MODIFICATION OF HUMAN U4 RNA REQUIRES U6 RNA AND MULTIPLE PSEUDOURIDINE SYNTHASES

Small nuclear RNAs (snRNA), cofactors in the splicing of pre-mRNA, are highly modified. In this report the modification of human U4 RNA was studied using cell extracts and in vitro synthesized, and therefore un modified, U4 RNA, The formation of pseudouridine (Psi) at positions 4, 72 and 79 in U4 RNA was dependent on an RNA-containing cofactor, sinc e the activities in the extracts were micrococcal nuclease (MN) sensit ive, Extracts were fractionated on glycerol gradients and there was a broad peak of reconstitution activity centered at 14 S. Reconstitution was not due to additional enzymatic activity, since the peak fraction was MN sensitive. Oligodeoxynucleotide-mediated RNase H digestion of U6 RNA in the extracts inhibited formation of Psi in U4 RNA. From glyc erol gradient analysis we determined that exogenously added U4 RNA tha t is associated with U6 RNA (sedimentation velocity 16 S) was signific antly higher in Psi content than U4 RNA not associated with U6 RNA (8 S). Competitive inhibitors of Psi synthases, 5-fluorouridine-containin g (5-FU) wild-type and mutant U4 RNAs, were used to investigate format ion of Psi in U4 RNA. Deletions and point mutations in these 5-FU-cont aining U4 RNAs affected their ability to inhibit Psi synthase in vitro . With the aid of these potent inhibitors it was determined that at le ast two separate activities modify the uridines at these positions.