Domains of yeast U4 spliceosomal RNA required for PRP4 protein binding, snRNP-snRNP interactions, and pre-mRNA splicing in vivo.
U4 small nuclear RNA (snRNA) contains two intramolecular stem-loop structures, located near each end of the molecule. The 5' stem-loop is highly conserved in structure and separates two regions of U4 snRNA that base-pair with U6 snRNA in the U4/U6 small nuclear ribonucleoprotein particle (snRNP). The 3' stem-loop is highly divergent in structure among species and lies immediately upstream of the binding site for Sm proteins. To investigate the function of these two domains, mutants were constructed that delete the yeast U4 snRNA 5' stem-loop and that replace the yeast 3' stem-loop with that from trypanosome U4 snRNA. Both mutants fail to complement a null allele of the yeast U4 gene. The defects of the mutants have been examined in heterozygous strains by native gel electrophoresis, glycerol gradient centrifugation, and immunoprecipitation. The chimeric yeast-trypanosome RNA does not associate efficiently with U6 snRNA, suggesting that the 3' stem-loop of yeast U4 snRNA might be a binding site for a putative protein that facilitates assembly of the U4/U6 complex. In contrast, the 5' hairpin deletion mutant associates efficiently with U6 snRNA. However, it does not bind the U4/U6-specific protein PRP4 and does not assemble into a U4/U5/U6 snRNA. Thus, we propose that the role of the PRP4 protein is to promote interactions between the U4/U6 snRNP and the U5 snRNP.
Bordonné
R
R
Department of Biochemistry and Biophysics, University of California, San Francisco 94143.
Banroques
J
J
Abelson
J
J
Guthrie
C
C
D001483:Base Sequence; D005656:Fungal Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010455:Peptides / Q000378:metabolism*; D055232:Proline-Rich Protein Domains; D011485:Protein Binding; D012322:RNA Precursors / Q000378:metabolism; D012326:RNA Splicing; D012331:RNA, Fungal / Q000378:metabolism* / Q000648:ultrastructure; D012333:RNA, Messenger / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000378:metabolism* / Q000648:ultrastructure; D012261:Ribonucleoproteins / Q000378:metabolism*; D017411:Ribonucleoproteins, Small Nuclear; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D005656:Fungal Proteins; D010455:Peptides; D012322:RNA Precursors; D012331:RNA, Fungal; D012333:RNA, Messenger; D012342:RNA, Small Nuclear; D012261:Ribonucleoproteins; D017411:Ribonucleoproteins, Small Nuclear