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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM064729-01
Application #
6417797
Study Section
Physiological Chemistry Study Section (PC)
Program Officer
Rhoades, Marcus M
Project Start
2002-04-01
Project End
2006-03-31
Budget Start
2002-04-01
Budget End
2003-03-31
Support Year
1
Fiscal Year
2002
Total Cost
$226,940
Indirect Cost
Name
Saint Louis University
Department
Pathology
Type
Schools of Medicine
DUNS #
City
Saint Louis
State
MO
Country
United States
Zip Code
63103
Eliceiri, George L (2006) The vertebrate E1/U17 small nucleolar ribonucleoprotein particle. J Cell Biochem 98:486-95
Smith, James L; Walton, Andrew H; Eliceiri, George L (2005) UV-crosslinking of E1 small nucleolar RNA to proteins in frog oocytes. J Cell Physiol 203:202-8