Chou M Y, Rooke N, Turck C W, Black D L
Howard Hughes Medical Institute, University of California, Los Angeles, Los Angeles, California 90095, USA.
Mol Cell Biol. 1999 Jan;19(1):69-77. doi: 10.1128/MCB.19.1.69.
The regulation of the c-src N1 exon is mediated by an intronic splicing enhancer downstream of the N1 5' splice site. Previous experiments showed that a set of proteins assembles onto the most conserved core of this enhancer sequence specifically in neuronal WERI-1 cell extracts. The most prominent components of this enhancer complex are the proteins hnRNP F, KSRP, and an unidentified protein of 58 kDa (p58). This p58 protein was purified from the WERI-1 cell nuclear extract by ammonium sulfate precipitation, Mono Q chromatography, and immunoprecipitation with anti-Sm antibody Y12. Peptide sequence analysis of purified p58 protein identified it as hnRNP H. Immunoprecipitation of hnRNP H cross-linked to the N1 enhancer RNA, as well as gel mobility shift analysis of the enhancer complex in the presence of hnRNP H-specific antibodies, confirmed that hnRNP H is a protein component of the splicing enhancer complex. Immunoprecipitation of splicing intermediates from in vitro splicing reactions with anti-hnRNP H antibody indicated that hnRNP H remains bound to the src pre-mRNA after the assembly of spliceosome. Partial immunodepletion of hnRNP H from the nuclear extract partially inactivated the splicing of the N1 exon in vitro. This inhibition of splicing can be restored by the addition of recombinant hnRNP H, indicating that hnRNP H is an important factor for N1 splicing. Finally, in vitro binding assays demonstrate that hnRNP H can interact with the related protein hnRNP F, suggesting that hnRNPs H and F may exist as a heterodimer in a single enhancer complex. These two proteins presumably cooperate with each other and with other enhancer complex proteins to direct splicing to the N1 exon upstream.
c-src N1外显子的调控由N1 5'剪接位点下游的内含子剪接增强子介导。先前的实验表明，一组蛋白质特异性地在神经元WERI-1细胞提取物中组装到该增强子序列最保守的核心区域。该增强子复合物最主要的成分是蛋白质hnRNP F、KSRP和一种未鉴定的58 kDa蛋白质（p58）。通过硫酸铵沉淀、Mono Q色谱法以及用抗Sm抗体Y12进行免疫沉淀，从WERI-1细胞核提取物中纯化出了这种p58蛋白质。对纯化的p58蛋白质进行肽序列分析，确定其为hnRNP H。与N1增强子RNA交联的hnRNP H的免疫沉淀，以及在存在hnRNP H特异性抗体的情况下对增强子复合物的凝胶迁移率变动分析，证实hnRNP H是剪接增强子复合物的一种蛋白质成分。用抗hnRNP H抗体对体外剪接反应中的剪接中间体进行免疫沉淀表明，在剪接体组装后，hnRNP H仍与src前体mRNA结合。从核提取物中部分免疫去除hnRNP H会使体外N1外显子的剪接部分失活。通过添加重组hnRNP H可以恢复这种剪接抑制，表明hnRNP H是N1剪接的一个重要因子。最后，体外结合试验表明hnRNP H可以与相关蛋白质hnRNP F相互作用，提示hnRNPs H和F可能以异二聚体形式存在于单个增强子复合物中。这两种蛋白质大概相互协作，并与其他增强子复合物蛋白质共同引导剪接至上游的N1外显子。
