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The N-Terminal Domain That Distinguishes Yeast from Bacterial RNase III Contains a Dimerization Signal Required for Efficient Double-Stranded RNA Cleavage

2000; Taylor & Francis; Volume: 20; Issue: 4 Linguagem: Inglês

10.1128/mcb.20.4.1104-1115.2000

1098-5549

Bruno Lamontagne, Annie Tremblay, Sherif Abou Elela,

Bacteriophages and microbial interactions

AbstractYeast Rnt1 is a member of the double-stranded RNA (dsRNA)-specific RNase III family identified by conserved dsRNA binding (dsRBD) and nuclease domains. Comparative sequence analyses have revealed an additional N-terminal domain unique to the eukaryotic homologues of RNase III. The deletion of this domain from Rnt1 slowed growth and led to mild accumulation of unprocessed 25S pre-rRNA. In vitro, deletion of the N-terminal domain reduced the rate of RNA cleavage under physiological salt concentration. Size exclusion chromatography and cross-linking assays indicated that the N-terminal domain and the dsRBD self-interact to stabilize the Rnt1 homodimer. In addition, an interaction between the N-terminal domain and the dsRBD was identified by a two-hybrid assay. The results suggest that the eukaryotic N-terminal domain of Rnt1 ensures efficient dsRNA cleavage by mediating the assembly of optimum Rnt1-RNA ribonucleoprotein complex. ACKNOWLEDGMENTSWe thank Guillaume Chanfreau for the ΔRNT1 yeast strain and for suggesting the in-gel cleavage experiment, James Hu for the λ repressor kit, Dennis Thiele and Simon Labbé for the copper expression vectors, and Philip James for the two-hybrid plasmids and strains. We also thank Allen Nicholson for communicating unpublished results. We are indebted for Benoit Chabot, April Colosimo, Skip Fournier, Christine Gagnon, Michael Katze, Allen Nicholson, and Raymond Wellinger for critical reading of the manuscript.This work was supported by grant MT-14305 from the Medical Research Council of Canada to S.A. The fast protein liquid chromatography apparatus used for protein purification was purchased by a grant from Canada Foundation for Innovation. S.A. is a Chercheur-Boursier Junior I of the Fonds de la Recherche en Santé du Québec.