Luz Ambrósio Daniela, Lee Ju Huck, Panigrahi Aswini K, Nguyen Tu Ngoc, Cicarelli Regina Maria Barretto, Günzl Arthur
Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, Connecticut 06030-3301, USA.
Eukaryot Cell. 2009 Jul;8(7):990-1000. doi: 10.1128/EC.00075-09. Epub 2009 May 8.
In trypanosomatid parasites, spliced leader (SL) trans splicing is an essential nuclear mRNA maturation step which caps mRNAs posttranscriptionally and, in conjunction with polyadenylation, resolves individual mRNAs from polycistronic precursors. While all trypanosomatid mRNAs are trans spliced, intron removal by cis splicing is extremely rare and predicted to occur in only four pre-mRNAs. trans- and cis-splicing reactions are carried out by the spliceosome, which consists of U-rich small nuclear ribonucleoprotein particles (U snRNPs) and of non-snRNP factors. Mammalian and yeast spliceosome complexes are well characterized and found to be associated with up to 170 proteins. Despite the central importance of trans splicing in trypanosomatid gene expression, only the core RNP proteins and a few snRNP-specific proteins are known. To characterize the trypanosome spliceosomal protein repertoire, we conducted a proteomic analysis by tagging and tandem affinity-purifying the canonical core RNP protein SmD1 in Trypanosoma brucei and by identifying copurified proteins by mass spectrometry. The set of 47 identified proteins harbored nearly all spliceosomal snRNP factors characterized in trypanosomes thus far and 21 proteins lacking a specific annotation. A bioinformatic analysis combined with protein pull-down assays and immunofluorescence microscopy identified 10 divergent orthologues of known splicing factors, including the missing U1-specific protein U1A. In addition, a novel U5-specific, and, as we show, an essential splicing factor was identified that shares a short, highly conserved N-terminal domain with the yeast protein Cwc21p and was thus tentatively named U5-Cwc21. Together, these data strongly indicate that most of the identified proteins are components of the spliceosome.
在锥虫寄生虫中，剪接前导序列（SL）反式剪接是细胞核内mRNA成熟的关键步骤，该步骤在转录后为mRNA加上帽结构，并与多聚腺苷酸化一起，将单个mRNA从多顺反子前体中分离出来。虽然所有锥虫的mRNA都进行反式剪接，但顺式剪接去除内含子的情况极为罕见，预计仅在四种前体mRNA中发生。反式剪接和顺式剪接反应均由剪接体完成，剪接体由富含尿嘧啶的小核核糖核蛋白颗粒（U snRNP）和非snRNP因子组成。哺乳动物和酵母的剪接体复合物已得到充分表征，发现与多达170种蛋白质相关。尽管反式剪接在锥虫基因表达中至关重要，但目前仅知道核心RNP蛋白和少数snRNP特异性蛋白。为了表征锥虫剪接体的蛋白质组成，我们通过对布氏锥虫中典型的核心RNP蛋白SmD1进行标记和串联亲和纯化，并通过质谱鉴定共纯化的蛋白质，开展了蛋白质组学分析。鉴定出的47种蛋白质几乎包含了迄今为止在锥虫中表征的所有剪接体snRNP因子，以及21种缺乏特定注释的蛋白质。生物信息学分析结合蛋白质下拉实验和免疫荧光显微镜检查，鉴定出10种已知剪接因子的不同直系同源物，包括缺失的U1特异性蛋白U1A。此外，还鉴定出一种新的U5特异性且必不可少的剪接因子，该因子与酵母蛋白Cwc21p共享一个短的、高度保守的N端结构域，因此暂命名为U5-Cwc21。总之，这些数据有力地表明，大多数鉴定出的蛋白质是剪接体的组成成分。
