PROVIDED. The objective of this proposal is to determine the structural basis of how the Stem-Loop Binding Protein (SLBP) functions to regulate histone mRNA processing and translation. Previous biochemical, and genetic studies have demonstrated that SLBP is the single most important trans-acting factor that plays an essential role in histone mRNA metabolism by forming a high affinity complex with a conserved stem-loop at the 3' end of replication-dependent histone mRNAs. The SLBP/RNA complex is important for the recruitment and assembly of multi-protein-RNA complexes that regulate pre-mRNA processing, translation, and degradation of histone mRNAs. Although a wealth of biochemical and genetic information exists for SLBP, the molecular basis for the SLBP-RNA interaction remains to be elucidated. We will take a multi-disciplinary approach to structurally characterize the RNA binding and processing domain (RPD) of SLBP both free and bound to histone mRNA, using NMR and mass spectrometry. The experiments described in this proposal will complement ongoing functional studies in my laboratory, provide mechanistic information relating to SLBP function, and test currently proposed models as to how SLBP regulates histone metabolism in vivo.
The specific aims of the proposal are 1) to determine the structural and dynamic properties of the Drosophila SLBP RNA binding and processing domain (dSLBP RPD) in the absence of RNA using high resolution NMR spectroscopy and FT-ICR H/D exchange mass spectrometry, 2) to determine the structural and dynamic properties of the dSLBP RPD stem-loop histone mRNA complex using high resolution NMR spectroscopy and FT-ICR H/D exchange mass spectrometry, 3) to characterize the biological and biochemical properties of sSLBP RPD mutants impaired in RNA binding and RNA processing, 4) to structurally characterize dSLBP RPD mutants by NMR Spectroscopy, and 5) to provide a structural basis for sequence specific recognition of the translation initiation factors AD2 and elF4G by SLBP. These studies will provide new information on structure/function relationships for this biological important protein and also lay the groundwork for long-term goals which are (i) to understand the molecular determinants for assembly of multi-protein complexes at the 3'end of histone mRNAs and (ii)to develop RNA binding domains with novel specificities that can be used as biosensors or reagents for cell biological studies.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM076660-04
Application #
7546526
Study Section
Macromolecular Structure and Function B Study Section (MSFB)
Program Officer
Preusch, Peter C
Project Start
2006-01-01
Project End
2010-12-31
Budget Start
2009-01-01
Budget End
2010-12-31
Support Year
4
Fiscal Year
2009
Total Cost
$240,433
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Biochemistry
Type
Schools of Medicine
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Thapar, Roopa (2015) Roles of Prolyl Isomerases in RNA-Mediated Gene Expression. Biomolecules 5:974-99
Thapar, Roopa (2015) Structural basis for regulation of RNA-binding proteins by phosphorylation. ACS Chem Biol 10:652-66
Thapar, Roopa (2015) Structure-specific nucleic acid recognition by L-motifs and their diverse roles in expression and regulation of the genome. Biochim Biophys Acta 1849:677-87
Thapar, Roopa (2014) Contribution of protein phosphorylation to binding-induced folding of the SLBP-histone mRNA complex probed by phosphorus-31 NMR. FEBS Open Bio 4:853-7
Krishnan, Nithya; Titus, Mark A; Thapar, Roopa (2014) The prolyl isomerase pin1 regulates mRNA levels of genes with short half-lives by targeting specific RNA binding proteins. PLoS One 9:e85427
Thapar, Roopa; Denmon, Andria P; Nikonowicz, Edward P (2014) Recognition modes of RNA tetraloops and tetraloop-like motifs by RNA-binding proteins. Wiley Interdiscip Rev RNA 5:49-67
Thapar, Roopa; Denmon, Andria P (2013) Signaling pathways that control mRNA turnover. Cell Signal 25:1699-710
Bansal, Nitin; Zhang, Minyou; Bhaskar, Aishwarya et al. (2013) Assembly of the SLIP1-SLBP complex on histone mRNA requires heterodimerization and sequential binding of SLBP followed by SLIP1. Biochemistry 52:520-36
Krishnan, Nithya; Lam, Tukiet T; Fritz, Andrew et al. (2012) The prolyl isomerase Pin1 targets stem-loop binding protein (SLBP) to dissociate the SLBP-histone mRNA complex linking histone mRNA decay with SLBP ubiquitination. Mol Cell Biol 32:4306-22
Zhang, Minyou; Lam, TuKiet T; Tonelli, Marco et al. (2012) Interaction of the histone mRNA hairpin with stem-loop binding protein (SLBP) and regulation of the SLBP-RNA complex by phosphorylation and proline isomerization. Biochemistry 51:3215-31

Showing the most recent 10 out of 11 publications