A uridylate tract mediates efficient heterogeneous nuclear ribonucleoprotein C protein-RNA cross-linking and functionally substitutes for the downstream element of the polyadenylation signal.
Every RNA added to an in vitro polyadenylation extract became stably associated with both the heterogeneous nuclear ribonucleoprotein (hnRNP) A and C proteins, as assayed by immunoprecipitation analysis using specific monoclonal antibodies. UV-cross-linking analysis, however, which assays the specific spatial relationship of certain amino acids and RNA bases, indicated that the hnRNP C proteins, but not the A proteins, were associated with downstream sequences of the simian virus 40 late polyadenylation signal in a sequence-mediated manner. A tract of five consecutive uridylate residues was required for this interaction. The insertion of a five-base U tract into a pGEM4 polylinker-derived transcript was sufficient to direct sequence-specific cross-linking of the C proteins to RNA. Finally, the five-base uridylate tract restored efficient in vitro processing to several independent poly(A) signals in which it substituted for downstream element sequences. The role of the downstream element in polyadenylation efficiency, therefore, may be mediated by sequence-directed alignment or phasing of an hnRNP complex.
Department of Microbiology and Molecular Genetics, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark 07103.
J
J
Wilusz
T
T
Shenk
D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism / Q000528:radiation effects; D006367:HeLa Cells / Q000378:metabolism / Q000528:radiation effects; D034482:Heterogeneous-Nuclear Ribonucleoprotein Group C; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010957:Plasmids; D011061:Poly A / Q000235:genetics*; D011485:Protein Binding; D012313:RNA / Q000235:genetics*; D012332:RNA, Heterogeneous Nuclear / Q000378:metabolism*; D012333:RNA, Messenger; D015183:Restriction Mapping; D012261:Ribonucleoproteins / Q000378:metabolism*; D014158:Transcription, Genetic; D014466:Ultraviolet Rays; D014498:Uracil; D014544:Uridine Triphosphate / Q000378:metabolism*
D034482:Heterogeneous-Nuclear Ribonucleoprotein Group C; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D046911:Macromolecular Substances; D015345:Oligonucleotide Probes; D012332:RNA, Heterogeneous Nuclear; D012333:RNA, Messenger; D012261:Ribonucleoproteins; D011061:Poly A; D014498:Uracil; D012313:RNA; D014544:Uridine Triphosphate