The tRNA synthetases are most prominently known for their aminoacylation of tRNA during protein synthesis. However, these enzymes play a number of diverse roles that are also essential to cells. One of these examples is tRNA synthetase-dependent RNA splicing. A novel ternary splicing complex has been identified in yeast where a group I intron called b14 is aided by two proteins, a maturase and a leucyl (Leu)-tRNA synthetase. Tyr-tRNA synthetase (CYT- 18) is the only other tRNA synthetase that has been found to facilitate RNA splicing. However, our preliminary data suggests that the molecular mechanisms by which these two related enzymes promote ribozyme self-splicing activity are quite distinct. Moreover, the Leu-tRNA synthetase-dependent ribozyme is the first example of a two protein: one RNA splicing complex and presents an intriguing elementary model to not only investigate RNA-protein interactions, but also potentially protein-protein interactions which might aid a ribozyme self-splicing reaction. We propose to investigate the Leu-tRNA synthetase-dependent ribozyme splicing reaction and identify discrete determinants that confer this protein synthesis enzyme's unique role in RNA splicing. We have established three-hybrid and RNA-dependent two-hybrid models that show for the first time that the Leu-tRNA synthetase and bI4 maturase can directly, independently, and simultaneously interact with the b14 intron. We have also developed a yeast nucleus-based assay via RT-PCR methods demonstrating that at least one of these protein partners must be bound to faciliate ribozyme splicing activity. We propose to map and identify specific interactions between Leu-tRNA synthetase and the b14 intron that dictate complex assembly and RNA splicing activity. Completion of the proposed specific aims will delineate the undefined splicing role of Leu-tRNA synthetase at the molecular level and provide insight into its inherent determinants that solicited cellular recruitment of this secondary, but also essential activity. Characterization of this simple ternary model offers an important stepping stone in understanding how catalytic ribozymes evolved to more complicated RNP complexes to enhance function and maintain essential biological processes. Moreover, since protein-dependent RNA splicing is critical to the health of human cells as well as to the life cycles of viral, protozoa, and fungal pathogens, elucidation of important molecular determinants required for RNA processing may identify new drug targets for the treatment of infectious disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM063107-04
Application #
6735679
Study Section
Physiological Chemistry Study Section (PC)
Program Officer
Rhoades, Marcus M
Project Start
2001-05-01
Project End
2005-04-30
Budget Start
2004-05-01
Budget End
2005-04-30
Support Year
4
Fiscal Year
2004
Total Cost
$215,534
Indirect Cost
Name
University of Houston
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
036837920
City
Houston
State
TX
Country
United States
Zip Code
77204
Pang, Yan Ling Joy; Poruri, Kiranmai; Martinis, Susan A (2014) tRNA synthetase: tRNA aminoacylation and beyond. Wiley Interdiscip Rev RNA 5:461-80
Sarkar, Jaya; Poruri, Kiranmai; Boniecki, Michal T et al. (2012) Yeast mitochondrial leucyl-tRNA synthetase CP1 domain has functionally diverged to accommodate RNA splicing at expense of hydrolytic editing. J Biol Chem 287:14772-81
Boniecki, Michal T; Rho, Seung Bae; Tukalo, Mikhail et al. (2009) Leucyl-tRNA synthetase-dependent and -independent activation of a group I intron. J Biol Chem 284:26243-50
Boniecki, Michal T; Vu, Michael T; Betha, Aswini K et al. (2008) CP1-dependent partitioning of pretransfer and posttransfer editing in leucyl-tRNA synthetase. Proc Natl Acad Sci U S A 105:19223-8
Hsu, Jennifer L; Martinis, Susan A (2008) A Flexible peptide tether controls accessibility of a unique C-terminal RNA-binding domain in leucyl-tRNA synthetases. J Mol Biol 376:482-91
Nawaz, Mir Hussain; Pang, Yan Ling Joy; Martinis, Susan A (2007) Molecular and functional dissection of a putative RNA-binding region in yeast mitochondrial leucyl-tRNA synthetase. J Mol Biol 367:384-94
Karkhanis, Vrajesh A; Boniecki, Michal T; Poruri, Kiranmai et al. (2006) A viable amino acid editing activity in the leucyl-tRNA synthetase CP1-splicing domain is not required in the yeast mitochondria. J Biol Chem 281:33217-25
Hsu, Jennifer L; Rho, Seung Bae; Vannella, Kevin M et al. (2006) Functional divergence of a unique C-terminal domain of leucyl-tRNA synthetase to accommodate its splicing and aminoacylation roles. J Biol Chem 281:23075-82
Jones, Christopher I; Spencer, Angela C; Hsu, Jennifer L et al. (2006) A counterintuitive Mg2+-dependent and modification-assisted functional folding of mitochondrial tRNAs. J Mol Biol 362:771-86
Tukalo, Michael; Yaremchuk, Anna; Fukunaga, Ryuya et al. (2005) The crystal structure of leucyl-tRNA synthetase complexed with tRNALeu in the post-transfer-editing conformation. Nat Struct Mol Biol 12:923-30

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