In vivo characterization of the Drosophila mRNA 3' end processing core cleavage complex.
In vivo characterization of the Drosophila mRNA 3' end processing core cleavage complex.
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DOI：
10.1261/rna.049551.115
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发表时间：
2015-08
期刊：
影响因子：
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通讯作者：
Steiniger M
中科院分区：
文献类型：
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作者：
Michalski D;Steiniger M
由CPSF73、CPSF100和Symplekin组成的核心切割复合体（CCC）是所有后生动物前mrna共转录3 '端加工所必需的，但对该复合体内部的分子相互作用知之甚少。CCC是两个不同复合物的组成部分，即切割/聚腺苷化复合物和处理非聚腺苷化组蛋白前mrna的复合物。果蝇培养细胞中CCC因子的rnai缺失导致CCC对组蛋白mrna加工活性降低，导致读通转录。相比之下，组蛋白mRNA加工所需的rnai缺失允许使用下游隐聚腺苷化信号来产生聚腺苷化的组蛋白mRNA。我们使用稳定表达标记CCC成分的Dmel-2组织培养细胞来确定simplekin的272-1080氨基酸和CPSF73和CPSF100的c端大约200个氨基酸是体内有效形成CCC所必需的。进一步的实验表明，CPSF100的c端241个氨基酸对组蛋白mRNA的加工是足够的，表明CPSF100的前524个氨基酸对CCC的形成和组蛋白mRNA 3 '端加工都是必不可少的。含有缺少前271个氨基酸的Symplekin缺失的CCCs导致组蛋白mRNA 3 ‘端加工中下游聚adenylation位点的使用急剧增加，类似于rnai -组蛋白特异性3 ’端加工因子FLASH， SLBP和U7 snRNA的缺失。我们提出了一个模型，其中CCC的形成是由CPSF73、CPSF100和辛普金c端介导的，辛普金的n端区域促进组蛋白mrna的共转录3 '端加工。
A core cleavage complex (CCC) consisting of CPSF73, CPSF100, and Symplekin is required for cotranscriptional 3′ end processing of all metazoan pre-mRNAs, yet little is known about the in vivo molecular interactions within this complex. The CCC is a component of two distinct complexes, the cleavage/polyadenylation complex and the complex that processes nonpolyadenylated histone pre-mRNAs. RNAi-depletion of CCC factors in Drosophila culture cells causes reduction of CCC processing activity on histone mRNAs, resulting in read through transcription. In contrast, RNAi-depletion of factors only required for histone mRNA processing allows use of downstream cryptic polyadenylation signals to produce polyadenylated histone mRNAs. We used Dmel-2 tissue culture cells stably expressing tagged CCC components to determine that amino acids 272–1080 of Symplekin and the C-terminal approximately 200 amino acids of both CPSF73 and CPSF100 are required for efficient CCC formation in vivo. Additional experiments reveal that the C-terminal 241 amino acids of CPSF100 are sufficient for histone mRNA processing indicating that the first 524 amino acids of CPSF100 are dispensable for both CCC formation and histone mRNA 3′ end processing. CCCs containing deletions of Symplekin lacking the first 271 amino acids resulted in dramatic increased use of downstream polyadenylation sites for histone mRNA 3′ end processing similar to RNAi-depletion of histone-specific 3′ end processing factors FLASH, SLBP, and U7 snRNA. We propose a model in which CCC formation is mediated by CPSF73, CPSF100, and Symplekin C-termini, and the N-terminal region of Symplekin facilitates cotranscriptional 3′ end processing of histone mRNAs.