Splicing Calls Back.

Removal of introns from eukaryotic messenger RNA precursors often occurs co-transcriptionally. In this issue of Cell, Fiszbein et al. report that evolutionary or tissue-specific activation of an internal exon can enhance gene expression by promoting the use of alternative transcription initiation sites.

Combinatorial Genetics Reveals a Scaling Law for the Effects of Mutations on Splicing.

Despite a wealth of molecular knowledge, quantitative laws for accurate prediction of biological phenomena remain rare. Alternative pre-mRNA splicing is an important regulated step in gene expression frequently perturbed in human disease. To understand the combined effects of mutations during evolution, we quantified the effects of all possible combinations of exonic mutations accumulated during the emergence of an alternatively spliced human exon. This revealed that mutation effects scale non-monotonically with the inclusion level of an exon, with each mutation having maximum effect at a predictable intermediate inclusion level. This scaling is observed genome-wide for cis and trans perturbations of splicing, including for natural and disease-associated variants. Mathematical modeling suggests that competition between alternative splice sites is sufficient to cause this non-linearity in the genotype-phenotype map. Combining the global scaling law with specific pairwise interactions between neighboring mutations allows accurate prediction of the effects of complex genotype changes involving >10 mutations.

Minority report: The minor spliceosome as a novel cancer vulnerability factor.

The minor spliceosome regulates the removal of a conserved subset of introns present in genes with regulatory functions. In this issue of Molecular Cell, Augspach et al.1 report that elevated levels of U6atac snRNA, a key minor spliceosome component, contribute to prostate cancer cell growth and can be a novel therapeutic target.

AI-assisted proofreading of RNA splicing.

RNA helicases orchestrate proofreading mechanisms that facilitate accurate intron removal from pre-mRNAs. How these activities are recruited to spliceosome/pre-mRNA complexes remains poorly understood. In this issue of Genes & Development, Zhang and colleagues (pp. 968-983) combine biochemical experiments with AI-based structure prediction methods to generate a model for the interaction between SF3B1, a core splicing factor essential for the recognition of the intron branchpoint, and SUGP1, a protein that bridges SF3B1 with the helicase DHX15. Interaction with SF3B1 exposes the G-patch domain of SUGP1, facilitating binding to and activation of DHX15. The model can explain the activation of cryptic 3' splice sites induced by mutations in SF3B1 or SUGP1 frequently found in cancer.

Regulation of pre-mRNA splicing: roles in physiology and disease, and therapeutic prospects.

The removal of introns from mRNA precursors and its regulation by alternative splicing are key for eukaryotic gene expression and cellular function, as evidenced by the numerous pathologies induced or modified by splicing alterations. Major recent advances have been made in understanding the structures and functions of the splicing machinery, in the description and classification of physiological and pathological isoforms and in the development of the first therapies for genetic diseases based on modulation of splicing. Here, we review this progress and discuss important remaining challenges, including predicting splice sites from genomic sequences, understanding the variety of molecular mechanisms and logic of splicing regulation, and harnessing this knowledge for probing gene function and disease aetiology and for the design of novel therapeutic approaches.

Synonymous mutations frequently act as driver mutations in human cancers.

Synonymous mutations change the sequence of a gene without directly altering the sequence of the encoded protein. Here, we present evidence that these "silent" mutations frequently contribute to human cancer. Selection on synonymous mutations in oncogenes is cancer-type specific, and although the functional consequences of cancer-associated synonymous mutations may be diverse, they recurrently alter exonic motifs that regulate splicing and are associated with changes in oncogene splicing in tumors. The p53 tumor suppressor (TP53) also has recurrent synonymous mutations, but, in contrast to those in oncogenes, these are adjacent to splice sites and inactivate them. We estimate that between one in two and one in five silent mutations in oncogenes have been selected, equating to ~6%- 8% of all selected single-nucleotide changes in these genes. In addition, our analyses suggest that dosage-sensitive oncogenes have selected mutations in their 3' UTRs.

Empowering MYC carcinogenesis via RNA loops.

Ciesla et al. (2021) uncover intricate circuits of post-transcriptional regulation induced by the Myc oncogene, including alternative splicing and translational control, which are relevant for breast cancer prognosis and contribute to metabolic reprogramming and stem cell-like features of cancer cells.

RNA-binding proteins in human genetic disease.

RNA-binding proteins (RBPs) are critical effectors of gene expression, and as such their malfunction underlies the origin of many diseases. RBPs can recognize hundreds of transcripts and form extensive regulatory networks that help to maintain cell homeostasis. System-wide unbiased identification of RBPs has increased the number of recognized RBPs into the four-digit range and revealed new paradigms: from the prevalence of structurally disordered RNA-binding regions with roles in the formation of membraneless organelles to unsuspected and potentially pervasive connections between intermediary metabolism and RNA regulation. Together with an increasingly detailed understanding of molecular mechanisms of RBP function, these insights are facilitating the development of new therapies to treat malignancies. Here, we provide an overview of RBPs involved in human genetic disorders, both Mendelian and somatic, and discuss emerging aspects in the field with emphasis on molecular mechanisms of disease and therapeutic interventions.

Alternative splicing regulation of cell-cycle genes by SPF45/SR140/CHERP complex controls cell proliferation.

The regulation of pre-mRNA processing has important consequences for cell division and the control of cancer cell proliferation, but the underlying molecular mechanisms remain poorly understood. We report that three splicing factors, SPF45, SR140, and CHERP, form a tight physical and functionally coherent complex that regulates a variety of alternative splicing events, frequently by repressing short exons flanked by suboptimal 3' splice sites. These comprise alternative exons embedded in genes with important functions in cell-cycle progression, including the G2/M key regulator FOXM1 and the spindle regulator SPDL1. Knockdown of either of the three factors leads to G2/M arrest and to enhanced apoptosis in HeLa cells. Promoting the changes in FOXM1 or SPDL1 splicing induced by SPF45/SR140/CHERP knockdown partially recapitulates the effects on cell growth, arguing that the complex orchestrates a program of alternative splicing necessary for efficient cell proliferation.

Genome-wide identification of Fas/CD95 alternative splicing regulators reveals links with iron homeostasis.

Alternative splicing of Fas/CD95 exon 6 generates either a membrane-bound receptor that promotes, or a soluble isoform that inhibits, apoptosis. Using an automatized genome-wide siRNA screening for alternative splicing regulators of endogenous transcripts in mammalian cells, we identified 200 genes whose knockdown modulates the ratio between Fas/CD95 isoforms. These include classical splicing regulators; core spliceosome components; and factors implicated in transcription and chromatin remodeling, RNA transport, intracellular signaling, and metabolic control. Coherent effects of genes involved in iron homeostasis and pharmacological modulation of iron levels revealed a link between intracellular iron and Fas/CD95 exon 6 inclusion. A splicing regulatory network linked iron levels with reduced activity of the Zinc-finger-containing splicing regulator SRSF7, and in vivo and in vitro assays revealed that iron inhibits SRSF7 RNA binding. Our results uncover numerous links between cellular pathways and RNA processing and a mechanism by which iron homeostasis can influence alternative splicing.

Functional splicing network reveals extensive regulatory potential of the core spliceosomal machinery.

Pre-mRNA splicing relies on the poorly understood dynamic interplay between >150 protein components of the spliceosome. The steps at which splicing can be regulated remain largely unknown. We systematically analyzed the effect of knocking down the components of the splicing machinery on alternative splicing events relevant for cell proliferation and apoptosis and used this information to reconstruct a network of functional interactions. The network accurately captures known physical and functional associations and identifies new ones, revealing remarkable regulatory potential of core spliceosomal components, related to the order and duration of their recruitment during spliceosome assembly. In contrast with standard models of regulation at early steps of splice site recognition, factors involved in catalytic activation of the spliceosome display regulatory properties. The network also sheds light on the antagonism between hnRNP C and U2AF, and on targets of antitumor drugs, and can be widely used to identify mechanisms of splicing regulation.

Competition by the masses.

Is the activity of the complex molecular machineries in charge of gene expression saturated in the cell? In this issue, Munding et al. (2013) report that titration of spliceosomal components by abundant ribosomal protein transcripts controls splicing of other genes and contributes to meiosis-specific splicing in budding yeast.

RBM5, 6, and 10 differentially regulate NUMB alternative splicing to control cancer cell proliferation.

RBM5, a regulator of alternative splicing of apoptotic genes, and its highly homologous RBM6 and RBM10 are RNA-binding proteins frequently deleted or mutated in lung cancer. We report that RBM5/6 and RBM10 antagonistically regulate the proliferative capacity of cancer cells and display distinct positional effects in alternative splicing regulation. We identify the Notch pathway regulator NUMB as a key target of these factors in the control of cell proliferation. NUMB alternative splicing, which is frequently altered in lung cancer, can regulate colony and xenograft tumor formation, and its modulation recapitulates or antagonizes the effects of RBM5, 6, and 10 in cell colony formation. RBM10 mutations identified in lung cancer cells disrupt NUMB splicing regulation to promote cell growth. Our results reveal a key genetic circuit in the control of cancer cell proliferation.

The Spliceosome: The Ultimate RNA Chaperone and Sculptor.

The spliceosome, one of the most complex machineries of eukaryotic cells, removes intronic sequences from primary transcripts to generate functional messenger and long noncoding RNAs (lncRNA). Genetic, biochemical, and structural data reveal that the spliceosome is an RNA-based enzyme. Striking mechanistic and structural similarities strongly argue that pre-mRNA introns originated from self-catalytic group II ribozymes. However, in the spliceosome, protein components organize and activate the catalytic-site RNAs, and recognize and pair together splice sites at intron boundaries. The spliceosome is a dynamic, reversible, and flexible machine that chaperones small nuclear (sn) RNAs and a variety of pre-mRNA sequences into conformations that enable intron removal. This malleability likely contributes to the regulation of alternative splicing, a prevalent process contributing to cell differentiation, homeostasis, and disease.

hnRNP A1 proofreads 3' splice site recognition by U2AF.

One of the earliest steps in metazoan pre-mRNA splicing involves binding of U2 snRNP auxiliary factor (U2AF) 65 KDa subunit to the polypyrimidine (Py) tract and of the 35 KDa subunit to the invariant AG dinucleotide at the intron 3' end. Here we use in vitro and in vivo depletion, as well as reconstitution assays using purified components, to identify hnRNP A1 as an RNA binding protein that allows U2AF to discriminate between pyrimidine-rich RNA sequences followed or not by a 3' splice site AG. Biochemical and NMR data indicate that hnRNP A1 forms a ternary complex with the U2AF heterodimer on AG-containing/uridine-rich RNAs, while it displaces U2AF from non-AG-containing/uridine-rich RNAs, an activity that requires the glycine-rich domain of hnRNP A1. Consistent with the functional relevance of this activity for splicing, proofreading assays reveal a role for hnRNP A1 in U2AF-mediated recruitment of U2 snRNP to the pre-mRNA.

Site-Specific mRNA Cleavage for Selective and Quantitative Profiling of Alternative Splicing with Label-Free Optical Biosensors.

Alternative splicing of mRNA precursors is a key process in gene regulation, contributing to the diversity of proteomes by the alternative selection of exonic sequences. Alterations in this mechanism are associated with most cancers, enhancing their proliferation and survival, and can be employed as cancer biomarkers. Label-free optical biosensors are ideal tools for the highly sensitive and label-free analysis of nucleic acids. However, their application for alternative splicing analysis has been hampered due to the formation of complex and intricate long-range base-pairing interactions which make the direct detection in mRNA isoforms difficult. To solve this bottleneck, we introduce a methodology for the generation of length-controlled RNA fragments from purified total RNA, which can be easily detected by the biosensor. The methodology seizes RNase H enzyme activity to degrade the upstream and downstream RNA segments flanking the target sequence upon hybridization to specific DNA oligos. It allows the fast and direct monitoring of Fas gene alternative splicing in real time, employing a surface plasmon resonance biosensor. We demonstrate the selective and specific detection of mRNA fragments in the pM-nM concentration range, reducing quantification errors and showing 81% accuracy when compared to RT-qPCR. The site-specific cleavage outperformed random RNA hydrolysis by increasing the detection accuracy by 20%, making this methodology particularly appropriate for label-free quantification of alternative splicing events in complex samples.

Large-scale analysis of genome and transcriptome alterations in multiple tumors unveils novel cancer-relevant splicing networks.

Alternative splicing is regulated by multiple RNA-binding proteins and influences the expression of most eukaryotic genes. However, the role of this process in human disease, and particularly in cancer, is only starting to be unveiled. We systematically analyzed mutation, copy number, and gene expression patterns of 1348 RNA-binding protein (RBP) genes in 11 solid tumor types, together with alternative splicing changes in these tumors and the enrichment of binding motifs in the alternatively spliced sequences. Our comprehensive study reveals widespread alterations in the expression of RBP genes, as well as novel mutations and copy number variations in association with multiple alternative splicing changes in cancer drivers and oncogenic pathways. Remarkably, the altered splicing patterns in several tumor types recapitulate those of undifferentiated cells. These patterns are predicted to be mainly controlled by MBNL1 and involve multiple cancer drivers, including the mitotic gene NUMA1 We show that NUMA1 alternative splicing induces enhanced cell proliferation and centrosome amplification in nontumorigenic mammary epithelial cells. Our study uncovers novel splicing networks that potentially contribute to cancer development and progression.

CPEB1 coordinates alternative 3'-UTR formation with translational regulation.

More than half of mammalian genes generate multiple messenger RNA isoforms that differ in their 3' untranslated regions (3' UTRs) and therefore in regulatory sequences, often associated with cell proliferation and cancer; however, the mechanisms coordinating alternative 3'-UTR processing for specific mRNA populations remain poorly defined. Here we report that the cytoplasmic polyadenylation element binding protein 1 (CPEB1), an RNA-binding protein that regulates mRNA translation, also controls alternative 3'-UTR processing. CPEB1 shuttles to the nucleus, where it co-localizes with splicing factors and mediates shortening of hundreds of mRNA 3' UTRs, thereby modulating their translation efficiency in the cytoplasm. CPEB1-mediated 3'-UTR shortening correlates with cell proliferation and tumorigenesis. CPEB1 binding to pre-mRNAs not only directs the use of alternative polyadenylation sites, but also changes alternative splicing by preventing U2AF65 recruitment. Our results reveal a novel function of CPEB1 in mediating alternative 3'-UTR processing, which is coordinated with regulation of mRNA translation, through its dual nuclear and cytoplasmic functions.

The Ewing sarcoma protein regulates DNA damage-induced alternative splicing.

The Ewing sarcoma (EWS) protein is a member of the TET (TLS/EWS/TAF15) family of RNA- and DNA-binding proteins whose expression is altered in cancer. We report that EWS depletion results in alternative splicing changes of genes involved in DNA repair and genotoxic stress signaling, including ABL1, CHEK2, and MAP4K2. Chromatin and RNA crosslinking immunoprecipitation results indicate that EWS cotranscriptionally binds to its target RNAs. This association is reduced upon irradiation of cells with ultraviolet light, concomitant with transient enrichment of EWS in nucleoli and with alternative splicing changes that parallel those induced by EWS depletion and that lead to reduced c-ABL protein expression. Consistent with the functional relevance of EWS-mediated alternative splicing regulation in DNA damage response, EWS depletion reduces cell viability and proliferation upon UV irradiation, effects that are attenuated by restoring c-ABL expression. These results provide insights into posttranscriptional mechanisms of DNA damage response by a TET protein.

Reduced fidelity of branch point recognition and alternative splicing induced by the anti-tumor drug spliceostatin A.

Spliceostatin A (SSA) is a stabilized derivative of a Pseudomonas bacterial fermentation product that displays potent anti-proliferative and anti-tumor activities in cancer cells and animal models. The drug inhibits pre-mRNA splicing in vitro and in vivo and binds SF3b, a protein subcomplex of U2 small nuclear ribonucleoprotein (snRNP), which is essential for recognition of the pre-mRNA branch point. We report that SSA prevents interaction of an SF3b 155-kDa subunit with the pre-mRNA, concomitant with nonproductive recruitment of U2 snRNP to sequences 5' of the branch point. Differences in base-pairing potential with U2 snRNA in this region lead to different sensitivity of 3' splice sites to SSA, and to SSA-induced changes in alternative splicing. Indeed, rather than general splicing inhibition, splicing-sensitive microarray analyses reveal specific alternative splicing changes induced by the drug that significantly overlap with those induced by knockdown of SF3b 155. These changes lead to down-regulation of genes important for cell division, including cyclin A2 and Aurora A kinase, thus providing an explanation for the anti-proliferative effects of SSA. Our results reveal a mechanism that prevents nonproductive base-pairing interactions in the spliceosome, and highlight the regulatory and cancer therapeutic potential of perturbing the fidelity of splice site recognition.