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首页> 外文期刊>Molecular and Cellular Biology >hnRNP F Influences Binding of a 64-Kilodalton Subunit of Cleavage Stimulation Factor to mRNA Precursors in Mouse B Cells
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hnRNP F Influences Binding of a 64-Kilodalton Subunit of Cleavage Stimulation Factor to mRNA Precursors in Mouse B Cells

机译：hnRNP F影响卵裂刺激因子的64千达尔顿亚基与小鼠B细胞中mRNA前体的结合。

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Previous studies on the regulation of polyadenylation of the immunoglobulin (Ig) heavy-chain pre-mRNA argued fortrans-acting modifiers of the cleavage-polyadenylation reaction operating differentially during B-cell developmental stages. Using four complementary approaches, we demonstrate that a change in the level of hnRNP F is an important determinant in the regulated use of alternative polyadenylation sites between memory and plasma stage B cells. First, by Western analyses of cellular proteins, the ratio of hnRNP F to H or H′ was found to be higher in memory B cells than in plasma cells. In memory B cells the activity of CstF-64 binding to pre-mRNA, but not its amount, was reduced. Second, examination of the complexes formed on input pre-mRNA in nuclear extracts revealed large assemblages containing hnRNP H, H′, and F but deficient in CstF-64 in memory B-cell extracts but not in plasma cells. Formation of these large complexes is dependent on the region downstream of the AAUAAA in pre-mRNA, suggesting that CstF-64 and the hnRNPs compete for a similar region. Third, using a recombinant protein we showed that hnRNP F could bind to the region downstream of a poly(A) site, block CstF-64 association with RNA, and inhibit the cleavage reaction. Fourth, overexpression of recombinant hnRNP F in plasma cells resulted in a decrease in the endogenous Ig heavy-chain mRNA secretory form-to-membrane ratio. These results demonstrate that mammalian hnRNP F can act as a negative regulator in the pre-mRNA cleavage reaction and that increased expression of F in memory B cells contributes to the suppression of the Ig heavy-chain secretory poly(A) site.
机译：先前关于免疫球蛋白（Ig）重链前mRNA的聚腺苷酸化调控的研究认为，在B细胞发育阶段，裂解-聚腺苷酸化反应的反式作用修饰剂不同。使用四种互补的方法，我们证明hnRNP F水平的变化是在记忆和血浆B期细胞之间替代性多腺苷酸化位点的调节使用中的重要决定因素。首先，通过细胞蛋白的Western分析，发现记忆B细胞中hnRNP F与H或H'的比例高于浆细胞。在记忆B细胞中，CstF-64与pre-mRNA结合的活性降低了，但没有降低。其次，检查核提取物中输入前mRNA上形成的复合物，发现含有hnRNP H，H'和F的大集合，但记忆B细胞提取物中CstF-64缺乏，但浆细胞中缺乏。这些大复合物的形成取决于前mRNA中AAUAAA的下游区域，表明CstF-64和hnRNP竞争相似区域。第三，使用重组蛋白，我们显示hnRNP F可以结合到poly（A）位点下游的区域，阻断CstF-64与RNA的结合，并抑制裂解反应。第四，重组hnRNP F在浆细胞中的过表达导致内源性Ig重链mRNA分泌形式与膜的比率降低。这些结果表明，哺乳动物的hnRNP F可以作为前mRNA裂解反应中的负调节剂，并且记忆B细胞中F的表达增加有助于抑制Ig重链分泌性poly（A）位点。

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