Determination of the RNA Binding Specificity of the Heterogeneous Nuclear Ribonucleoprotein (hnRNP) H/H′/F/2H9 Family

Abstract

Members of the heterogeneous nuclear ribonucleoprotein (hnRNP) H protein family, H, H, F, and 2H9, are involved in pre-mRNA processing. We analyzed the assembly of these proteins from splicing extracts onto four RNA regulatory elements as follows: a high affinity hnRNP A1-binding site (WA1), a sequence involved in Rev-dependent export (p17gag INS), an exonic splicing silencer from the ␤-tropomyosin gene, and an intronic splicing regulator (downstream control sequence (DCS) from the c-src gene. The entire family binds the WA1, instability (INS), and ␤-tropomyosin substrates, and the core-binding site for each is a run of three G residues followed by an A. Transfer of small regions containing this sequence to a substrate lacking hnRNP H binding activity is sufficient to promote binding of all family members. The c-src DCS has been shown to assemble hnRNP H, not hnRNP F, from HeLa cell extracts, and we show that hnRNP 2H9 does not bind this element. The DCS contains five G residues followed by a C. Mutation of the C to an A changes the specificity of the DCS from a substrate that binds only hnRNP H/H to a binding site for all hnRNP H family members. We conclude that the sequence GGGA is recognized by all hnRNP H family proteins.Some proteins of the heterogeneous nuclear ribonucleoprotein (hnRNP) 1 class were initially described as components of a nucleosome-like structure that protects and organizes nascent RNA polymerase II transcripts (1-3). Our understanding of hnRNP functions has broadened from this purely structural vision to include multiple aspects of mRNA biogenesis such as transcription, splicing, capping, polyadenylation, nuclear transport, and mRNA stability. More than 30 hnRNPs have been identified so far. Many hnRNPs have a modular structure characterized by one or more RNA binding domains and by one or more auxiliary domains that are frequently enriched in a few amino acids, mainly glycines. The RNA recognition motif (RRM), the arginine-rich motif, the KH domain, and the RGG box are some of the main motifs that are found in hnRNP proteins (4).The precise roles of hnRNP proteins in gene expression are likely to be reflected by their RNA binding specificity. Therefore, it is important to have a clear understanding of the RNA binding specificities and affinities of the different family members. Early studies demonstrated that several hnRNPs have affinities for immobilized ribohomopolymers such as poly(G) bound by hnRNPs E, H, F, and M, poly(C) bound by hnRNPs K and J, and poly(U) bound by hnRNPs C and M (5). Subsequently, several hnRNPs were observed to bind specific RNA sequences by UV-mediated cross-linking. Furthermore, utilizing a selection and amplification approach from pools of random RNAs, high affinity binding sequences for hnRNPs A1 (6) and C (7), have been identified. The binding properties of hnRNP A1 are the best characterized so far. Several reports (1, 8 -12) have shown that hnRNP A1 and other members of the hnRNP A/B group (hnRNP A1b, A2, and B1) share common RNA substrate specificities...

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

“…G-tracts are also abundant downstream of mammalian polyadenylation signals 7 and in 5' and 3' untranslated regions (UTR) 8 . 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] .…”

Mechanisms of activation and repression by the alternative splicing factors RBFOX1/2

“…As hnRNP H1 and hnRNP F are 78% identical and appear to have redundant functions (Caputi and Zahler 2001), we knocked down both proteins simultaneously. We transfected siRNAs against luciferase, hnRNP H/F, RALY, or TFG into HeLa cells, and extracted both total RNA and protein after 72 h. All of the proteins were efficiently knocked down without significant changes in endogenous RBFOX2 levels (Fig.…”

Involvement of Heterogeneous Ribonucleoprotein F in the Regulation of Cell Proliferation via the Mammalian Target of Rapamycin/S6 Kinase 2 Pathway

“…Both hnRNPs F and H have three RNA recognition motifs with binding preferences of GGGA (37) or DGGGD (where D represents a U, G, or A nucleotide) motifs (38). In addition, hnRNP F/H have an extensive glycine-rich domain near the C terminus, which may facilitate hetero-or homodimerization between hnRNP F and H (39).…”