The pivotal roles of TIA proteins in 5' splice-site selection of alu exons and across evolution.

More than 5% of alternatively spliced internal exons in the human genome are derived from Alu elements in a process termed exonization. Alus are comprised of two homologous arms separated by an internal polypyrimidine tract (PPT). In most exonizations, splice sites are selected from within the same arm. We hypothesized that the internal PPT may prevent selection of a splice site further downstream. Here, we demonstrate that this PPT enhanced the selection of an upstream 5' splice site (5'ss), even in the presence of a stronger 5'ss downstream. Deletion of this PPT shifted selection to the stronger downstream 5'ss. This enhancing effect depended on the strength of the downstream 5'ss, on the efficiency of base-pairing to U1 snRNA, and on the length of the PPT. This effect of the PPT was mediated by the binding of TIA proteins and was dependent on the distance between the PPT and the upstream 5'ss. A wide-scale evolutionary analysis of introns across 22 eukaryotes revealed an enrichment in PPTs within approximately 20 nt downstream of the 5'ss. For most metazoans, the strength of the 5'ss inversely correlated with the presence of a downstream PPT, indicative of the functional role of the PPT. Finally, we found that the proteins that mediate this effect, TIA and U1C, and in particular their functional domains, are highly conserved across evolution. Overall, these findings expand our understanding of the role of TIA1/TIAR proteins in enhancing recognition of exons, in general, and Alu exons, in particular.

Alu exonization events reveal features required for precise recognition of exons by the splicing machinery.

Despite decades of research, the question of how the mRNA splicing machinery precisely identifies short exonic islands within the vast intronic oceans remains to a large extent obscure. In this study, we analyzed Alu exonization events, aiming to understand the requirements for correct selection of exons. Comparison of exonizing Alus to their non-exonizing counterparts is informative because Alus in these two groups have retained high sequence similarity but are perceived differently by the splicing machinery. We identified and characterized numerous features used by the splicing machinery to discriminate between Alu exons and their non-exonizing counterparts. Of these, the most novel is secondary structure: Alu exons in general and their 5' splice sites (5'ss) in particular are characterized by decreased stability of local secondary structures with respect to their non-exonizing counterparts. We detected numerous further differences between Alu exons and their non-exonizing counterparts, among others in terms of exon-intron architecture and strength of splicing signals, enhancers, and silencers. Support vector machine analysis revealed that these features allow a high level of discrimination (AUC = 0.91) between exonizing and non-exonizing Alus. Moreover, the computationally derived probabilities of exonization significantly correlated with the biological inclusion level of the Alu exons, and the model could also be extended to general datasets of constitutive and alternative exons. This indicates that the features detected and explored in this study provide the basis not only for precise exon selection but also for the fine-tuned regulation thereof, manifested in cases of alternative splicing.

Alternative splicing of Alu exons--two arms are better than one.

Alus, primate-specific retroelements, are the most abundant repetitive elements in the human genome. They are composed of two related but distinct monomers, left and right arms. Intronic Alu elements may acquire mutations that generate functional splice sites, a process called exonization. Most exonizations occur in right arms of antisense Alu elements, and are alternatively spliced. Here we show that without the left arm, exonization of the right arm shifts from alternative to constitutive splicing. This eliminates the evolutionary conserved isoform and may thus be selected against. We further show that insertion of the left arm downstream of a constitutively spliced non-Alu exon shifts splicing from constitutive to alternative. Although the two arms are highly similar, the left arm is characterized by weaker splicing signals and lower exonic splicing regulatory (ESR) densities. Mutations that improve these potential splice signals activate exonization and shift splicing from the right to the left arm. Collaboration between two or more putative splice signals renders the intronic left arm with a pseudo-exon function. Thus, the dimeric form of the Alu element fortuitously provides it with an evolutionary advantage, allowing enrichment of the primate transcriptome without compromising its original repertoire.

Abnormalities in A-to-I RNA editing patterns in CNS injuries correlate with dynamic changes in cell type composition.

Adenosine to Inosine (A-to-I) RNA editing is a co- or post-transcriptional mechanism that modifies genomically encoded nucleotides at the RNA level. A-to-I RNA editing is abundant in the brain, and altered editing levels have been reported in various neurological pathologies and following spinal cord injury (SCI). The prevailing concept is that the RNA editing process itself is dysregulated by brain pathologies. Here we analyzed recent RNA-seq data, and found that, except for few mammalian conserved editing sites, editing is significantly higher in neurons than in other cell populations of the brain. We studied A-to-I RNA editing in stab wound injury (SWI) and SCI models and showed that the apparent under-editing observed after injury correlates with an approximately 20% reduction in the relative density of neurons, due to cell death and immune cell infiltration that may account for the observed under-editing. Studies of neuronal and astrocyte cultures and a computational analysis of SCI RNA-seq data further supported the possibility that a reduction in neuronal density is responsible for alterations in the tissue-wide editing patterns upon injury. Thus, our data suggest that the case for a mechanistic linkage between A-to-I RNA editing and brain pathologies should be revisited.

Comparative analysis of transposed element insertion within human and mouse genomes reveals Alu's unique role in shaping the human transcriptome.

BACKGROUND: Transposed elements (TEs) have a substantial impact on mammalian evolution and are involved in numerous genetic diseases. We compared the impact of TEs on the human transcriptome and the mouse transcriptome. RESULTS: We compiled a dataset of all TEs in the human and mouse genomes, identifying 3,932,058 and 3,122,416 TEs, respectively. We than extracted TEs located within human and mouse genes and, surprisingly, we found that 60% of TEs in both human and mouse are located in intronic sequences, even though introns comprise only 24% of the human genome. All TE families in both human and mouse can exonize. TE families that are shared between human and mouse exhibit the same percentage of TE exonization in the two species, but the exonization level of Alu, a primate-specific retroelement, is significantly greater than that of other TEs within the human genome, leading to a higher level of TE exonization in human than in mouse (1,824 exons compared with 506 exons, respectively). We detected a primate-specific mechanism for intron gain, in which Alu insertion into an exon creates a new intron located in the 3' untranslated region (termed 'intronization'). Finally, the insertion of TEs into the first and last exons of a gene is more frequent in human than in mouse, leading to longer exons in human. CONCLUSION: Our findings reveal many effects of TEs on these two transcriptomes. These effects are substantially greater in human than in mouse, which is due to the presence of Alu elements in human.