Yang Xiao-cui, Torres Matthew P, Marzluff William F, Dominski Zbigniew
Program in Molecular Biology and Biotechnology, University of North Carolina, Chapel Hill, NC 27599, USA.
Mol Cell Biol. 2009 Aug;29(15):4045-56. doi: 10.1128/MCB.00296-09. Epub 2009 May 26.
Cleavage of histone pre-mRNAs at the 3' end is guided by the U7 snRNP, which is a component of a larger 3'-end processing complex. To identify other components of this complex, we isolated proteins that stably associate with a fragment of histone pre-mRNA containing all necessary processing elements and a biotin affinity tag at the 5' end. Among the isolated proteins, we identified three well-characterized processing factors: the stem-loop binding protein (SLBP), which interacts with the stem-loop structure upstream of the cleavage site, and both Lsm11 and SmB, which are components of the U7-specific Sm ring. We also identified 3'hExo/Eri-1, a multifunctional 3' exonuclease that is known to trim the 3' end of 5.8S rRNA. 3'hExo primarily binds to the downstream portion of the stem-loop structure in mature histone mRNA, with the upstream portion being occupied by SLBP. The two proteins bind their respective RNA sites in a cooperative manner, and 3'hExo can recruit SLBP to a mutant stem-loop that itself does not interact with SLBP. UV-cross-linking studies used to characterize interactions within the processing complex demonstrated that 3'hExo also interacts in a U7-dependent manner with unprocessed histone pre-mRNA. However, this interaction is not required for the cleavage reaction. The region between the cleavage site and the U7-binding site interacts with three low-molecular-weight proteins, which were identified as components of the U7-specific Sm core: SmB, SmD3, and Lsm10. These proteins likely rigidify the substrate and function as the molecular ruler in determining the site of cleavage.
组蛋白前体mRNA在3'端的切割由U7 snRNP引导，U7 snRNP是一个更大的3'端加工复合体的组成部分。为了鉴定该复合体的其他成分，我们分离了与一段组蛋白前体mRNA片段稳定结合的蛋白质，该片段包含所有必要的加工元件且在5'端带有生物素亲和标签。在分离出的蛋白质中，我们鉴定出了三个特征明确的加工因子：茎环结合蛋白（SLBP），它与切割位点上游的茎环结构相互作用；以及Lsm11和SmB，它们是U7特异性Sm环的组成部分。我们还鉴定出了3'hExo/Eri-1，一种多功能3'核酸外切酶，已知它能修剪5.8S rRNA的3'端。3'hExo主要结合成熟组蛋白mRNA中茎环结构的下游部分，上游部分则被SLBP占据。这两种蛋白质以协同方式结合各自的RNA位点，并且3'hExo可以将SLBP招募到一个自身不与SLBP相互作用的突变茎环上。用于表征加工复合体内相互作用的紫外线交联研究表明，3'hExo还以U7依赖的方式与未加工的组蛋白前体mRNA相互作用。然而，这种相互作用对于切割反应并非必需。切割位点与U7结合位点之间的区域与三种低分子量蛋白质相互作用，这些蛋白质被鉴定为U7特异性Sm核心的组成部分：SmB、SmD3和Lsm10。这些蛋白质可能会使底物变硬，并在确定切割位点时起到分子标尺的作用。
