RNA-BINDING BY SXL PROTEINS IN-VITRO AND IN-VIVO
RNA-BINDING BY SXL PROTEINS IN-VITRO AND IN-VIVO
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DOI：
10.1128/mcb.14.7.4975
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发表时间：
1994-07-01
影响因子：
5.3
通讯作者：
SCHEDL, P
中科院分区：
文献类型：
--
作者：
SAMUELS, ME;BOPP, D;SCHEDL, P
已经提出Sri通过直接与它们的前mRNA相互作用来调节特定靶基因的剪接。因此，我们研究了RNA结合特性的Sri蛋白在体外和体内。用纯化的重组MBP-Sxl融合蛋白进行的凝胶位移和W交联测定证明了与含有聚（U）区的RNA的优先结合，并且蛋白质足迹遍布聚（U）区。该蛋白似乎不识别分支点或AG二核苷酸序列，但多聚（U）束5'端的腺苷残基增强结合数倍。MBP-Sxl在反式调节受体位点RNA上形成两个移位复合物;双移位形式可能已通过蛋白质-蛋白质相互作用稳定。与其在前mRNA加工中的作用一致，在核提取物中，在大的核糖核蛋白（RNP）复合物中发现了Sri，其沉降速度明显快于大体积异质核RNP和小核RNP。多线染色体的抗Sxl染色显示，在许多染色体位置的Sri蛋白，其中是Sri基因座本身。在转录诱导后，Sri蛋白也可以靶向携带含有来自Sd基因的剪接调控序列的转基因的新染色体位点。长时间热休克后，所有的Sri蛋白被限制在热诱导的puff在hs 93 D位点。与此相反，一个假定的小核RNP蛋白，观察到在几个热休克喷后休克。
Sri has been proposed to regulate splicing of specific target genes by directly interacting with their pre-mRNAs. We have therefore examined the RNA-binding properties of Sri protein in vitro and in vivo. Gel shift and W cross-linking assays with a purified recombinant MBP-Sxl fusion protein demonstrated preferential binding to RNAs containing poly(U) tracts, and the protein footprinted over the poly(U) region. The protein did not appear to recognize either branch point or AG dinucleotide sequences, but an adenosine residue at the 5' end of the poly(U) tract enhanced binding severalfold. MBP-Sxl formed two shifted complexes on a tra regulated acceptor site RNA; the doubly shifted form may have been stabilized by protein-protein interactions. Consistent with its proposed role in pre-mRNA processing, in nuclear extracts Sri was found in large ribonucleoprotein (RNP) complexes which sedimented significantly faster than bulk heterogeneous nuclear RNP and small nuclear RNPs. Anti-Sxl staining of polytene chromosomes showed Sri protein at a number of chromosomal locations, among which was the Sri Locus itself. Sri protein could also be targeted to a new chromosomal site carrying a transgene containing splicing regulatory sequences from the Sd gene, following transcriptional induction. After prolonged heat shock, all Sri protein was restricted to the heat-induced puff at the hs93D locus. In contrast, a presumptive small nuclear RNP protein was observed at several heat shock puffs following shock.