A DISCRETE 3'-REGION OF U6 SMALL NUCLEAR-RNA MODULATES THE PHOSPHORYLATION CYCLE OF THE C1 HETEROGENEOUS NUCLEAR RIBONUCLEOPROTEIN PARTICLEPROTEIN

The C heterogeneous ribonucleoprotein particle (hnRNP) proteins bind t o nascent pre-mRNA and may participate in assembly of the early prespl iceosome. Ser/Thr phosphorylation of the C1 hnRNP protein in HeLa nucl ear 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 hnRN P protein, with emphasis on its regulation, Pretreatment of nuclear ex tracts with micrococcal nuclease eliminated the phosphorylation of C1 hnRNP protein, but pretreatment with DNase did not, suggesting a depen dence on RNA, Oligodeoxynucleotide-targeted RNase H cleavage of U1, U2 , and U4 small nuclear RNAs did not affect the phosphorylation of C1 h nRNP protein, However, cleavage of nucleotides 78 to 95, but not other regions, of U6 small nuclear RNA resulted in an inhibition of the dep hosphorylation step of the C1 hnRNP protein phosphorylation cycle, Thi s inhibition was as pronounced as that seen with the serine/threonine protein phosphatase inhibitor okadaic acid, C1 hnRNP protein dephospho rylation could be completely restored by the addition of intact U6 RNA , Add-back experiments,vith mutant RNAs further delineated the minimal region essential for C1 protein dephosphorylation as residing in nucl eotides 85 to 92 of U6 RNA, These results illuminate a hitherto unanti cipated function of U6 RNA: the modulation of a phosphorylation-dephos phorylation cycle of C1 hnRNP protein that influences the binding affi nity 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.