Intron sequences and the length of the downstream second exon affect the binding of hnRNP C proteins in an in vitro splicing reaction.
Intron sequences and the length of the downstream second exon affect the binding of hnRNP C proteins in an in vitro splicing reaction.
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内含子序列和下游第二外显子的长度影响体外剪接反应中 hnRNP C 蛋白的结合。
DOI：
10.1093/nar/16.11.4995
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发表时间：
1988
影响因子：
14.9
通讯作者：
Goldenberg,CJ
中科院分区：
文献类型：
--
作者：
Goswami,P;Goldenberg,CJ
通过紫外交联实验分析了蛋白体外剪接反应中与前体mrna直接接触的蛋白。检测到6个主要蛋白（120、55、44、42、39和38 KD）和3个次要多肽（84、72和63 KD）。优势蛋白44,42 KD属于hnRNP C蛋白类，因为它们是由针对hnRNP C蛋白的单克隆抗体免疫沉淀的。在没有Mg2+、ATP或在剪接反应中使用缺乏内含子的RNA作为底物时，未检测到交联蛋白。研究了外显子序列对光交联蛋白结合效率的影响。与“全长”前mrna相比，含有24个核苷酸的β-珠蛋白或107个核苷酸的小鼠胰岛素第二外显子的转录本产生的交联蛋白数量减少。β-珠蛋白前mrna第一外显子内的序列不影响这些蛋白的结合效率。交联蛋白对截断的β-珠蛋白或小鼠胰岛素前mrna的结合效率降低与体外剪接效率降低相关。在β-珠蛋白第二外显子中替换不相关的序列可以恢复交联蛋白的结合，这表明第二外显子的长度而不是特定序列与这些蛋白的结合效率有关。分离并测序了与hnRNP - C交联的SP6/小鼠胰岛素寡核苷酸。一个17-mer位于第二外显子（位于3 ‘剪接位点下游134个核苷酸），一个14-mer位于内含子区域（位于5 ’剪接位点下游25个核苷酸）。与hnRNP - C蛋白交联的β-珠蛋白寡核苷酸在第二外显子（3 ‘剪接位点下游28个核苷酸）和第一个外显子（pre-mRNA 5 ’端下游81个核苷酸）分别为13个mer和8个mer。我们的研究结果表明，hnRNP C蛋白与位于mrna前体不同区域的寡核苷酸相互作用。然而，这些蛋白质的结合效率取决于第二外显子的长度和内含子序列的存在（二级和/或三级mrna前结构）。
The proteins that are in direct contact with the pre-mRNA in anin vitrosplicing reaction were analyzed by UV cross-linking experiments. Six major proteins (120, 55, 44, 42, 39 and 38 KD) and three minor polypeptides (84, 72 and 63 KD) were detected. The predominant proteins 44, 42 KD belong to the class of hnRNP C proteins since they were immunoprecipitated by monoclonal antibodies directed against hnRNP C proteins. The cross-linked proteins were not detected in the absence of Mg2+, ATP or when RNA lacking introns were used as substrates in the splicing reactions. The effect of exon sequences on the binding efficiency for the photocrosslinked proteins was investigated. Transcripts containing a second exon of 24 nucleotides for theβ-globin or 107 nucleotides for the mouse insulin, yielded a reduced amount of cross-linked proteins when compared with “full length” pre-mRNAs. Sequences within the first exon of theβ-globin pre-mRNA did not affect the binding efficiency of these proteins. The reduced binding efficiency of the cross-linked proteins for the truncatedβ-globin or mouse insulin pre-mRNAs correlated with the lower efficiency forinvitrosplicing. Substitutions with unrelated sequences in theβ-globin second exon restore the binding of the cross-linked proteins indicating that the length of the second exon and not specific sequences are relevant for the binding efficiency of these proteins. The SP6/mouse insulin oligonu-cleotides cross-linked to the hnRNP C proteins were isolated and sequenced. A 17-mer was located in the second exon (134 nucleotides downstream from the 3′ splice site) and a 14-mer in the intron region (25 nucleotides downstream the 5′ splice site). Theβ-globin oligonucleotides cross-linked to the hnRNP C proteins were a 13-mer in the second exon (28 nucleotides downstream the 3′ splice site) and an 8-mer in the first exon (81 nucleotides downstream the 5′ end of the pre-mRNA). Our results indicate that the hnRNP C proteins interact with those oligonucleotides located in different regions of the pre-mRNA. The binding efficiency of those proteins, however, depends on the length of the second exon and the presence of intron sequences (secondary and/or tertiary pre-mRNA structure).