Schneider Claudia, Leung Eileen, Brown Jeremy, Tollervey David
Wellcome Trust Centre for Cell Biology, University of Edinburgh, Edinburgh EH9 3JR, UK.
Nucleic Acids Res. 2009 Mar;37(4):1127-40. doi: 10.1093/nar/gkn1020. Epub 2009 Jan 7.
Nuclear and cytoplasmic forms of the yeast exosome share 10 components, of which only Rrp44/Dis3 is believed to possess 3' exonuclease activity. We report that expression only of Rrp44 lacking 3'-exonuclease activity (Rrp44-exo) supports growth in S288c-related strains (BY4741). In BY4741, rrp44-exo was synthetic-lethal with loss of the cytoplasmic 5'-exonuclease Xrn1, indicating block of mRNA turnover, but not with loss of the nuclear 3'-exonuclease Rrp6. The RNA processing phenotype of rrp44-exo was milder than that seen on Rrp44 depletion, indicating that Rrp44-exo retains important functions. Recombinant Rrp44 was shown to possess manganese-dependent endonuclease activity in vitro that was abolished by four point mutations in the putative metal binding residues of its N-terminal PIN domain. Rrp44 lacking both exonuclease and endonuclease activity failed to support growth in strains depleted of endogenous Rrp44. Strains expressing Rrp44-exo and Rrp44-endo-exo exhibited different RNA processing patterns in vivo suggesting Rrp44-dependent endonucleolytic cleavages in the 5'-ETS and ITS2 regions of the pre-rRNA. Finally, the N-terminal PIN domain was shown to be necessary and sufficient for association with the core exosome, indicating its dual function as a nuclease and structural element.
酵母外切体的核形式和细胞质形式共有10个组分，其中只有Rrp44/Dis3被认为具有3'核酸外切酶活性。我们报道，仅表达缺乏3'-核酸外切酶活性的Rrp44（Rrp44-exo）能支持S288c相关菌株（BY4741）的生长。在BY4741中，rrp44-exo与细胞质5'-核酸外切酶Xrn1缺失呈合成致死，表明mRNA周转受阻，但与核3'-核酸外切酶Rrp6缺失不呈合成致死。rrp44-exo的RNA加工表型比Rrp44缺失时更轻，表明Rrp44-exo保留了重要功能。重组Rrp44在体外显示具有锰依赖性内切酶活性，其N端PIN结构域的假定金属结合残基中的四个点突变可消除该活性。缺乏核酸外切酶和内切酶活性的Rrp44不能支持内源性Rrp44缺失菌株的生长。表达Rrp44-exo和Rrp44-endo-exo的菌株在体内表现出不同的RNA加工模式，表明在pre-rRNA的5'-ETS和ITS2区域存在Rrp44依赖性内切酶切割。最后，N端PIN结构域被证明对于与核心外切体结合是必要且充分的，表明其作为核酸酶和结构元件的双重功能。
