Heterogeneous nuclear ribonucleoprotein H is required for optimal U11 small nuclear ribonucleoprotein binding to a retroviral RNA-processing control element: implications for U12-dependent RNA splicing.
An RNA-processing element from Rous sarcoma virus, the negative regulator of splicing (NRS), represses splicing to generate unspliced RNA that serves as mRNA and as genomic RNA for progeny virions and also promotes polyadenylation of the unspliced RNA. Integral to NRS function is the binding of U1 small nuclear ribonucleoprotein (snRNP), but its binding is controlled by U11 snRNP that binds to an overlapping site. U11 snRNP, the U1 counterpart for splicing of U12-dependent introns, binds the NRS remarkably well and requires G-rich elements just downstream of the consensus U11 binding site. We present evidence that heterogeneous nuclear ribonucleoprotein (hnRNP) H binds to the NRS G-rich elements and that hnRNP H is required for optimal U11 binding in vitro. It is further shown that hnRNP H (but not hnRNP F) can promote U11 binding and splicing from the NRS in vivo when tethered to the RNA as an MS2 fusion protein. Interestingly, 17% of the naturally occurring U12-dependent introns have at least two potential hnRNP H binding sites positioned similarly to the NRS. For two such introns from the SCN4A and P120 genes, we show that hnRNP H binds to each in a G-tract-dependent manner, that G-tract mutations strongly reduce splicing of minigene RNA, and that tethered hnRNP H restores splicing to mutant RNA. In support of a role for hnRNP H in both splicing pathways, hnRNP H antibodies co-precipitate U1 and U11 small nuclear ribonucleoproteins. These results indicate that hnRNP H is an auxiliary factor for U11 binding to the NRS and that, more generally, hnRNP H is a splicing factor for a subset of U12-dependent introns that harbor G-rich elements.
McNally
Lisa M
LM
Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226.
Yee
Lily
L
McNally
Mark T
MT
D001358:Avian Sarcoma Viruses / Q000235:genetics*; D001665:Binding Sites; D006367:HeLa Cells; D034541:Heterogeneous-Nuclear Ribonucleoprotein Group F-H / Q000502:physiology*; D006801:Humans; D007438:Introns; D009154:Mutation; D012323:RNA Processing, Post-Transcriptional; D012326:RNA Splicing; D012342:RNA, Small Nuclear / Q000502:physiology*; D011993:Recombinant Fusion Proteins; D038621:Regulatory Sequences, Ribonucleic Acid / Q000235:genetics*; D017411:Ribonucleoproteins, Small Nuclear / Q000235:genetics / Q000378:metabolism*
D034541:Heterogeneous-Nuclear Ribonucleoprotein Group F-H; D012342:RNA, Small Nuclear; D011993:Recombinant Fusion Proteins; D038621:Regulatory Sequences, Ribonucleic Acid; D017411:Ribonucleoproteins, Small Nuclear; C098762:U11 small nuclear ribonucleoprotein, human; C096986:U12 small nuclear RNA