In addition, although a high frequency of alternative splicing events in the testis are species-specific[61], the high conservation of binding sites in theTra2apoison-exon suggests regulation by Tra2 has been conserved since the radiation of vertebrates

In addition, although a high frequency of alternative splicing events in the testis are species-specific[61], the high conservation of binding sites in theTra2apoison-exon suggests regulation by Tra2 has been conserved since the radiation of vertebrates. 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 O4I1 proteins. BothNasp-Tand theTra2apoison exon are evolutionarily conserved, suggesting they might control fundamental developmental processes. Tra2 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 Tra2. Versions of Tra2 lacking this N-terminal RS1 domain potently repressed the same target exons activated by full-length Tra2 protein. == Author Summary == Alternative splicing amplifies the informational content of the genome, making multiple mRNA isoforms from single genes. Tra2 proteins bind and activate alternative exons, and in mice Tra2 is essential for embryonic development through unknown target RNAs. Here we report the first target exons that are physiologically regulated by Tra2 in developing mice. Normal activation of these regulated exons depends on multiple Tra2 binding sites, and significant mis-regulation of these exons is observed during mouse development when Tra2 is removed. As expected, Tra2 activates splicing of some target exons through direct RNA binding via its RNA Recognition O4I1 Motif. Surprisingly, for some exons Tra2 can also activate splicing independent of direct RNA binding through two domains enriched in arginine and serine residues (called RS domains). The N-terminal RS1 domain of Tra2 is absolutely essential for splicing activation of physiological target exons, explaining why this domain is conserved between vertebrates and invertebrates. Surprisingly, Tra2 proteins without RS1 operate as splicing repressors, suggesting the possibility that endogenous Tra2 protein isoforms may differentially regulate the same target exons. == Introduction == Almost all transcripts from genes encoding multiple exons are alternatively spliced, and correct patterns of alternative splicing are important for health and normal development[1],[2],[3]. Alternative splicing introduces new coding information into mRNAs, thereby increasing genome capacity to encode an expanded number of mRNAs and proteins from a finite number of genes[3]. Poison exons which introduce premature stop codons can also be alternatively spliced to target mRNAs for degradation through Nonsense Mediated Decay (NMD)[4],[5],[6],[7], . Alternative splice events are controlled in part bytrans-acting RNA binding proteins which help establish patterns of alternative splicing through deciphering a splicing code embedded within the pre-mRNA sequence[9],[10],[11]. Tra2 proteins bind directly to target exons thereby activating splicing inclusion[12], and have a modular organisation comprising a single central RNA recognition motif (RRM) which binds to target RNA sequences, flanked by arginine-serine rich (RS1 and RS2) domains[13],[14]. The N-terminal Tra2 RS1 domain is longer and contains more RS Rabbit Polyclonal to PDLIM1 dipeptides than RS2. The reason for this unique modular organisation is unknown, but is conserved in vertebrate and invertebrate Tra2 proteins and different from the classical SR super-family which have a single C-terminal RS domain[15]. Also unlike classical SR proteins, Tra2 proteins do not restore splicing activity to S100 extracts[12]. A single Tra2 protein is conserved in fruit flies, where it is essential for spermatogenesis and sex determination[16]. There are two mammalian Tra2 proteins called Tra2 (encoded by theTra2agene on mouse chromosome 6) and Tra2 (encoded by theSfrs10gene on mouse chromosome 16) which share 63% amino acid identity and similar RNA binding specificities[12]. NMR analyses have recently shown that the optimal core RNA target sequence for binding full length Tra2 protein is an AGAA motif, with each of the nucleotide residues being specifically recognized by the Tra2 RRM[17],[18]. A key priority to understand the biological functions of Tra2 is to identify target O4I1 RNAs which are functionally regulated within animal cells, and associated pathways of gene activity. Mice with ubiquitous deficiency of.