The C-terminal region of the exosome-associated protein Rrp47 is specifically required for box C/D small nucleolar RNA 3'-maturation.
Cells lacking the exosome-associated protein Rrp47 show similar defects in stable RNA processing to those observed in the absence of the catalytic subunit Rrp6, but the precise mechanism(s) by which Rrp47 functions together with Rrp6 remains unclear. Deletion complementation analyses defined an N-terminal region of Rrp47, largely coincident with the bioinformatically defined Sas10/C1D domain, which was sufficient for protein function in vivo. In vitro protein interaction studies demonstrated that this domain of Rrp47 binds the PMC2NT domain of Rrp6. Expression of the N-terminal domain of Rrp47 in yeast complemented most RNA-processing defects associated with the rrp47Δ mutant but failed to complement the defect observed in 3'-end maturation of box C/D small nucleolar RNAs. Consistent with these results, protein capture assays revealed an interaction between the C-terminal region of Rrp47 and the small nucleolar ribonucleoproteins Nop56 and Nop58. Filter binding assays demonstrated that deletion of the lysine-rich sequence at the C terminus of Rrp47 blocked RNA binding in vitro. Furthermore, a protein mutated both at the C terminus and within the N-terminal domain showed a synergistic defect in RNA binding without impacting on its ability to interact with Rrp6. These studies provide evidence for a role of Rrp47 in registering a small nucleolar ribonucleoprotein particle assembly, functionally characterize the Sas10/C1D domain of Rrp47, and show that both the C terminus of Rrp47 and the N-terminal domain contribute to its RNA-binding activity.
Department of Molecular Biology and Biotechnology, The University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, United Kingdom.
Joe L
JL
Costello
Jonathan A
JA
Stead
Monika
M
Feigenbutz
Rebecca M
RM
Jones
Phil
P
Mitchell
D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005095:Exoribonucleases / Q000235:genetics / Q000378:metabolism; D063326:Exosome Multienzyme Ribonuclease Complex; D005816:Genetic Complementation Test; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012323:RNA Processing, Post-Transcriptional / Q000502:physiology*; D020537:RNA, Small Nucleolar / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D020718:Ribonucleoproteins, Small Nucleolar / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; C494789:LRP1 protein, S cerevisiae; C430407:NOP56 protein, S cerevisiae; C438199:NOP58 protein, S cerevisiae; D009687:Nuclear Proteins; D020537:RNA, Small Nucleolar; D016601:RNA-Binding Proteins; D020718:Ribonucleoproteins, Small Nucleolar; D029701:Saccharomyces cerevisiae Proteins; D005095:Exoribonucleases; D063326:Exosome Multienzyme Ribonuclease Complex; C112610:RRP6 protein, S cerevisiae