Chowdhury Ashis, Mukhopadhyay Jaba, Tharun Sundaresan
Department of Biochemistry and Molecular Biology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814-4799, USA.
RNA. 2007 Jul;13(7):998-1016. doi: 10.1261/rna.502507. Epub 2007 May 18.
Decapping is a critical step in mRNA decay. In the 5'-to-3' mRNA decay pathway conserved in all eukaryotes, decay is initiated by poly(A) shortening, and oligoadenylated mRNAs (but not polyadenylated mRNAs) are selectively decapped allowing their subsequent degradation by 5' to 3' exonucleolysis. The highly conserved heptameric Lsm1p-7p complex (made up of the seven Sm-like proteins, Lsm1p-Lsm7p) and its interacting partner Pat1p activate decapping by an unknown mechanism and localize with other decapping factors to the P-bodies in the cytoplasm. The Lsm1p-7p-Pat1p complex also protects the 3'-ends of mRNAs in vivo from trimming, presumably by binding to the 3'-ends. In order to determine the intrinsic RNA-binding properties of this complex, we have purified it from yeast and carried out in vitro analyses. Our studies revealed that it directly binds RNA at/near the 3'-end. Importantly, it possesses the intrinsic ability to distinguish between oligoadenylated and polyadenylated RNAs such that the former are bound with much higher affinity than the latter. These results indicate that the intrinsic RNA-binding characteristics of this complex form a critical determinant of its in vivo interactions and functions.
去帽是mRNA降解过程中的关键步骤。在所有真核生物中保守的5'至3'mRNA降解途径中，降解由多聚腺苷酸（poly(A)）缩短引发，寡聚腺苷酸化的mRNA（而非多聚腺苷酸化的mRNA）被选择性去帽，从而使其随后通过5'至3'核酸外切酶降解。高度保守的七聚体Lsm1p-7p复合物（由七个Sm样蛋白Lsm1p-Lsm7p组成）及其相互作用伴侣Pat1p通过未知机制激活去帽，并与其他去帽因子一起定位于细胞质中的P小体。Lsm1p-7p-Pat1p复合物在体内还可能通过与mRNA的3'末端结合来保护其3'末端不被修剪。为了确定该复合物的内在RNA结合特性，我们从酵母中纯化了它并进行了体外分析。我们的研究表明，它直接在3'末端或其附近结合RNA。重要的是，它具有区分寡聚腺苷酸化和多聚腺苷酸化RNA的内在能力，使得前者的结合亲和力远高于后者。这些结果表明，该复合物的内在RNA结合特性是其体内相互作用和功能的关键决定因素。
