Ribosome Biogenesis: Small Nucleolar RNPs

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DESCRIPTION (provided by applicant): Ribosome biogenesis is a fundamental process in the cell but there are major gaps in what is known about its mechanism. We are excited about the finding that specific depletion ofE1/U17 small nucleolar RNA (snoRNA) produces a unique pre-rRNA cleavage processing phenotype that is specifically reversed by U17 RNA in frog oocytes. U17 RNA is essential for 18S rRNA formation. U17 RNA interacts directly (psoralen-crosslinks) with unexpected sites in ribosomal RNA precursor (pre-rRNA) in vivo. Long-term goal: To contribute to our understanding of the roles of small nucleolar ribonucleoprotein particles (snoRNPs) in ribosome formation, focusing first on the U 17 snoRNP. For brevity, "U 17 RNP role A" refers in this text to the step(s) in 18S rRNA formation that is blocked in U17-depleted cells, this block being reversed by injection of wild-type U17 RNA. "RP element" refers here to a U17 RNA segment whose substitution mutation interferes with "U17 RNP role A." SpeqfIc Aims: #1) Which is the U1 7-dependent pre-rRNA processing step(s) in 18S rRNA formation?: Mapping of termini and pulse-chase labeling of "20S" pre-rRNA in U17-depleted frog oocytes. #2) Which are ihe RP elements of U1 7 RNA?: U17 RNA mutation analysis in vivo. #3) Identification ofproteins that interact specifically with U17 RNA. Which U17 RNA RP element (identified in Aim 2) interacts specifically with which protein?: Affinity chromatography purification of the U1 7 snoRNP using antisense, biotinylated, 2 -O-methy1 oligoribonucleotides; protein identification by MALDI and ESI mass spectrometry, or partial microsequencing; U17 RNA footprinting; RNA competition electrophoretic mobility shift assays and RNA-protein UV crosslinking, both in vivo and/or in vitro. #4) Which U17 RNA -protein specific interactions (identify led in Aim 3) are involved in U17 RNP role A?: Point mutations in and next to a U17 RNA RP element: do they have tightly coupled effects on U17 RNP role A and a U17 RNA-protein interaction? We will test models ofUl 7 RNP role A, focused on these U17 RNA RP elements and their interactions. Most of the experiments in vivo will be done by injection into frog oocytes.