Investigation of multifunctional proteins that integrate packaging RNPs, RNA export, and translation

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Project Summary Eukaryotic gene expression depends on many steps in both the nucleus and cytoplasm. In the nucleus, large nascent RNAs, such as ribosomal RNAs and mRNAs, are manufactured and assembled into ribonucleoprotein particles (RNPs) that are exported to the cytoplasm for protein synthesis. The proteins involved in RNP assembly and export play critical roles throughout gene expression from co-transcriptional RNA processing to translation. AAA+ ATPases are a large and functionally diverse family of proteins that use the energy of ATP binding and hydrolysis to induce conformational changes and remodeling in various protein substrates. Loss of Elf1 (Elongation-Like Factor 1), an AAA+ superfamily ATPase implicated in RNA nuclear export, causes severe growth defects that can be mitigated by spontaneous suppressor mutations. We confirmed and isolated two suppressor mutants: an endonuclease, Cue2, and a large ribosomal subunit protein, Rpl2702. Elf1 co-purifies with these mutants, providing additional support for their functional connection. Using affinity purification and mass spectrometry analysis of Elf1 and Cue2, we have developed a molecular framework to systematically investigate their roles in the multifaceted regulation of posttranscriptional gene expression from RNP export to translation to ribosome-associated quality control. In addition, we have observed RNA export defects with loss of Elf1. The resulting nuclear retention of RNA destabilizes the genome, probably because abnormal DNA-RNA hybrids (R-loops) form. We hypothesize that Elf1 is associated with RNPs and functions in RNA/ribosome export and translation in different cellular compartments, antagonistically regulated by Cue2. To test this hypothesis, we will investigate the integrated roles of Elf1, Cue2, and Rpl2702 in maintaining genome stability (Aim 1), RNA and/or ribosome nuclear export (Aim 2), and translation elongation and ribosome-associated quality control (Aim 3). We will use traditional molecular biology and biochemical approaches, and also develop new genetic tools to analyze transcription-dependent hyper-recombination and examine various types of ribosome- associated mRNA decays. Our hypotheses and research strategy are based on a host of preliminary findings. Results are expected to advance the fields of RNA biology, protein synthesis, and genomic instability. Through implementation of research and student training, we will generate new quantitative genetic tools that will be appreciated in the fission yeast community.