Splice site strength–dependent activity and genetic buffering by poly-G runs

Abstract

Pre-mRNA splicing is regulated through combinatorial activity of RNA motifs including splice sites and splicing regulatory elements (SREs). Here, we show that the activity of the G-run class of SREs is ∼4-fold higher when adjacent to intermediate strength 5'ss relative to weak 5'ss, and ∼1.3-fold higher relative to strong 5'ss. This dependence on 5'ss strength was observed in splicing reporters and in global microarray and mRNA-Seq analyses of splicing changes following RNAi against heterogeneous nuclear ribonucleoprotein (hnRNP) H, which crosslinked to G-runs adjacent to many regulated exons. An exon’s responsiveness to changes in hnRNP H levels therefore depends in a complex way on G-run abundance and 5'ss strength, and other splicing factors may function similarly. This pattern of activity enables G-runs and hnRNP H to buffer the effects of 5'ss mutations, augmenting the frequency of 5'ss polymorphism and the evolution of new splicing patterns.

Structural basis of G-tract recognition and encaging by hnRNP F quasi-RRMs

“…When located in introns, G-tracts generally act as splicing enhancers 4 while when located in exons, they mainly act as splicing silencers 5 . G-tracts located in introns were recently shown to be able to buffer genetic variations of 5' splice sites by potentially acting as evolutionary capacitors of splicing changes 6 . G-tracts are also abundant downstream of mammalian polyadenylation signals 7 and in 5' and 3' untranslated regions (UTR) 8 .…”

HnRNPH1/H2, U1 snRNP, and U11 snRNP cooperate to regulate the stability of the U11-48K pre-mRNA

“…1). Such G-runs are typical binding sites for members of the hnRNPF/H protein family (Caputi and Zahler 2001;Schaub et al 2007), which have often been found to inhibit splicing when bound to exonic splicing silencers (ESSs) (Chen et al 1999;Buratti et al 2004;Xiao et al 2009;LeFave et al 2011;Huelga et al 2012;Wang et al 2012). To test whether hnRNPF/H proteins bind to the G-run in exon 4i, we performed protein-RNA crosslinking experiments with RNA substrates containing the conserved 48K element and part of the preceding intron (Verbeeren et al 2010).…”

“…To study this, we knocked down hnRNPH1 in HeLa cells by using increasing amounts of an siRNA that efficiently targets hnRNPH1 (Xiao et al 2009). Efficient reduction of hnRNPH1/H2 levels by 40%-75% (with increasing concentration of siRNA against hnRNPH1) was verified by Western blotting and hnRNPH1 mRNA levels by RT-PCR analysis (Fig.…”

Aberrantly splicedHTT,a new player in Huntington’s disease pathogenesis

“…8 Clustering of G and C repeats in the adjacent downstream intronic sequence probably helps to increase splice site strength, as has been demonstrated for other introns. 9 In fact, mouse Htt and human HTT have > 75% GC content in the first 55 bases downstream of the intron 1 5′-splice site. Similarly upstream, between the CAG repeat and 5′-splice site, there is a tract of > 80% GC bases in mouse Htt and human HTT.…”