A nuclear 3′-5′ exonuclease involved in mRNA degradation interacts with poly(A) polymerase and the hnRNA protein Npl3p
A nuclear 3′-5′ exonuclease involved in mRNA degradation interacts with poly(A) polymerase and the hnRNA protein Npl3p
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DOI：
10.1128/mcb.20.2.604-616.2000
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发表时间：
2000-01-01
影响因子：
5.3
通讯作者：
Butler, JS
中科院分区：
文献类型：
--
作者：
Burkard, KTD;Butler, JS
在携带温度敏感、致命性Pap1-1突变的酿酒酵母细胞中，PAP1编码的聚(A)聚合酶失活会导致聚(A)(+)mRNAs水平降低。对PAP1-1抑制基因的遗传选择产生了RRP6基因的两个隐性冷敏感等位基因。这些抑制子，RRP6-1和RRP6-2，以及RRP6的缺失，允许PAP1-1菌株在高温下生长，并以一种不同于细胞质mRNA周转途径的方式部分恢复Poly(A)(+)mRNA水平，而不会减缓mRNA衰退的限速步骤。Rrp6p-绿色荧光蛋白融合的亚细胞定位表明，该酶残基位于细胞核内。系统发育分析和RRP6-1突变的性质表明，Rrp6p内存在一个高度保守的3‘-5’外切核酸酶核心区，正如预测的那样，重组Rrp6p以与3‘-5’外核溶解机制一致的方式催化合成的放射性标记RNA的水解。遗传和生化实验表明，Rrp6p与Poly(A)聚合酶和Npl3p相互作用，Npl3p是一种Poly(A)(+)mRNA结合蛋白，参与mRNA前加工和mRNA核输出。这些发现表明，Rrp6p可能与mRNA多聚腺苷化系统相互作用，从而在降解异常加工的前体mRNAs的核途径中发挥作用。
Inactivation of poly(A) polymerase (encoded by PAP1) in Saccharomyces cerevisiae cells carrying the temperature-sensitive, lethal pap1-1 mutation results in reduced levels of poly(A)(+) mRNAs. Genetic selection for suppressors of pap1-1 yielded two recessive, cold-sensitive alleles of the gene RRP6. These suppressors, rrp6-1 and rrp6-2, as well as a deletion of RRP6, allow growth of pap1-1 strains at high temperature and partially restore the levels of poly(A)(+) mRNA in a manner distinct from the cytoplasmic mRNA turnover pathway and without slowing a rate-limiting step in mRNA decay. Subcellular localization of an Rrp6p-green fluorescent protein fusion shows that the enzyme residues in the nucleus. Phylogenetic analysis and the nature of the rrp6-1 mutation suggest the existence of a highly conserved 3'-5' exonuclease core domain within Rrp6p, As predicted, recombinant Rrp6p catalyzes the hydrolysis of a synthetic radiolabeled RNA in a manner consistent with a 3'-5' exonucleolytic mechanism. Genetic and biochemical experiments indicate that Rrp6p interacts with poly(A) polymerase and with Npl3p, a poly(A)(+) mRNA binding protein implicated in pre-mRNA processing and mRNA nuclear export. These findings suggest that Rrp6p may interact with the mRNA polyadenylation system and thereby play a role in a nuclear pathway for the degradation of aberrantly processed precursor mRNAs.