Rrp5p, a trans-acting factor in yeast ribosome biogenesis, is an RNA-binding protein with a pronounced preference for U-rich sequences.
Rrp5p is a trans-acting factor important for biogenesis of both the 40S and 60S subunit of the Saccharomyces cerevisiae ribosome. The protein contains 12 tandemly repeated S1 RNA binding motifs in its N-terminal region, suggesting the ability to interact directly with the pre-rRNA. In vitro binding studies, using immunopurified Rrp5p and in vitro transcribed, 32P-UTP-labeled RNA fragments, revealed that Rrp5p is a general RNA-binding protein with a strong preference for single-stranded sequences rich in uridines. Co-immunoprecipitation studies in yeast cells expressing ProtA-tagged Rrp5p showed that the protein is still associated with pre-ribosomal particles containing 27SA2 pre-rRNA but not with particles containing the 27SB precursor. Thus, Rrp5p appears to dissociate from the 66S pre-ribosome upon or immediately after further processing of 27SA2 pre-rRNA, suggesting the presence of (an) important binding site(s) within the 3'-terminal portion of ITS1. The location of these possible binding site(s) was further delimited using rrp2-1 mutant cells, which accumulate the 5'-extended 5.8S pre-rRNA species. The results indicate that association of Rrp5p with the pre-ribosome is abolished upon removal of a 30-nt region downstream from site A2, which contains two short, single-stranded U stretches. Sequence comparison shows that only the most 5' of these two U-rich stretches is conserved among yeast species whose ITS1 can functionally replace the S. cerevisiae spacer. The implications for the role of Rrp5p in yeast ribosome biogenesis are discussed.
de Boer
Paulo
P
Section of Biochemistry and Molecular Biology, FEW, Vrije Universiteit, de Boelelaan 1083, 1081 HV Amsterdam, The Netherlands.
Vos
Harmjan R
HR
Faber
Alex W
AW
Vos
Jan C
JC
Raué
Hendrik A
HA
D001665:Binding Sites; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012322:RNA Precursors / Q000235:genetics / Q000378:metabolism; D012335:RNA, Ribosomal / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000235:genetics* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014529:Uridine / Q000378:metabolism
D009687:Nuclear Proteins; D012322:RNA Precursors; D012335:RNA, Ribosomal; D016601:RNA-Binding Proteins; C102025:RRP5 protein, S cerevisiae; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014529:Uridine