Lamontagne B, Tremblay A, Abou Elela S
Département de Microbiologie et d'Infectiologie, Faculté de Médecine, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4.
Mol Cell Biol. 2000 Feb;20(4):1104-15. doi: 10.1128/MCB.20.4.1104-1115.2000.
Yeast 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.
酵母Rnt1是双链RNA(dsRNA)特异性RNase III家族的成员,通过保守的dsRNA结合(dsRBD)和核酸酶结构域鉴定。比较序列分析揭示了RNase III真核同源物特有的额外N端结构域。从Rnt1中缺失该结构域会减缓生长并导致未加工的25S前体rRNA轻度积累。在体外,N端结构域的缺失降低了生理盐浓度下RNA切割的速率。尺寸排阻色谱和交联分析表明,N端结构域和dsRBD自身相互作用以稳定Rnt1同型二聚体。此外,通过双杂交试验鉴定了N端结构域与dsRBD之间的相互作用。结果表明,Rnt1的真核N端结构域通过介导最佳Rnt1-RNA核糖核蛋白复合物的组装来确保有效的dsRNA切割。