Ghetti A, Piñol-Roma S, Michael W M, Morandi C, Dreyfuss G
Universita di Verona, Facolta di Medicina e Chirurgia, Istituto di Scienze Biologiche, Italy.
Nucleic Acids Res. 1992 Jul 25;20(14):3671-8. doi: 10.1093/nar/20.14.3671.
Many hnRNP proteins and snRNPs interact with hnRNA in the nucleus of eukaryotic cells and affect the fate of hnRNA and its processing into mRNA. There are at least 20 abundant proteins in vertebrate cell hnRNP complexes and their structure and arrangement on specific hnRNAs is likely to be important for the processing of pre-mRNAs. hnRNP I, a basic protein of ca. 58,000 daltons by SDS-PAGE, is one of the abundant hnRNA-binding proteins. Monoclonal antibodies to hnRNP I were produced and full length cDNA clones for hnRNP I were isolated and sequenced. The sequence of hnRNP I (59,632 daltons and pI 9.86) demonstrates that it is identical to the previously described polypyrimidine tract-binding protein (PTB) and shows that it is highly related to hnRNP L. The sequences of these two proteins, I and L, define a new family of hnRNP proteins within the large superfamily of the RNP consensus RNA-binding proteins. Here we describe experiments which reveal new and unique properties on the association of hnRNP I/PTB with hnRNP complexes and on its cellular localization. Micrococcal nuclease digestions show that hnRNP I, along with hnRNP S and P, is released from hnRNP complexes by nuclease digestion more readily than most other hnRNP proteins. This nuclease hypersensitivity suggests that hnRNP I is bound to hnRNA regions that are particularly exposed in the complexes. Immunofluorescence microscopy shows that hnRNP I is found in the nucleoplasm but in addition high concentrations are detected in a discrete perinucleolar structure. Thus, the PTB is one of the major proteins that bind pre-mRNAs; it is bound to nuclease-hypersensitive regions of the hnRNA-protein complexes and shows a novel pattern of nuclear localization.
许多不均一核糖核蛋白（hnRNP）和小核核糖核蛋白（snRNP）在真核细胞的细胞核中与核内不均一RNA（hnRNA）相互作用，并影响hnRNA的命运及其加工成信使核糖核酸（mRNA）的过程。脊椎动物细胞的hnRNP复合物中至少有20种丰富的蛋白质，它们在特定hnRNA上的结构和排列可能对前体mRNA的加工很重要。hnRNP I是一种通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）显示分子量约为58,000道尔顿的碱性蛋白质，是丰富的hnRNA结合蛋白之一。制备了针对hnRNP I的单克隆抗体，并分离和测序了hnRNP I的全长互补DNA（cDNA）克隆。hnRNP I（59,632道尔顿，等电点9.86）的序列表明它与先前描述的多嘧啶序列结合蛋白（PTB）相同，并显示出它与hnRNP L高度相关。这两种蛋白质（I和L）的序列在RNP共有RNA结合蛋白的大型超家族中定义了一个新的hnRNP蛋白家族。在此，我们描述了一些实验，这些实验揭示了hnRNP I/PTB与hnRNP复合物结合及其细胞定位的新的独特特性。微球菌核酸酶消化表明，hnRNP I与hnRNP S和P一起，比大多数其他hnRNP蛋白更容易通过核酸酶消化从hnRNP复合物中释放出来。这种核酸酶超敏感性表明hnRNP I与复合物中特别暴露的hnRNA区域结合。免疫荧光显微镜显示，hnRNP I存在于核质中，但此外在离散的核仁周围结构中检测到高浓度。因此，PTB是结合前体mRNA的主要蛋白质之一；它与hnRNA-蛋白质复合物的核酸酶超敏区域结合，并显示出一种新的核定位模式。
