Intronic Binding Sites for hnRNP A/B and hnRNP F/H Proteins Stimulate Pre-mRNA Splicing

Abstract

hnRNP A/B proteins modulate the alternative splicing of several mammalian and viral pre-mRNAs, and are typically viewed as proteins that enforce the activity of splicing silencers. Here we show that intronic hnRNP A/B–binding sites (ABS) can stimulate the in vitro splicing of pre-mRNAs containing artificially enlarged introns. Stimulation of in vitro splicing could also be obtained by providing intronic ABS in trans through the use of antisense oligonucleotides containing a non-hybridizing ABS-carrying tail. ABS-tailed oligonucleotides also improved the in vivo inclusion of an alternative exon flanked by an enlarged intron. Notably, binding sites for hnRNP F/H proteins (FBS) replicate the activity of ABS by improving the splicing of an enlarged intron and by modulating 5′ splice-site selection. One hypothesis formulated to explain these effects is that bound hnRNP proteins self-interact to bring in closer proximity the external pair of splice sites. Consistent with this model, positioning FBS or ABS at both ends of an intron was required to stimulate splicing of some pre-mRNAs. In addition, a computational analysis of the configuration of putative FBS and ABS located at the ends of introns supports the view that these motifs have evolved to support cooperative interactions. Our results document a positive role for the hnRNP A/B and hnRNP F/H proteins in generic splicing, and suggest that these proteins may modulate the conformation of mammalian pre-mRNAs.

Coupling between alternative polyadenylation and alternative splicing is limited to terminal introns

“…Instead, hnRNP A2/B1's was identified to be one of several splicing regulatory proteins that alter APA or AS patterns upon CstF64 KD. Given hnRNP A2/B1's important role in regulating AS 42 and mRNA stability, 41,60 these observations strongly suggest that CstF64 influences AS decisions through indirect mechanisms. Investigating the mechanisms that lead to 3 0 UTR diversification and alternative terminal exon definition will therefore be an important future research endeavor.…”

“…5D). As hnRNP A2/B1 has also been implicated in the regulation of AS, 42 it is highly possible that many of the observed AS changes activated upon CstF64 KD were caused by the altered expression of this or other splicing regulatory proteins. Indeed, our analysis of CstF64 KD cells also identified hnRNP AB, hnRNP C, hnRNP H3, SRSF5 and SRSF6 as AS target genes (GEO submission GSE79157).…”

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

“…Some of the trans-acting factors that bind to these G-tracts belong to the heterogeneous nuclear ribonucleoprotein (hnRNP) F/H family of proteins that consists of five members (hnRNP F, hnRNP H, hnRNP H', hnRNP 2H9, and GRich Sequence Factor (GRSF) 1) 9,10 . GRSF-1 is mainly involved in translation regulation 11,12 and Internal Ribosome Entry Site (IRES) mediated translation 13 , while hnRNP H, H' and F are mostly involved in the regulation of alternative splicing [14][15][16][17][18][19][20][21][22][23] and polyadenylation [24][25][26] . The posttranscriptional regulation by hnRNP F/H proteins is a crucial event since mutations in G-tracts often result in aberrant splicing patterns and can be associated with various diseases [27][28][29][30][31][32] .…”

“…In some cases, hnRNP F/H proteins regulate splicing by preventing or enhancing the binding of spliceosomal components to the pre-mRNA 17,27,37 or by competing with other trans-acting factors 38 . HnRNP F/H members were also proposed to influence splice site selection through self association that would loop out introns and bring specific pairs of splice sites in closer proximity 22 . Additionally, hnRNP F/H members can help the formation of spliceosomal complexes but not the formation of the ATP-independent pre-spliceosomal early E complex 39 .…”

The search for alternative splicing regulators: new approaches offer a path to a splicing code

“…2). This mechanism has been suggested for some instances of splicing enhancement mediated by hnRNPs binding to intronic splicing enhancers, as well as for the Nova proteins (Martinez-Contreras et al 2006;Ule et al 2006). If this is the case in let-2 regulation, other conserved regions of intron 10 that do not bind ASD-2b may come into play, along with (an) additional factor(s) not identified in the screen, offering another conceivable explanation for why ASD-2b expression throughout development is insufficient to affect splicing early in development.…”