Clustered variants in the 5' coding region of TRA2B cause a distinctive neurodevelopmental syndrome.

PURPOSE: Transformer2 proteins (Tra2α and Tra2ß) control splicing patterns in human cells, and no human phenotypes have been associated with germline variants in these genes. The aim of this work was to associate germline variants in the TRA2B gene to a novel neurodevelopmental disorder. METHODS: A total of 12 individuals from 11 unrelated families who harbored predicted loss-of-function monoallelic variants, mostly de novo, were recruited. RNA sequencing and western blot analyses of Tra2ß-1 and Tra2ß-3 isoforms from patient-derived cells were performed. Tra2ß1-GFP, Tra2ß3-GFP and CHEK1 exon 3 plasmids were transfected into HEK-293 cells. RESULTS: All variants clustered in the 5' part of TRA2B, upstream of an alternative translation start site responsible for the expression of the noncanonical Tra2ß-3 isoform. All affected individuals presented intellectual disability and/or developmental delay, frequently associated with infantile spasms, microcephaly, brain anomalies, autism spectrum disorder, feeding difficulties, and short stature. Experimental studies showed that these variants decreased the expression of the canonical Tra2ß-1 isoform, whereas they increased the expression of the Tra2ß-3 isoform, which is shorter and lacks the N-terminal RS1 domain. Increased expression of Tra2ß-3-GFP were shown to interfere with the incorporation of CHEK1 exon 3 into its mature transcript, normally incorporated by Tra2ß-1. CONCLUSION: Predicted loss-of-function variants clustered in the 5' portion of TRA2B cause a new neurodevelopmental syndrome through an apparently dominant negative disease mechanism involving the use of an alternative translation start site and the overexpression of a shorter, repressive Tra2ß protein.

Binding site density enables paralog-specific activity of SLM2 and Sam68 proteins in Neurexin2 AS4 splicing control.

SLM2 and Sam68 are splicing regulator paralogs that usually overlap in function, yet only SLM2 and not Sam68 controls the Neurexin2 AS4 exon important for brain function. Herein we find that SLM2 and Sam68 similarly bind to Neurexin2 pre-mRNA, both within the mouse cortex and in vitro. Protein domain-swap experiments identify a region including the STAR domain that differentiates SLM2 and Sam68 activity in splicing target selection, and confirm that this is not established via the variant amino acids involved in RNA contact. However, far fewer SLM2 and Sam68 RNA binding sites flank the Neurexin2 AS4 exon, compared with those flanking the Neurexin1 and Neurexin3 AS4 exons under joint control by both Sam68 and SLM2. Doubling binding site numbers switched paralog sensitivity, by placing the Neurexin2 AS4 exon under joint splicing control by both Sam68 and SLM2. Our data support a model where the density of shared RNA binding sites around a target exon, rather than different paralog-specific protein-RNA binding sites, controls functional target specificity between SLM2 and Sam68 on the Neurexin2 AS4 exon. Similar models might explain differential control by other splicing regulators within families of paralogs with indistinguishable RNA binding sites.

The tissue-specific RNA binding protein T-STAR controls regional splicing patterns of neurexin pre-mRNAs in the brain.

The RNA binding protein T-STAR was created following a gene triplication 520-610 million years ago, which also produced its two parologs Sam68 and SLM-1. Here we have created a T-STAR null mouse to identify the endogenous functions of this RNA binding protein. Mice null for T-STAR developed normally and were fertile, surprisingly, given the high expression of T-STAR in the testis and the brain, and the known infertility and pleiotropic defects of Sam68 null mice. Using a transcriptome-wide search for splicing targets in the adult brain, we identified T-STAR protein as a potent splicing repressor of the alternatively spliced segment 4 (AS4) exons from each of the Neurexin1-3 genes, and exon 23 of the Stxbp5l gene. T-STAR protein was most highly concentrated in forebrain-derived structures like the hippocampus, which also showed maximal Neurexin1-3 AS4 splicing repression. In the absence of endogenous T-STAR protein, Nrxn1-3 AS4 splicing repression dramatically decreased, despite physiological co-expression of Sam68. In transfected cells Neurexin3 AS4 alternative splicing was regulated by either T-STAR or Sam68 proteins. In contrast, Neurexin2 AS4 splicing was only regulated by T-STAR, through a UWAA-rich response element immediately downstream of the regulated exon conserved since the radiation of bony vertebrates. The AS4 exons in the Nrxn1 and Nrxn3 genes were also associated with distinct patterns of conserved UWAA repeats. Consistent with an ancient mechanism of splicing control, human T-STAR protein was able to repress splicing inclusion of the zebrafish Nrxn3 AS4 exon. Although Neurexin1-3 and Stxbp5l encode critical synaptic proteins, T-STAR null mice had no detectable spatial memory deficits, despite an almost complete absence of AS4 splicing repression in the hippocampus. Our work identifies T-STAR as an ancient and potent tissue-specific splicing regulator that uses a concentration-dependent mechanism to co-ordinately regulate regional splicing patterns of the Neurexin1-3 AS4 exons in the mouse brain.

The splicing landscape is globally reprogrammed during male meiosis.

Meiosis requires conserved transcriptional changes, but it is not known whether there is a corresponding set of RNA splicing switches. Here, we used RNAseq of mouse testis to identify changes associated with the progression from mitotic spermatogonia to meiotic spermatocytes. We identified ∼150 splicing switches, most of which affect conserved protein-coding exons. The expression of many key splicing regulators changed in the course of meiosis, including downregulation of polypyrimidine tract binding protein (PTBP1) and heterogeneous nuclear RNP A1, and upregulation of nPTB, Tra2ß, muscleblind, CELF proteins, Sam68 and T-STAR. The sequences near the regulated exons were significantly enriched in target sites for PTB, Tra2ß and STAR proteins. Reporter minigene experiments investigating representative exons in transfected cells showed that PTB binding sites were critical for splicing of a cassette exon in the Ralgps2 mRNA and a shift in alternative 5' splice site usage in the Bptf mRNA. We speculate that nPTB might functionally replace PTBP1 during meiosis for some target exons, with changes in the expression of other splicing factors helping to establish meiotic splicing patterns. Our data suggest that there are substantial changes in the determinants and patterns of alternative splicing in the mitotic-to-meiotic transition of the germ cell cycle.

Tra2 protein biology and mechanisms of splicing control.

Tra2 proteins regulate pre-mRNA splicing in vertebrates and invertebrates, and are involved in important processes ranging from brain development in mice to sex determination in fruitflies. In structure Tra2 proteins contain two RS domains (domains enriched in arginine and serine residues) flanking a central RRM (RNA recognition motif). Understanding the mechanisms of how Tra2 proteins work to control splicing is one of the key requirements to understand their biology. In the present article, we review what is known about how Tra2 proteins regulate splicing decisions in mammals and fruitflies.

Identification of evolutionarily conserved exons as regulated targets for the splicing activator tra2ß in development.

Alternative splicing amplifies the information content of the genome, creating multiple mRNA isoforms from single genes. The evolutionarily conserved splicing activator Tra2ß (Sfrs10) is essential for mouse embryogenesis and implicated in spermatogenesis. Here we find that Tra2ß is up-regulated as the mitotic stem cell containing population of male germ cells differentiate into meiotic and post-meiotic cells. Using CLIP coupled to deep sequencing, we found that Tra2ß binds a high frequency of exons and identified specific G/A rich motifs as frequent targets. Significantly, for the first time we have analysed the splicing effect of Sfrs10 depletion in vivo by generating a conditional neuronal-specific Sfrs10 knock-out mouse (Sfrs10(fl/fl); Nestin-Cre(tg/+)). This mouse has defects in brain development and allowed correlation of genuine physiologically Tra2ß regulated exons. These belonged to a novel class which were longer than average size and importantly needed multiple cooperative Tra2ß binding sites for efficient splicing activation, thus explaining the observed splicing defects in the knockout mice. Regulated exons included a cassette exon which produces a meiotic isoform of the Nasp histone chaperone that helps monitor DNA double-strand breaks. We also found a previously uncharacterised poison exon identifying a new pathway of feedback control between vertebrate Tra2 proteins. Both Nasp-T and the Tra2a poison exon are evolutionarily conserved, suggesting they might control fundamental developmental processes. Tra2ß protein isoforms lacking the RRM were able to activate specific target exons indicating an additional functional role as a splicing co-activator. Significantly the N-terminal RS1 domain conserved between flies and humans was essential for the splicing activator function of Tra2ß. Versions of Tra2ß lacking this N-terminal RS1 domain potently repressed the same target exons activated by full-length Tra2ß protein.

Molecular design of a splicing switch responsive to the RNA binding protein Tra2ß.

Tra2ß regulates a number of splicing switches including activation of the human testis-specific exon HIPK3-T in the Homeodomain Interacting Protein Kinase 3 gene. By testing HIPK3-T exons of different intrinsic strengths, we found Tra2ß most efficiently activated splicing inclusion of intrinsically weak exons, although these were spliced at a lower overall level. Both the RRM and N-terminal RS-rich region of Tra2ß were required for splicing activation. Bioinformatic searches for splicing enhancers and repressors mapped four physically distinct exonic splicing enhancers (ESEs) within HIPK3-T, each containing the known Tra2ß AGAA-rich binding site. Surprisingly disruption of each single ESE prevented Tra2ß-mediated activation, although single mutated exons could still bind Tra2ß protein by gel shifts and functional splicing analyses. Titration experiments indicate an additive model of HIPK3-T splicing activation, requiring availability of an array of four distinct ESEs to enable splicing activation. To enable this efficient Tra2ß-mediated splicing switch to operate, a closely adjacent downstream and potentially competitive stronger 5'-splice site is actively repressed. Our data indicate that a novel arrangement of multiple mono-specific AGAA-rich ESEs coupled to a weak 5'-splice site functions as a responsive gauge. This gauge monitors changes in the specific nuclear concentration of the RNA binding protein Tra2ß, and co-ordinately regulates HIPK3-T exon splicing inclusion.

Epithelial specific splicing regulator proteins as emerging oncogenes in aggressive prostate cancer.

Prostate cancer progression is connected to the activity of conventional oncogenes and tumour suppressors and driven by circulating steroid hormones. A key issue has been how to identify and care for aggressively developing prostate tumours. Here we discuss how expression of the splicing regulators ESRP1 and ESRP2, and how their role as "masterminds" of epithelial splicing patterns, have been identified as markers of aggressively proliferating prostate primary tumours. We suggest that the origin of prostate cancer within epithelial cells, and the subsequent association of ESRP1 and ESRP2 expression with more aggressive disease progression, identify ESRP1 and ESRP2 as lineage survival oncogenes. To move this field on in the future it will be important to identify the gene expression targets controlled by ESRP1/2 that regulate prostate cancer proliferation. Potential future therapies could be designed to target ESRP1 and ESRP2 protein activity or their regulated splice isoforms in aggressive prostate tumours. Design of these therapies is potentially complicated by the risk of producing a more mesenchymal splicing environment that might promote tumour metastasis.

How does Tra2ß protein regulate tissue-specific RNA splicing?

The splicing regulator protein Tra2ß is conserved between humans and insects and is essential for mouse development. Recent identification of physiological RNA targets has started to uncover molecular targets and mechanisms of action of Tra2ß. At a transcriptome-wide level, Tra2ß protein binds a matrix of AGAA-rich sequences mapping frequently to exons. Particular tissue-specific alternatively spliced exons contain high concentrations of high scoring Tra2ß-binding sites and bind Tra2ß strongly in vitro. These top exons were also activated for splicing inclusion in cellulo by co-expression of Tra2ß protein and were significantly down-regulated after genetic depletion of Tra2ß. Tra2ß itself seems to be fairly evenly expressed across several different mouse tissues. In the present paper, we review the properties of Tra2ß and its regulated target exons, and mechanisms through which this fairly evenly expressed alternative splicing regulator might drive tissue-specific splicing patterns.

Cryptic splicing: common pathological mechanisms involved in male infertility and neuronal diseases.

High levels of transcription and alternative splicing are recognized hallmarks of gene expression in the testis and largely driven by cells in meiosis. Because of this, the male meiosis stage of the cell cycle is often viewed as having a relatively permissive environment for gene expression. In this review, we highlight recent findings that identify the RNA binding protein RBMXL2 as essential for male meiosis. RBMXL2 functions as a "guardian of the transcriptome" that protects against the use of aberrant (or "cryptic") splice sites that would disrupt gene expression. This newly discovered protective role during meiosis links with a wider field investigating mechanisms of cryptic splicing control that protect neurons from amyotrophic lateral sclerosis and Alzheimer's disease. We discuss how the mechanism repressing cryptic splicing patterns during meiosis evolved, and why it may be essential for sperm production and male fertility.

Haploinsufficiency of the germ cell-specific nuclear RNA binding protein hnRNP G-T prevents functional spermatogenesis in the mouse.

Human HNRNPGT, encoding the protein hnRNP G-T, is one of several autosomal retrogenes derived from RBMX. It has been suggested that HNRNPGT functionally replaces the sex-linked RBMX and RBMY genes during male meiosis. We show here that during normal mouse germ cell development, hnRNP G-T protein is strongly expressed during and after meiosis when proteins expressed from Rbmx or Rbmx-like genes are absent. Amongst these Rbmx-like genes, DNA sequence analyses indicate that two other mouse autosomal Rbmx-derived retrogenes have evolved recently in rodents and one already shows signs of degenerating into a non-expressed pseudogene. In contrast, orthologues of Hnrnpgt are present in all four major groups of placental mammals. The sequence of Hnrnpgt is under considerable positive selection suggesting it performs an important germ cell function in eutherians. To test this, we inactivated Hnrnpgt in ES cells and studied its function during spermatogenesis in chimaeric mice. Although germ cells heterozygous for this targeted allele could produce sperm, they did not contribute to the next generation. Chimaeric mice with a high level of mutant germ cells were infertile with low sperm counts and a high frequency of degenerate seminiferous tubules and abnormal sperm. Chimaeras made from a 1:1 mix of targeted and wild-type ES cell clones transmitted wild-type germ cells only. Our data show that haploinsufficiency of Hnrnpgt results in abnormal sperm production in the mouse. Genetic defects resulting in reduced levels of HNRNPGT could, therefore, be a cause of male infertility in humans.

Proteomic identification of heterogeneous nuclear ribonucleoprotein L as a novel component of SLM/Sam68 Nuclear Bodies.

BACKGROUND: Active pre-mRNA splicing occurs co-transcriptionally, and takes place throughout the nucleoplasm of eukaryotic cells. Splicing decisions are controlled by networks of nuclear RNA-binding proteins and their target sequences, sometimes in response to signalling pathways. Sam68 (Src-associated in mitosis 68 kDa) is the prototypic member of the STAR (Signal Transduction and Activation of RNA) family of RNA-binding proteins, which regulate splicing in response to signalling cascades. Nuclear Sam68 protein is concentrated within subnuclear organelles called SLM/Sam68 Nuclear Bodies (SNBs), which also contain some other splicing regulators, signalling components and nucleic acids. RESULTS: We used proteomics to search for the major interacting protein partners of nuclear Sam68. In addition to Sam68 itself and known Sam68-associated proteins (heterogeneous nuclear ribonucleoproteins hnRNP A1, A2/B1 and G), we identified hnRNP L as a novel Sam68-interacting protein partner. hnRNP L protein was predominantly present within small nuclear protein complexes approximating to the expected size of monomers and dimers, and was quantitatively associated with nucleic acids. hnRNP L spatially co-localised with Sam68 as a novel component of SNBs and was also observed within the general nucleoplasm. Localisation within SNBs was highly specific to hnRNP L and was not shared by the closely-related hnRNP LL protein, nor any of the other Sam68-interacting proteins we identified by proteomics. The interaction between Sam68 and hnRNP L proteins was observed in a cell line which exhibits low frequency of SNBs suggesting that this association also takes place outside SNBs. Although ectopic expression of hnRNP L and Sam68 proteins independently affected splicing of CD44 variable exon v5 and TJP1 exon 20 minigenes, these proteins did not, however, co-operate with each other in splicing regulation of these target exons. CONCLUSION: Here we identify hnRNP L as a novel SNB component. We show that, compared with other identified Sam68-associated hnRNP proteins and hnRNP LL, this co-localisation within SNBs is specific to hnRNP L. Our data suggest that the novel Sam68-hnRNP L protein interaction may have a distinct role within SNBs.

RBMX family proteins connect the fields of nuclear RNA processing, disease and sex chromosome biology.

RBMX is a ubiquitously expressed nuclear RNA binding protein that is encoded by a gene on the X chromosome. RBMX belongs to a small protein family with additional members encoded by paralogs on the mammalian Y chromosome and other chromosomes. These RNA binding proteins are important for normal development, and also implicated in cancer and viral infection. At the molecular level RBMX family proteins contribute to splicing control, transcription and genome integrity. Establishing what endogenous genes and pathways are controlled by RBMX and its paralogs will have important implications for understanding chromosome biology, DNA repair and mammalian development. Here we review what is known about this family of RNA binding proteins, and identify important current questions about their functions.

Androgen-regulated transcription of ESRP2 drives alternative splicing patterns in prostate cancer.

Prostate is the most frequent cancer in men. Prostate cancer progression is driven by androgen steroid hormones, and delayed by androgen deprivation therapy (ADT). Androgens control transcription by stimulating androgen receptor (AR) activity, yet also control pre-mRNA splicing through less clear mechanisms. Here we find androgens regulate splicing through AR-mediated transcriptional control of the epithelial-specific splicing regulator ESRP2. Both ESRP2 and its close paralog ESRP1 are highly expressed in primary prostate cancer. Androgen stimulation induces splicing switches in many endogenous ESRP2-controlled mRNA isoforms, including splicing switches correlating with disease progression. ESRP2 expression in clinical prostate cancer is repressed by ADT, which may thus inadvertently dampen epithelial splice programmes. Supporting this, treatment with the AR antagonist bicalutamide (Casodex) induced mesenchymal splicing patterns of genes including FLNB and CTNND1. Our data reveals a new mechanism of splicing control in prostate cancer with important implications for disease progression.

Structural basis of RNA recognition and dimerization by the STAR proteins T-STAR and Sam68.

Sam68 and T-STAR are members of the STAR family of proteins that directly link signal transduction with post-transcriptional gene regulation. Sam68 controls the alternative splicing of many oncogenic proteins. T-STAR is a tissue-specific paralogue that regulates the alternative splicing of neuronal pre-mRNAs. STAR proteins differ from most splicing factors, in that they contain a single RNA-binding domain. Their specificity of RNA recognition is thought to arise from their property to homodimerize, but how dimerization influences their function remains unknown. Here, we establish at atomic resolution how T-STAR and Sam68 bind to RNA, revealing an unexpected mode of dimerization different from other members of the STAR family. We further demonstrate that this unique dimerization interface is crucial for their biological activity in splicing regulation, and suggest that the increased RNA affinity through dimer formation is a crucial parameter enabling these proteins to select their functional targets within the transcriptome.

A SLM2 Feedback Pathway Controls Cortical Network Activity and Mouse Behavior.

The brain is made up of trillions of synaptic connections that together form neural networks needed for normal brain function and behavior. SLM2 is a member of a conserved family of RNA binding proteins, including Sam68 and SLM1, that control splicing of Neurexin1-3 pre-mRNAs. Whether SLM2 affects neural network activity is unknown. Here, we find that SLM2 levels are maintained by a homeostatic feedback control pathway that predates the divergence of SLM2 and Sam68. SLM2 also controls the splicing of Tomosyn2, LysoPLD/ATX, Dgkb, Kif21a, and Cask, each of which are important for synapse function. Cortical neural network activity dependent on synaptic connections between SLM2-expressing-pyramidal neurons and interneurons is decreased in Slm2-null mice. Additionally, these mice are anxious and have a decreased ability to recognize novel objects. Our data reveal a pathway of SLM2 homeostatic auto-regulation controlling brain network activity and behavior.

A novel androgen-regulated isoform of the TSC2 tumour suppressor gene increases cell proliferation.

TSC2 (Tuberous sclerosis complex 2) is an important tumour suppressor gene, mutations within which are linked to the development of tuberous sclerosis and implicated in multiple tumour types. TSC2 protein complexes with TSC1 and blocks the ability of the Rheb (Ras homolog enriched in brain) GTPase to activate mTOR (mammalian target of rapamycin), a crucial signal transducer which regulates protein synthesis and cell growth. Here, we report the characterisation of a novel isoform of TSC2 which is under direct control of the ligand-activated androgen receptor. TSC2 isoform A (TSC2A) is derived from an internal androgen-regulated alternative promoter and encodes a 508-amino acid cytoplasmic protein corresponding to the C-terminal region of full-length TSC2, lacking the interaction domain for TSC1 and containing an incomplete interaction domain required for Rheb inactivation. Expression of TSC2A is induced in response to androgens and full-length TSC2 is co-ordinately down-regulated, indicating an androgen-driven switch in TSC2 protein isoforms. In contrast to the well-characterised suppressive effect on cell proliferation of full-length TSC2 protein, both LNCaP and HEK293 cells over-expressing TSC2 isoform A proliferate more rapidly (measured by MTT assays) and have increased levels of cells in S-phase (measured by both Edu staining and FACS analysis). Our work indicates, for the first time, a novel role for this well-known tumour suppressor gene, which encodes an activator of cell proliferation in response to androgen stimulation.

Human Tra2 proteins jointly control a CHEK1 splicing switch among alternative and constitutive target exons.

Alternative splicing--the production of multiple messenger RNA isoforms from a single gene--is regulated in part by RNA binding proteins. While the RBPs transformer2 alpha (Tra2α) and Tra2ß have both been implicated in the regulation of alternative splicing, their relative contributions to this process are not well understood. Here we find simultaneous--but not individual--depletion of Tra2α and Tra2ß induces substantial shifts in splicing of endogenous Tra2ß target exons, and that both constitutive and alternative target exons are under dual Tra2α-Tra2ß control. Target exons are enriched in genes associated with chromosome biology including CHEK1, which encodes a key DNA damage response protein. Dual Tra2 protein depletion reduces expression of full-length CHK1 protein, results in the accumulation of the DNA damage marker γH2AX and decreased cell viability. We conclude Tra2 proteins jointly control constitutive and alternative splicing patterns via paralog compensation to control pathways essential to the maintenance of cell viability.

Identification of novel androgen-regulated pathways and mRNA isoforms through genome-wide exon-specific profiling of the LNCaP transcriptome.

Androgens drive the onset and progression of prostate cancer (PCa) by modulating androgen receptor (AR) transcriptional activity. Although several microarray-based studies have identified androgen-regulated genes, here we identify in-parallel global androgen-dependent changes in both gene and alternative mRNA isoform expression by exon-level analyses of the LNCaP transcriptome. While genome-wide gene expression changes correlated well with previously-published studies, we additionally uncovered a subset of 226 novel androgen-regulated genes. Gene expression pathway analysis of this subset revealed gene clusters associated with, and including the tyrosine kinase LYN, as well as components of the mTOR (mammalian target of rapamycin) pathway, which is commonly dysregulated in cancer. We also identified 1279 putative androgen-regulated alternative events, of which 325 (∼25%) mapped to known alternative splicing events or alternative first/last exons. We selected 30 androgen-dependent alternative events for RT-PCR validation, including mRNAs derived from genes encoding tumour suppressors and cell cycle regulators. Of seven positively-validating events (∼23%), five events involved transcripts derived from alternative promoters of known AR gene targets. In particular, we found a novel androgen-dependent mRNA isoform derived from an alternative internal promoter within the TSC2 tumour suppressor gene, which is predicted to encode a protein lacking an interaction domain required for mTOR inhibition. We confirmed that expression of this alternative TSC2 mRNA isoform was directly regulated by androgens, and chromatin immunoprecipitation indicated recruitment of AR to the alternative promoter region at early timepoints following androgen stimulation, which correlated with expression of alternative transcripts. Together, our data suggest that alternative mRNA isoform expression might mediate the cellular response to androgens, and may have roles in clinical PCa.

Regulation of gene expression by the RNA-binding protein Sam68 in cancer.

Sam68 (Src-associated in mitosis 68 kDa) is the prototypical member of the STAR (signal transducer and activator of RNA) family of RNA-binding proteins. Sam68 is implicated in a number of cellular processes including signal transduction, transcription, RNA metabolism, cell cycle regulation and apoptosis. In the present review, we summarize the functions of Sam68 as a transcriptional and post-transcriptional regulator of gene expression, with particular relevance to cancer.