Prp43 bound at different sites on the pre-rRNA performs distinct functions in ribosome synthesis.
Yeast ribosome synthesis requires 19 different RNA helicases, but none of their pre-rRNA-binding sites were previously known, making their precise functions difficult to determine. Here we identify multiple binding sites for the helicase Prp43 in the 18S and 25S rRNA regions of pre-rRNAs, using UV crosslinking. Binding in 18S was predominantly within helix 44, close to the site of 18S 3' cleavage, in which Prp43 is functionally implicated. Four major binding sites were identified in 25S, including helix 34. In strains depleted of Prp43 or expressing only catalytic point mutants, six snoRNAs that guide modifications close to helix 34 accumulated on preribosomes, implicating Prp43 in their release, whereas other snoRNAs showed reduced preribosome association. Prp43 was crosslinked to snoRNAs that target sequences close to its binding sites, indicating direct interactions. We propose that Prp43 acts on preribosomal regions surrounding each binding site, with distinct functions at different locations.
Bohnsack
Markus T
MT
Wellcome Trust Centre for Cell Biology, University of Edinburgh, UK. bohnsack@bio.uni-frankfurt.de
Martin
Roman
R
Granneman
Sander
S
Ruprecht
Maike
M
Schleiff
Enrico
E
Tollervey
David
D
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000378:metabolism; D053487:DEAD-box RNA Helicases / Q000172:deficiency / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012322:RNA Precursors / Q000378:metabolism*; D012331:RNA, Fungal / Q000737:chemistry / Q000235:genetics; D012335:RNA, Ribosomal / Q000378:metabolism; D012342:RNA, Small Nuclear / Q000378:metabolism; D020537:RNA, Small Nucleolar / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*
D003432:Cross-Linking Reagents; D012322:RNA Precursors; D012331:RNA, Fungal; D012335:RNA, Ribosomal; D012342:RNA, Small Nuclear; D020537:RNA, Small Nucleolar; D029701:Saccharomyces cerevisiae Proteins; C405486:U6 small nuclear RNA; C109034:PRP43 protein, S cerevisiae; D053487:DEAD-box RNA Helicases