Chen Yen-I G, Moore Roger E, Ge Helen Y, Young Mary K, Lee Terry D, Stevens Scott W
Graduate program in Microbiology, City of Hope Beckman Research Institute, Duarte, CA 91010, USA.
Nucleic Acids Res. 2007;35(12):3928-44. doi: 10.1093/nar/gkm347. Epub 2007 May 30.
Previous compositional studies of pre-mRNA processing complexes have been performed in vitro on synthetic pre-mRNAs containing a single intron. To provide a more comprehensive list of polypeptides associated with the pre-mRNA splicing apparatus, we have determined the composition of the bulk pre-mRNA processing machinery in living cells. We purified endogenous nuclear pre-mRNA processing complexes from human and chicken cells comprising the massive (>200S) supraspliceosomes (a.k.a. polyspliceosomes). As expected, RNA components include a heterogeneous mixture of pre-mRNAs and the five spliceosomal snRNAs. In addition to known pre-mRNA splicing factors, 5' end binding factors, 3' end processing factors, mRNA export factors, hnRNPs and other RNA binding proteins, the protein components identified by mass spectrometry include RNA adenosine deaminases and several novel factors. Intriguingly, our purified supraspliceosomes also contain a number of structural proteins, nucleoporins, chromatin remodeling factors and several novel proteins that were absent from splicing complexes assembled in vitro. These in vivo analyses bring the total number of factors associated with pre-mRNA to well over 300, and represent the most comprehensive analysis of the pre-mRNA processing machinery to date.
以往对前体mRNA加工复合体的组成研究是在体外对含有单个内含子的合成前体mRNA进行的。为了提供与前体mRNA剪接装置相关的更全面的多肽列表，我们确定了活细胞中大量前体mRNA加工机制的组成。我们从人和鸡细胞中纯化了内源性核前体mRNA加工复合体，这些复合体包含大量（>200S）的超级剪接体（也称为多剪接体）。正如预期的那样，RNA成分包括前体mRNA的异质混合物和五种剪接体snRNA。除了已知的前体mRNA剪接因子、5'端结合因子、3'端加工因子、mRNA输出因子、hnRNP和其他RNA结合蛋白外，通过质谱鉴定的蛋白质成分还包括RNA腺苷脱氨酶和几种新因子。有趣的是，我们纯化的超级剪接体还含有许多结构蛋白、核孔蛋白、染色质重塑因子以及一些在体外组装的剪接复合体中不存在的新蛋白质。这些体内分析使与前体mRNA相关的因子总数超过300个，代表了迄今为止对前体mRNA加工机制最全面的分析。
