dsRID provides an effective approach to capture global dsRNA profiles using long-read RNA-seq data. AVAILABILITY AND IMPLEMENTATION: Software implementation of dsRID, and genomic coordinates of regions predicted by dsRID in all samples are available at the GitHub repository: https://github.com/gxiaolab/dsRID.

Genetics of white color and iridophoroma in "Lemon Frost" leopard geckos.

The squamates (lizards and snakes) are close relatives of birds and mammals, with more than 10,000 described species that display extensive variation in a number of important biological traits, including coloration, venom production, and regeneration. Due to a lack of genomic tools, few genetic studies in squamates have been carried out. The leopard gecko, Eublepharis macularius, is a popular companion animal, and displays a variety of coloration patterns. We took advantage of a large breeding colony and used linkage analysis, synteny, and homozygosity mapping to investigate a spontaneous semi-dominant mutation, "Lemon Frost", that produces white coloration and causes skin tumors (iridophoroma). We localized the mutation to a single locus which contains a strong candidate gene, SPINT1, a tumor suppressor implicated in human skin cutaneous melanoma (SKCM) and over-proliferation of epithelial cells in mice and zebrafish. Our work establishes the leopard gecko as a tractable genetic system and suggests that a tumor suppressor in melanocytes in humans can also suppress tumor development in iridophores in lizards.

Cell type-restricted activity of hnRNPM promotes breast cancer metastasis via regulating alternative splicing.

Tumor metastasis remains the major cause of cancer-related death, but its molecular basis is still not well understood. Here we uncovered a splicing-mediated pathway that is essential for breast cancer metastasis. We show that the RNA-binding protein heterogeneous nuclear ribonucleoprotein M (hnRNPM) promotes breast cancer metastasis by activating the switch of alternative splicing that occurs during epithelial-mesenchymal transition (EMT). Genome-wide deep sequencing analysis suggests that hnRNPM potentiates TGFß signaling and identifies CD44 as a key downstream target of hnRNPM. hnRNPM ablation prevents TGFß-induced EMT and inhibits breast cancer metastasis in mice, whereas enforced expression of the specific CD44 standard (CD44s) splice isoform overrides the loss of hnRNPM and permits EMT and metastasis. Mechanistically, we demonstrate that the ubiquitously expressed hnRNPM acts in a mesenchymal-specific manner to precisely control CD44 splice isoform switching during EMT. This restricted cell-type activity of hnRNPM is achieved by competition with ESRP1, an epithelial splicing regulator that binds to the same cis-regulatory RNA elements as hnRNPM and is repressed during EMT. Importantly, hnRNPM is associated with aggressive breast cancer and correlates with increased CD44s in patient specimens. These findings demonstrate a novel molecular mechanism through which tumor metastasis is endowed by the hnRNPM-mediated splicing program.

RNA editing in nascent RNA affects pre-mRNA splicing.

In eukaryotes, nascent RNA transcripts undergo an intricate series of RNA processing steps to achieve mRNA maturation. RNA editing and alternative splicing are two major RNA processing steps that can introduce significant modifications to the final gene products. By tackling these processes in isolation, recent studies have enabled substantial progress in understanding their global RNA targets and regulatory pathways. However, the interplay between individual steps of RNA processing, an essential aspect of gene regulation, remains poorly understood. By sequencing the RNA of different subcellular fractions, we examined the timing of adenosine-to-inosine (A-to-I) RNA editing and its impact on alternative splicing. We observed that >95% A-to-I RNA editing events occurred in the chromatin-associated RNA prior to polyadenylation. We report about 500 editing sites in the 3' acceptor sequences that can alter splicing of the associated exons. These exons are highly conserved during evolution and reside in genes with important cellular function. Furthermore, we identified a second class of exons whose splicing is likely modulated by RNA secondary structures that are recognized by the RNA editing machinery. The genome-wide analyses, supported by experimental validations, revealed remarkable interplay between RNA editing and splicing and expanded the repertoire of functional RNA editing sites.

Statistical inference of differential RNA-editing sites from RNA-sequencing data by hierarchical modeling.

MOTIVATION: RNA-sequencing (RNA-seq) enables global identification of RNA-editing sites in biological systems and disease. A salient step in many studies is to identify editing sites that statistically associate with treatment (e.g. case versus control) or covary with biological factors, such as age. However, RNA-seq has technical features that incumbent tests (e.g. t-test and linear regression) do not consider, which can lead to false positives and false negatives. RESULTS: In this study, we demonstrate the limitations of currently used tests and introduce the method, RNA-editing tests (REDITs), a suite of tests that employ beta-binomial models to identify differential RNA editing. The tests in REDITs have higher sensitivity than other tests, while also maintaining the type I error (false positive) rate at the nominal level. Applied to the GTEx dataset, we unveil RNA-editing changes associated with age and gender, and differential recoding profiles between brain regions. AVAILABILITY AND IMPLEMENTATION: REDITs are implemented as functions in R and freely available for download at https://github.com/gxiaolab/REDITs. The repository also provides a code example for leveraging parallelization using multiple cores.

Coregulation of alternative splicing by hnRNPM and ESRP1 during EMT.

The epithelial-mesenchymal transition (EMT) is a fundamental developmental process that is abnormally activated in cancer metastasis. Dynamic changes in alternative splicing occur during EMT. ESRP1 and hnRNPM are splicing regulators that promote an epithelial splicing program and a mesenchymal splicing program, respectively. The functional relationships between these splicing factors in the genome scale remain elusive. Comparing alternative splicing targets of hnRNPM and ESRP1 revealed that they coregulate a set of cassette exon events, with the majority showing discordant splicing regulation. Discordant splicing events regulated by hnRNPM show a positive correlation with splicing during EMT; however, concordant events do not, indicating the role of hnRNPM in regulating alternative splicing during EMT is more complex than previously understood. Motif enrichment analysis near hnRNPM-ESRP1 coregulated exons identifies guanine-uridine rich motifs downstream from hnRNPM-repressed and ESRP1-enhanced exons, supporting a general model of competitive binding to these cis-elements to antagonize alternative splicing. The set of coregulated exons are enriched in genes associated with cell migration and cytoskeletal reorganization, which are pathways associated with EMT. Splicing levels of coregulated exons are associated with breast cancer patient survival and correlate with gene sets involved in EMT and breast cancer subtyping. This study identifies complex modes of interaction between hnRNPM and ESRP1 in regulation of splicing in disease-relevant contexts.

DeepPASTA: deep neural network based polyadenylation site analysis.

MOTIVATION: Alternative polyadenylation (polyA) sites near the 3' end of a pre-mRNA create multiple mRNA transcripts with different 3' untranslated regions (3' UTRs). The sequence elements of a 3' UTR are essential for many biological activities such as mRNA stability, sub-cellular localization, protein translation, protein binding and translation efficiency. Moreover, numerous studies in the literature have reported the correlation between diseases and the shortening (or lengthening) of 3' UTRs. As alternative polyA sites are common in mammalian genes, several machine learning tools have been published for predicting polyA sites from sequence data. These tools either consider limited sequence features or use relatively old algorithms for polyA site prediction. Moreover, none of the previous tools consider RNA secondary structures as a feature to predict polyA sites. RESULTS: In this paper, we propose a new deep learning model, called DeepPASTA, for predicting polyA sites from both sequence and RNA secondary structure data. The model is then extended to predict tissue-specific polyA sites. Moreover, the tool can predict the most dominant (i.e. frequently used) polyA site of a gene in a specific tissue and relative dominance when two polyA sites of the same gene are given. Our extensive experiments demonstrate that DeepPASTA signisficantly outperforms the existing tools for polyA site prediction and tissue-specific relative and absolute dominant polyA site prediction. AVAILABILITY AND IMPLEMENTATION: https://github.com/arefeen/DeepPASTA. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

The Sm protein methyltransferase PRMT5 is not required for primordial germ cell specification in mice.

PRMT5 is a type II protein arginine methyltransferase with roles in stem cell biology, reprograming, cancer and neurogenesis. During embryogenesis in the mouse, it was hypothesized that PRMT5 functions with the master germline determinant BLIMP1 to promote primordial germ cell (PGC) specification. Using a Blimp1-Cre germline conditional knockout, we discovered that Prmt5 has no major role in murine germline specification, or the first global epigenetic reprograming event involving depletion of cytosine methylation from DNA and histone H3 lysine 9 dimethylation from chromatin. Instead, we discovered that PRMT5 functions at the conclusion of PGC reprograming I to promote proliferation, survival and expression of the gonadal germline program as marked by MVH. We show that PRMT5 regulates gene expression by promoting methylation of the Sm spliceosomal proteins and significantly altering the spliced repertoire of RNAs in mammalian embryonic cells and primordial cells.

Alternative splicing modulated by genetic variants demonstrates accelerated evolution regulated by highly conserved proteins.

Identification of functional genetic variants and elucidation of their regulatory mechanisms represent significant challenges of the post-genomic era. A poorly understood topic is the involvement of genetic variants in mediating post-transcriptional RNA processing, including alternative splicing. Thus far, little is known about the genomic, evolutionary, and regulatory features of genetically modulated alternative splicing (GMAS). Here, we systematically identified intronic tag variants for genetic modulation of alternative splicing using RNA-seq data specific to cellular compartments. Combined with our previous method that identifies exonic tags for GMAS, this study yielded 622 GMAS exons. We observed that GMAS events are highly cell type independent, indicating that splicing-altering genetic variants could have widespread function across cell types. Interestingly, GMAS genes, exons, and single-nucleotide variants (SNVs) all demonstrated positive selection or accelerated evolution in primates. We predicted that GMAS SNVs often alter binding of splicing factors, with SRSF1 affecting the most GMAS events and demonstrating global allelic binding bias. However, in contrast to their GMAS targets, the predicted splicing factors are more conserved than expected, suggesting that cis-regulatory variation is the major driving force of splicing evolution. Moreover, GMAS-related splicing factors had stronger consensus motifs than expected, consistent with their susceptibility to SNV disruption. Intriguingly, GMAS SNVs in general do not alter the strongest consensus position of the splicing factor motif, except the more than 100 GMAS SNVs in linkage disequilibrium with polymorphisms reported by genome-wide association studies. Our study reports many GMAS events and enables a better understanding of the evolutionary and regulatory features of this phenomenon.

TAPAS: tool for alternative polyadenylation site analysis.

Motivation: The length of the 3' untranslated region (3' UTR) of an mRNA is essential for many biological activities such as mRNA stability, sub-cellular localization, protein translation, protein binding and translation efficiency. Moreover, correlation between diseases and the shortening (or lengthening) of 3' UTRs has been reported in the literature. This length is largely determined by the polyadenylation cleavage site in the mRNA. As alternative polyadenylation (APA) sites are common in mammalian genes, several tools have been published recently for detecting APA sites from RNA-Seq data or performing shortening/lengthening analysis. These tools consider either up to only two APA sites in a gene or only APA sites that occur in the last exon of a gene, although a gene may generally have more than two APA sites and an APA site may sometimes occur before the last exon. Furthermore, the tools are unable to integrate the analysis of shortening/lengthening events with APA site detection. Results: We propose a new tool, called TAPAS, for detecting novel APA sites from RNA-Seq data. It can deal with more than two APA sites in a gene as well as APA sites that occur before the last exon. The tool is based on an existing method for finding change points in time series data, but some filtration techniques are also adopted to remove change points that are likely false APA sites. It is then extended to identify APA sites that are expressed differently between two biological samples and genes that contain 3' UTRs with shortening/lengthening events. Our extensive experiments on simulated and real RNA-Seq data demonstrate that TAPAS outperforms the existing tools for APA site detection or shortening/lengthening analysis significantly. Availability and implementation: https://github.com/arefeen/TAPAS. Supplementary information: Supplementary data are available at Bioinformatics online.

Novel approaches for bioinformatic analysis of salivary RNA sequencing data for development.

Motivation: Analysis of RNA sequencing (RNA-Seq) data in human saliva is challenging. Lack of standardization and unification of the bioinformatic procedures undermines saliva's diagnostic potential. Thus, it motivated us to perform this study. Results: We applied principal pipelines for bioinformatic analysis of small RNA-Seq data of saliva of 98 healthy Korean volunteers including either direct or indirect mapping of the reads to the human genome using Bowtie1. Analysis of alignments to exogenous genomes by another pipeline revealed that almost all of the reads map to bacterial genomes. Thus, salivary exRNA has fundamental properties that warrant the design of unique additional steps while performing the bioinformatic analysis. Our pipelines can serve as potential guidelines for processing of RNA-Seq data of human saliva. Availability and implementation: Processing and analysis results of the experimental data generated by the exceRpt (v4.6.3) small RNA-seq pipeline (github.gersteinlab.org/exceRpt) are available from exRNA atlas (exrna-atlas.org). Alignment to exogenous genomes and their quantification results were used in this paper for the analyses of small RNAs of exogenous origin. Contact: dtww@ucla.edu.

Genome sequence-independent identification of RNA editing sites.

RNA editing generates post-transcriptional sequence changes that can be deduced from RNA-seq data, but detection typically requires matched genomic sequence or multiple related expression data sets. We developed the GIREMI tool (genome-independent identification of RNA editing by mutual information; https://www.ibp.ucla.edu/research/xiao/GIREMI.html) to predict adenosine-to-inosine editing accurately and sensitively from a single RNA-seq data set of modest sequencing depth. Using GIREMI on existing data, we observed tissue-specific and evolutionary patterns in editing sites in the human population.

Characterization of Human Salivary Extracellular RNA by Next-generation Sequencing.

BACKGROUND: It was recently discovered that abundant and stable extracellular RNA (exRNA) species exist in bodily fluids. Saliva is an emerging biofluid for biomarker development for noninvasive detection and screening of local and systemic diseases. Use of RNA-Sequencing (RNA-Seq) to profile exRNA is rapidly growing; however, no single preparation and analysis protocol can be used for all biofluids. Specifically, RNA-Seq of saliva is particularly challenging owing to high abundance of bacterial contents and low abundance of salivary exRNA. Given the laborious procedures needed for RNA-Seq library construction, sequencing, data storage, and data analysis, saliva-specific and optimized protocols are essential. METHODS: We compared different RNA isolation methods and library construction kits for long and small RNA sequencing. The role of ribosomal RNA (rRNA) depletion also was evaluated. RESULTS: The miRNeasy Micro Kit (Qiagen) showed the highest total RNA yield (70.8 ng/mL cell-free saliva) and best small RNA recovery, and the NEBNext library preparation kits resulted in the highest number of detected human genes [5649-6813 at 1 reads per kilobase RNA per million mapped (RPKM)] and small RNAs [482-696 microRNAs (miRNAs) and 190-214 other small RNAs]. The proportion of human RNA-Seq reads was much higher in rRNA-depleted saliva samples (41%) than in samples without rRNA depletion (14%). In addition, the transfer RNA (tRNA)-derived RNA fragments (tRFs), a novel class of small RNAs, were highly abundant in human saliva, specifically tRF-4 (4%) and tRF-5 (15.25%). CONCLUSIONS: Our results may help in selection of the best adapted methods of RNA isolation and small and long RNA library constructions for salivary exRNA studies.

Global analyses of endonucleolytic cleavage in mammals reveal expanded repertoires of cleavage-inducing small RNAs and their targets.

In mammals, small RNAs are important players in post-transcriptional gene regulation. While their roles in mRNA destabilization and translational repression are well appreciated, their involvement in endonucleolytic cleavage of target RNAs is poorly understood. Very few microRNAs are known to guide RNA cleavage. Endogenous small interfering RNAs are expected to induce target cleavage, but their target genes remain largely unknown. We report a systematic study of small RNA-mediated endonucleolytic cleavage in mouse through integrative analysis of small RNA and degradome sequencing data without imposing any bias toward known small RNAs. Hundreds of small cleavage-inducing RNAs and their cognate target genes were identified, significantly expanding the repertoire of known small RNA-guided cleavage events. Strikingly, both small RNAs and their target sites demonstrated significant overlap with retrotransposons, providing evidence for the long-standing speculation that retrotransposable elements in mRNAs are leveraged as signals for gene targeting. Furthermore, our analysis showed that the RNA cleavage pathway is also present in human cells but affecting a different repertoire of retrotransposons. These results show that small RNA-guided cleavage is more widespread than previously appreciated. Their impact on retrotransposons in non-coding regions shed light on important aspects of mammalian gene regulation.

Catabolic Defect of Branched-Chain Amino Acids Promotes Heart Failure.

BACKGROUND: Although metabolic reprogramming is critical in the pathogenesis of heart failure, studies to date have focused principally on fatty acid and glucose metabolism. Contribution of amino acid metabolic regulation in the disease remains understudied. METHODS AND RESULTS: Transcriptomic and metabolomic analyses were performed in mouse failing heart induced by pressure overload. Suppression of branched-chain amino acid (BCAA) catabolic gene expression along with concomitant tissue accumulation of branched-chain α-keto acids was identified as a significant signature of metabolic reprogramming in mouse failing hearts and validated to be shared in human cardiomyopathy hearts. Molecular and genetic evidence identified the transcription factor Krüppel-like factor 15 as a key upstream regulator of the BCAA catabolic regulation in the heart. Studies using a genetic mouse model revealed that BCAA catabolic defect promoted heart failure associated with induced oxidative stress and metabolic disturbance in response to mechanical overload. Mechanistically, elevated branched-chain α-keto acids directly suppressed respiration and induced superoxide production in isolated mitochondria. Finally, pharmacological enhancement of branched-chain α-keto acid dehydrogenase activity significantly blunted cardiac dysfunction after pressure overload. CONCLUSIONS: BCAA catabolic defect is a metabolic hallmark of failing heart resulting from Krüppel-like factor 15-mediated transcriptional reprogramming. BCAA catabolic defect imposes a previously unappreciated significant contribution to heart failure.