Physical and functional interaction between the methyltransferase Bud23 and the essential DEAH-box RNA helicase Ecm16.
The small ribosomal subunit assembles cotranscriptionally on the nascent primary transcript. Cleavage at site A2 liberates the pre-40S subunit. We previously identified Bud23 as a conserved eukaryotic methyltransferase that is required for efficient cleavage at A2. Here, we report that Bud23 physically and functionally interacts with the DEAH-box RNA helicase Ecm16 (also known as Dhr1). Ecm16 is also required for cleavage at A2. We identified mutations in ECM16 that suppressed the growth and A2 cleavage defects of a bud23Δ mutant. RNA helicases often require protein cofactors to provide substrate specificity. We used yeast (Saccharomyces cerevisiae) two-hybrid analysis to map the binding site of Bud23 on Ecm16. Despite the physical and functional interaction between these factors, mutations that disrupted the interaction, as assayed by two-hybrid analysis, did not display a growth defect. We previously identified mutations in UTP2 and UTP14 that suppressed bud23Δ. We suggest that a network of protein interactions may mask the loss of interaction that we have defined by two-hybrid analysis. A mutation in motif I of Ecm16 that is predicted to impair its ability to hydrolyze ATP led to accumulation of Bud23 in an ∼45S particle containing Ecm16. Thus, Bud23 enters the pre-40S pathway at the time of Ecm16 function.
Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas, USA.
Richa
R
Sardana
Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas, USA.
Jieyi
J
Zhu
Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas, USA.
Michael
M
Gill
Department of Molecular Biosciences and Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas, USA arlen@austin.utexas.edu.
Arlen W
AW
Johnson
D000595:Amino Acid Sequence; D002498:Centrifugation; D053487:DEAD-box RNA Helicases / Q000737:chemistry / Q000378:metabolism*; D005799:Genes, Dominant; D005816:Genetic Complementation Test; D008780:Methyltransferases / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D025941:Protein Interaction Mapping; D012323:RNA Processing, Post-Transcriptional; D012335:RNA, Ribosomal / Q000235:genetics; D012270:Ribosomes / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D013489:Suppression, Genetic
D050505:Mutant Proteins; D012335:RNA, Ribosomal; D029701:Saccharomyces cerevisiae Proteins; C578381:Bud23 protein, S cerevisiae; D008780:Methyltransferases; C414169:ECM16 protein, S cerevisiae; D053487:DEAD-box RNA Helicases