THE C-GROUP HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEIN PROTEINS BIND TO THE 5'-STEM-LOOP OF THE U2 SMALL NUCLEAR RIBONUCLEOPROTEIN PARTICLE

The C group heterogeneous nuclear ribonucleoprotein (hnRNP) proteins b ind to nascent pre-messenger RNA. In vitro studies have indicated that the C hnRNP proteins bind particularly strongly to the intron polypyr imidine tract of pre-mRNA and may be important for pre-mRNA splicing. In addition, there is evidence that the interaction of the C hnRNP pro teins with pre-mRNA is facilitated by the U1 and U2 small nuclear RNPs (snRNPs). In the present study, we have uncovered another feature of the C hnRNP proteins that may provide a unifying framework for these p revious observations; the C hnRNP proteins bind to the 5' stem-loop of the U2 snRNP. This was detected by incubating human P-32-labeled U2 s nRNP in micrococcal nuclease-treated HeLa nuclear extracts, followed b y UV-mediated protein-RNA cross-linking, which revealed that C hnRNP p roteins were cross-linked to P-32-nucleotides in the U2 snRNP. In simi lar experiments, no cross-linking of C hnRNP proteins to P-32-labeled U1 or U4 snRNPs was observed. The observed cross-linking of C hnRNP pr oteins to U2 snRNP was efficiently competed by excess U2 RNA and by po ly(U) but not by poly(A). No competition was observed with an RNA mole cule comprising U2 nucleotides 105-189, indicating that the C hnRNP pr otein interactive regions of U2 RNA reside solely in the 5' half of th e molecule. Oligodeoxynucleotide-mediated RNase H cleavage experiments revealed that a 5' region of U2 RNA including nucleotides 15-28 is es sential for the observed C hnRNP protein cross-linking. C hnRNP protei n cross-linking to U2 snRNP was efficiently competed by a mini-RNA cor responding to the first 29 nucleotides of U2 RNA, whereas no competiti on was observed with a variant of this mini-RNA in which the UUUU loop of stem-loop I was mutationally configured into a single-stranded RNA by replacing the stem with non-pairing nucleotides. Competition exper iments with another mutant mini-U2 RNA in which the UUUU loop was repl aced by AAAA indicated that both the UUUU loop and the stem are import ant for C hnRNP protein cross-linking, a finding consistent with other recent data on the RNA sequence specificity of C hnRNP protein bindin g.