FUNCTIONS OF HNRNP A1 AND PTB IN THE CYTOPLASM

有几种RNA结合蛋白，包括异质性核糖核蛋白(HnRNP A1)和多嘧啶结合蛋白(PTB/hnRNP 1)，最初被鉴定为核蛋白，但后来发现在细胞核和细胞质之间穿梭。这些蛋白在RNA剪接中发挥重要作用，与包括小鼠肝炎病毒(MHV)在内的几种RNA病毒的RNA相互作用，并可能参与病毒RNA在细胞质中的转录和翻译。有人假设MHV RNA的不连续转录可能涉及RNA剪接，这一功能以前从未被认为存在于细胞质中。作为核剪接因子，hnRNP A1和PTB在细胞质中可能具有相似的功能。因此，本提案的目的是以MHV为模型系统，研究这些蛋白在细胞质中剪接中的潜在作用。我将首先通过在体外转录系统中免疫耗尽hnRNP A1和PTB蛋白来确定这些蛋白在MHV RNA转录中的功能重要性。然后，我将把我的研究重点放在细胞质RNA剪接作为hnRNP A1和PTB调节MHV RNA转录的一种机制上。最后，我还将研究PTB和hnRNP A1以及PTB对MHV RNA转录的影响。最后，我还将研究PTB和hnRNP A1对MHV蛋白翻译的影响。随着上述研究的完成，我希望能够确定hnRNP A1和PTB在MHV感染细胞胞浆中的功能，这可能反映其正常的细胞质功能。

DESCRIPTION There are several RNA-binding proteins, including heterogeneous nuclear ribonucleoprotein (hnRNP A1) and polypyrimidine tract-binding protein (PTB/hnRNP 1), that were initially characterized as nuclear proteins but later found to shuttle between the nucleus and the cytoplasm. These proteins play important roles in RNA splicing shown to interact with RNAs of several RNA viruses including mouse hepatitis virus (MHV), and may possibly be involved in viral RNA transcription and translation in the cytoplasm. It has been hypothesized that the discontinuous transcription of MHV RNA may involve RNA splicing, a function that has never been thought to be present in the cytoplasm before. As nuclear splicing factors, hnRNP A1 and PTB may possess similar functions in the cytoplasm. Thus, the purpose of this proposal is to study the potential roles in splicing of these proteins in the cytoplasm using MHV as a model systems. I will first establish the functional importance of hnRNP A1 and PTB in MHV RNA transcription by immunodepletion of these proteins in an in vitro transcription system. I will then focus my study on the possible cytoplasmic RNA splicing as a mechanism by which hnRNP A1 and PTB regulate MHV RNA transcription. Finally, I will also investigate the effects of PTB and hnRNP A1 and PTB regulate MHV RNA transcription. Finally, I will also investigate the effects of PTB and hnRNP A1 on MHV protein translation. By the completion of the proposed studies, I hope to characterize the functions of hnRNP A1 and PTB in the cytoplasm of MHV-infected cells, which may reflect their normal cytoplasmic functions.