Ribonucleoprotein complex formation during pre-mRNA splicing in vitro
Ribonucleoprotein complex formation during pre-mRNA splicing in vitro
复制标题
体外前 mRNA 剪接过程中核糖核蛋白复合物的形成
DOI：
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复制
发表时间：
1986
影响因子：
5.3
通讯作者：
M. Green
中科院分区：
文献类型：
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作者：
A. Bindereif;M. Green
通过蔗糖梯度沉降分析，表征了在SP 6/β-珠蛋白前体mRNA的体外剪接过程中产生的前体mRNA和RNA加工产物的核糖核蛋白（RNP）结构。早期，在剪接反应的初始滞后期，前体mRNA不均匀地沉降，但在40 S和60 S RNP复合物中均检测到。缺少3'剪接位点共有序列的RNA底物不能组装到60 S RNP复合物中。两个剪接中间体，第一外显子RNA种类和RNA种类含有内含子和第二外显子在一个liscons配置（IVS 1-外显子2 RNA种类），被发现只在60 S RNP复合物。这两个剪接中间体在各种条件下共沉淀，表明它们包含在同一RNP复合物中。剪接反应的产物，准确剪接的RNA和切除的IVS 1干扰RNA物质，从60 S RNP复合物中释放出来，并在较小的RNP复合物中检测到。通过优先保护前mRNA分支点免受RNA酶A消化和保护IRNA物质的2 '-5'磷酸二酯键免受酶促脱支，证明了这些RNP复合物内的序列特异性RNA-因子相互作用。各种RNP复合物的进一步特点，并可以区分与抗Sm和抗（U1）RNP抗体的免疫沉淀。
The ribonucleoprotein (RNP) structures of the pre-mRNA and RNA processing products generated during in vitro splicing of an SP6/beta-globin pre-mRNA were characterized by sucrose gradient sedimentation analysis. Early, during the initial lag phase of the splicing reaction, the pre-mRNA sedimented heterogeneously but was detected in both 40S and 60S RNP complexes. An RNA substrate lacking a 3' splice site consensus sequence was not assembled into the 60S RNP complex. The two splicing intermediates, the first exon RNA species and an RNA species containing the intron and the second exon in a lariat configuration (IVS1-exon 2 RNA species), were found exclusively in a 60S RNP complex. These two splicing intermediates cosedimented under a variety of conditions, indicating that they are contained in the same RNP complex. The products of the splicing reaction, accurately spliced RNA and the excised IVS1 lariat RNA species, are released from the 60S RNP complex and detected in smaller RNP complexes. Sequence-specific RNA-factor interactions within these RNP complexes were evidenced by the preferential protection of the pre-mRNA branch point from RNase A digestion and protection of the 2'-5' phosphodiester bond of the lariat RNA species from enzymatic debranching. The various RNP complexes were further characterized and could be distinguished by immunoprecipitation with anti-Sm and anti-(U1)RNP antibodies.