The role of short RNA loops in recognition of a single-hairpin exon derived from a mammalian-wide interspersed repeat

Abstract

Splice-site selection is controlled by secondary structure through sequestration or approximation of splicing signals in primary transcripts but the exact role of even the simplest and most prevalent structural motifs in exon recognition remains poorly understood. Here we took advantage of a single-hairpin exon that was activated in a mammalian-wide interspersed repeat (MIR) by a mutation stabilizing a terminal triloop, with splice sites positioned close to each other in a lower stem of the hairpin. We first show that the MIR exon inclusion in mRNA correlated inversely with hairpin stabilities. Employing a systematic manipulation of unpaired regions without altering splice-site configuration, we demonstrate a high correlation between exon inclusion of terminal tri-and tetraloop mutants and matching tri-/ tetramers in splicing silencers/enhancers. Loop-specific exon inclusion levels and enhancer/silencer associations were preserved across primate cell lines, in 4 hybrid transcripts and also in the context of a distinct stem, but only if its loopclosing base pairs were shared with the MIR hairpin. Unlike terminal loops, splicing activities of internal loop mutants were predicted by their intramolecular Watson-Crick interactions with the antiparallel strand of the MIR hairpin rather than by frequencies of corresponding trinucleotides in splicing silencers/enhancers. We also show that splicing outcome of oligonucleotides targeting the MIR exon depend on the identity of the triloop adjacent to their antisense target. Finally, we identify proteins regulating MIR exon recognition and reveal a distinct requirement of adjacent exons for C-terminal extensions of Tra2a and Tra2b RNA recognition motifs.

Alternative splicing ofU2AF1reveals a shared repression mechanism for duplicated exons

“…Exon Ab was most promoted by exon 3 sequences that encode the U2AF35a RNP2 motif, as illustrated by mutation Ab-2 (Figure 4B, C). This Ab-to-3 swap changes glutamine 49 to leucine and creates a GAA trinucleotide, one of the most potent exonic splicing enhancer (33,67,68). The remaining insertions of exon 3 segments to exon Ab were closer to splice sites and promoted exon skipping.…”

“…Human embryonic kidney (HEK) 293 cells were grown as described (33). Transient transfections with plasmids and siRNAs were performed with jetPRIME (Polyplus) according to manufacturer's recommendations.…”

“…Transient transfections with plasmids and siRNAs were performed with jetPRIME (Polyplus) according to manufacturer's recommendations. siRNAs are listed in Supplementary Table S1 or were described previously (19,33). Knockdown of heterogeneous nuclear ribonucleoprotein C (hnRNP C) was achieved by the HSS179304 siRNA (Invitrogen) (13).…”

A birth of bipartite exon by intragenic deletion

“…Our XLAS case also highlights the perilous inadequacy of predicting phenotypic severity of Mendelian disorders from DNA changes alone. As with exonized mammalian-wide interspersed repeats (Kralovicova et al 2015), future systematic analyses of L1-derived 3 0 splice sites should help characterize RNA interactions that facilitate their recognition by the spliceosome. Finally, our results suggest that the fraction of disease-causing intragenic deletions that affect RNA processing could be much larger than anticipated and that such cases may provide valuable exon selection models for future studies.…”

“…Intronic splicing enhancers or silencers promote or inhibit recognition of cryptic splice sites, which have similar sequences as authentic splice sites but outnumber them by at least an order of magnitude in the genome (Fairbrother and Chasin 2000). Reports of disease-causing mutations that activate new exons without creating a new splice-site consensus have been sporadic, yet they have provided unique insights into our understanding of ancillary motifs required for inclusion of intronic sequences in mature transcripts (Ferlini et al 1998;Pagani et al 2002;Buratti et al 2007b;Vorechovsky 2010;Kralovicova et al 2015). Alport syndrome is characterized by a progressive kidney disease accompanied by hearing loss and ocular abnormalities (Kashtan 1999).…”