A Discrete 3′ Region of U6 Small Nuclear RNA Modulates the Phosphorylation Cycle of the C1 Heterogeneous Nuclear Ribonucleoprotein Particle Protein†

Abstract

The C heterogeneous ribonucleoprotein particle (hnRNP) proteins bind to nascent pre-mRNA and may participate in assembly of the early prespliceosome. Ser/Thr phosphorylation of the C1 hnRNP protein in HeLa nuclear extracts regulates its binding to pre-mRNA (S. H. Mayrand, P. Dwen, and T. Pederson, Proc. Natl. Acad. Sci. USA 90:7764-7768, 1993). We have now further investigated the phosphorylation cycle of the C1 hnRNP protein, with emphasis on its regulation. Pretreatment of nuclear extracts with micrococcal nuclease eliminated the phosphorylation of C1 hnRNP protein, but pretreatment with DNase did not, suggesting a dependence on RNA. Oligodeoxynucleotide-targeted RNase H cleavage of U1, U2, and U4 small nuclear RNAs did not affect the phosphorylation of C1 hnRNP protein. However, cleavage of nucleotides 78 to 95, but not other regions, of U6 small nuclear RNA resulted in an inhibition of the dephosphorylation step of the C1 hnRNP protein phosphorylation cycle. This inhibition was as pronounced as that seen with the serine/ threonine protein phosphatase inhibitor okadaic acid. C1 hnRNP protein dephosphorylation could be completely restored by the addition of intact U6 RNA. Add-back experiments with mutant RNAs further delineated the minimal region essential for C1 protein dephosphorylation as residing in nucleotides 85 to 92 of U6 RNA. These results illuminate a hitherto unanticipated function of U6 RNA: the modulation of a phosphorylation-dephosphorylation cycle of C1 hnRNP protein that influences the binding affinity of this protein for pre-mRNA. This newly revealed function of U6 RNA is likely to play a very early role in the prespliceosome assembly pathway, prior to U6 RNA's entry into the mature spliceosome's active center. The C heterogeneous nuclear ribonucleoprotein particle (hnRNP) proteins are a highly conserved set of 42,000 to 44,000-molecular-weight proteins (3, 23, 37, 41) that associate with pre-mRNA at a very early posttranscriptional stage in vivo (2, 14, 15, 29, 55) and interact tightly and specifically with pre-mRNA in nuclear extract pre-mRNA splicing systems (35, 46). We have previously reported that the binding of the C hnRNP proteins to pre-mRNA is negatively regulated by hy-perphosphorylation (34). A dynamic cycle of hnRNP C protein phosphorylation-dephosphorylation takes place in HeLa nuclear extracts, and inhibition of hnRNP C protein dephosphor-ylation by the serine/threonine protein phosphatase inhibitor okadaic acid leads to a markedly reduced affinity of C proteins for pre-mRNA (34). In the present investigation, we have explored the possibility that the phosphorylation-dephosphorylation cycle of C hnRNP proteins is itself regulated and have focused on components of the splicing machinery as potential control elements. We identify a discrete 3 region of U6 small nuclear RNA that modulates C hnRNP dephosphorylation. MATERIALS AND METHODS Nuclear extracts were prepared from HeLa cells as previously described (11). All extracts were prepared from cells harvested in logarithmic growth under the culture conditions detailed previously (40). Nuclear extracts (30%, vol/vol) were made 3.2 mM MgCl 2 , 400 M ATP, and 20 mM creatine phosphate prior to incubation with [-32 P]ATP and/or [-32 P]GTP at the concentrations specified in the figure legends. Where indicated, nuclear extracts were preincubated with the serine/threonine protein phosphatase inhibitor okadaic acid (1 M; LC Services, Woburn, Mass.), with micrococcal nuclease (400 U/ml; Worthington Biochemical Corp., Freehold, N.J.) or with oligodeoxynucleotides (600 ng/l unless otherwise specified; Integrated DNA Technologies, Coralville, Iowa) complementary to regions of U1, U2, U4, or U6 small nuclear RNA in the presence of 400 M ATP, as detailed in the figure legends. Oligodeoxynucleotide-mediated cleavage of U1, U2, U4, and U6 RNAs by endogenous RNase H was monitored by electrophoresis of deproteinized nuclear extract RNA on 10% polyacryl-amide-8.3 M urea gels followed by ethidium bromide staining (36, 49). Following pretreatments as described above, the nuclear extracts were incu-bated with [-32 P]ATP and/or [-32 P]GTP as specified in the figure legends. C hnRNP proteins were isolated from the reactions by immunoaffinity selection on protein A-Sepharose-bound monoclonal antibody 4F4 (6) and prepared for sodium dodecyl sulfate-polyacrylamide gel electrophoresis as we have described in detail previously (34, 36) except that the acrylamide/N,N-methylenebisacryl-amide ratio in the gels was 74:1. In some experiments, in vitro-transcribed human U6 small nuclear RNA or mutant U6 RNAs were added to nuclear extracts in which the endogenous U6 RNA had been destroyed by prior oligodeoxynucleotide-mediated RNase H cleavage. The wild-type U6 transcript was synthesized with T7 RNA polymerase from the human U6 plasmid, pHU6-1