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

• Published in: The Journal of biological chemistry 2001-11, Vol.276 (47), p.43850-43859
• Main Authors: Caputi, Massimo, Zahler, Alan M.
• Format: Article
• Language: English
• Subjects: Animals; Base Sequence; Chromatography, Affinity; Exons; Gene Products, gag - chemistry; Gene Products, gag - metabolism; HeLa Cells; Heterogeneous Nuclear Ribonucleoprotein A1; Heterogeneous-Nuclear Ribonucleoprotein Group A-B; Heterogeneous-Nuclear Ribonucleoprotein Group F-H; Heterogeneous-Nuclear Ribonucleoproteins; hnRNA; hnRNP H protein; Humans; Rats; Ribonucleoproteins - isolation & purification; Ribonucleoproteins - metabolism; RNA Splicing; RNA, Messenger - chemistry; RNA, Messenger - metabolism; RNA-Binding Proteins - isolation & purification; RNA-Binding Proteins - metabolism; Tropomyosin - genetics; Tropomyosin - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M102861200

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 β-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 β-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.