IDENTIFICATION, MOLECULAR-CLONING, EXPRESSION AND CHROMOSOME MAPPING OF A FAMILY OF TRANSFORMATION UP-REGULATED HNRNP-K PROTEINS DERIVED BY ALTERNATIVE SPLICING
IDENTIFICATION, MOLECULAR-CLONING, EXPRESSION AND CHROMOSOME MAPPING OF A FAMILY OF TRANSFORMATION UP-REGULATED HNRNP-K PROTEINS DERIVED BY ALTERNATIVE SPLICING
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DOI：
10.1006/jmbi.1994.1116
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发表时间：
1994-02-11
影响因子：
5.6
通讯作者：
CELIS, JE
中科院分区：
文献类型：
--
作者：
DEJGAARD, K;LEFFERS, H;CELIS, JE
酸性核蛋白（Mr 介于 64,000 和 66,000 之间；pI 4·9 到 5·5）在转化细胞中高度上调，属于 hnRNP-K 家族，已使用单克隆抗体 (mAB B4B6) 进行了鉴定，该抗体可区分静止和增殖的人类角质形成细胞。该家族由四种主要蛋白（hnRNPs-KA、B、C 和 D）及其修饰形式组成，在静止和增殖的正常角质形成细胞中以相似的总体水平存在，尽管在一些个体变体的水平中观察到明显差异。使用 mAB B4B6 对增殖的正常角质形成细胞进行免疫荧光染色显示，约 40% 的角质形成细胞（主要对应于 G1 期和一半 S 期细胞）与抗体发生反应，呈现出排除核仁的点状核质染色。只有 3% 至 4% 的静止角质形成细胞与抗体发生反应，而猿猴病毒 40 (SV40) 转化的角质形成细胞 (K14) 在整个细胞周期中持续染色。使用 mAB B4B6 作为探针，我们克隆了编码该家族一个成员 (hnRNP-K B) 的 cDNA，并将其用于筛选其他家族成员。阳性克隆的测序揭示了四种不同的 cDNA，所有这些都是由映射到 9 号染色体的基因的共同初级转录物的选择性剪接产生的。痘苗病毒系统中 cDNA 的表达证实了它们的身份为 hnRNP-K A、B、C 和 D，并显示它们的修饰形式被磷酸化。所有四种 hnRNP 在 NorthWestern 印迹上均与聚 (rC) 结合，尽管磷酸化形式的酸性更强，但其水平却大大降低。 hnRNP-K 与含有富含胞苷序列的转录物的前 mRNA 代谢有关，我们的结果表明其在细胞周期进程中发挥作用。
Acidic nuclear proteins (Mrbetween 64,000 and 66,000; pI 4·9 to 5·5) that are highly upregulated in transformed cells and that belong to the hnRNP-K family have been identified using a monoclonal antibody (mAB B4B6) that distinguish between quiescent and proliferating human keratinocytes. The family, which is composed of four major proteins (hnRNPs-K A, B, C and D) and their modified forms, is present in similar overall levels in quiescent and proliferating normal keratinocytes although clear differences were observed in the levels of some of the individual variants. Immunofluorescence staining of proliferating normal keratinocytes with mAB B4B6 showed that about 40% of the keratinocytes, corresponding mainly to G1and to half of the cells in S-phase, reacted with the antibody depicting a dotted, nucleoplasmic staining that excluded the nucleolus. Only 3 to 4% of the quiescent keratinocytes reacted with the antibody while simian virus 40 (SV40) transformed keratinocytes (K14) stained constitutively throughout the cell cycle.Using mAB B4B6 as a probe we cloned a cDNA coding for one member of the family (hnRNP-K B) and this was used to screen for additional family members. Sequencing of the positive clones revealed four different cDNAs, all resulting from alternative splicing of a common primary transcript of a gene that mapped to chromosome 9. Expression of the cDNAs in the vaccinia virus system confirmed their identity as hnRNPs-K A, B, C and D and showed that their modified forms are phosphorylated. All four hnRNPs bound poly(rC) on NorthWestern blots, although the more acidic of the phosphorylated forms, did so at a much reduced level. hnRNP-K has been implicated in pre-mRNA metabolism of transcripts containing cytidine-rich sequences and our results point towards a role during cell cycle progression.