This research aims to advance our understanding of eukaryotic RNA processing and ribonucleoprotein organization, coming at the problem from both molecular biology and cell biology approaches. One objective is to further define a model RNA processing reaction, viz. the 3' processing of U2 RNA. The enzymatic activity that carries out this reaction has been partially characterized and will be purified and studied mechanistically in the proposed research. The U2 RNA 3' processing activity bands in cesium sulfate at a density characteristic of nucleoprotein, and the possibility that it contains an essential RNA cofactor, like RNase P and MRP RNase, will be investigated. A second objective is to define the interactions between spliceosome proteins, specifically the A1 and C hnRNP proteins, and the U1 and U2 small nuclear RNPs (snRNPs). The hypothesis under investigation is that these hnRNP proteins assemble in the spliceosome partly due to affinity for the pre-mRNA but also through specific binding interactions with the U1 and U2 snRNPs. A third objective is to investigate the nuclear localization of specific RNAs by microinjection of reporter group-tagged RNAs into mammalian cells. The nuclear localization of pre-mRNA and the export of mRNA to the cytoplasm will be studied. The traffic of small nuclear RNAs between nucleus and cytoplasm will also be investigated, as will the nuclear sites of transfer RNA processing. These in vivo studies of RNA traffic and localization will complement concurrent biochemical investigations of RNA processing and ribonucleoprotein. The significance of this research lies in enhancing our understanding of gene I readout in eukaryotic cells. The pre-mRNA splicing step of gene expression is ubiquitous and subject to regulation during embryonic development and adult life. Retroviral proteins such as Rev and Rex (in HIV and HTLV-I, respectively) are known to operate at the levels of pre-mRNA splicing and export, aspects of gene expression that will be studied in this work. Finally the experiments on RNA localization in vivo will provide a biological footing for the results obtained from in vitro experiments on RNA processing.
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