Physical and Functional Interaction between the Methyltransferase Bud23 and the Essential DEAH-Box RNA Helicase Ecm16

Abstract

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.

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Correction for Sardana et al., “Physical and Functional Interaction between the Methyltransferase Bud23 and the Essential DEAH-Box RNA Helicase Ecm16”

ACKNOWLEDGMENTS

We thank M. Anjos for assistance with subcloning ecm16 mutations, C. Wang for anti-Bud23 antibody, and G. Dieci for Rps8 antibody.

This work was supported by NIH grants RO1GM53655 and RO1GM108823 to A.W.J.