Some picornaviral and cellular mRNAs initiate translation in a CAP-independent manner at internal ibosome entry sites (IRESes) contained within the mRNA. While extensively studied in picornaviruses, little is known about internal initiation in cellular mRNAs. The boundaries of cellular IRESes have been difficult to define and sequence comparisons show no obvious sequence similarities among cellular IRESes or between cellular and picomaviral IRESes. The Mauro laboratory has analyzed two cellular IRESes contained within the 5' UTRs of the mRNAs that encode Gtx, a homeodomain protein and Rbm3, a cold stress induced protein that is of special interest because it appears to enhance the activity of its own IRES. These studies indicated that some cellular IRE Ses were composed of shorter cis-acting regulatory sequences, some as short as 7-nucleotides, that could function independently to internally initiate translation (IRES-module), or to enhance internal initiation (enhancer element). In addition, a diversity of IRES-modules was selected from a library containing short random nucleotide sequences using a method that was developed in this laboratory. Multiple copies of some naturally-occurring and selected IRESnodules increased internal initiation synergistically. The working hypothesis is that some cellular IRESes are composed of shorter elements that can function independently. The proposed studies will identify naturally-occurring IRES-modules and regulatory elements from cellular mRNAs, while selection studies will attempt to identify sequences with different expression properties. These sequences will be analyzed to determine if they can be categorized, to investigate the rules governing their activity, and to examine if the3 recruit the translation machinery directly by interacting with rRNA or with ribosomal proteins, or indirectly through intermediary proteins. The Rbm3 protein will be studied as a potential example of a trans-acting protein. The proposed studies should provide new insights into our understanding of internal initiation. Inasmuch as several clinically important cellular genes contain IRESes, this increased understanding may allow useful therapeutic manipulations. Moreover, the use of IRE S-modules and enhancers has already resulted in synthetic IRESes that are shorter and more efficient than the large viral IRESes currently used ir dicistronic constructs and should have broad applications in research, gene therapy, and biotechnology.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM061725-04
Application #
6739044
Study Section
Physiological Chemistry Study Section (PC)
Program Officer
Rhoades, Marcus M
Project Start
2001-05-01
Project End
2006-04-30
Budget Start
2004-05-01
Budget End
2006-04-30
Support Year
4
Fiscal Year
2004
Total Cost
$296,320
Indirect Cost
Name
Scripps Research Institute
Department
Type
DUNS #
781613492
City
La Jolla
State
CA
Country
United States
Zip Code
92037
Mauro, Vincent P; Edelman, Gerald M (2007) The ribosome filter redux. Cell Cycle 6:2246-51
Mauro, Vincent P; Chappell, Stephen A; Dresios, John (2007) Analysis of ribosomal shunting during translation initiation in eukaryotic mRNAs. Methods Enzymol 429:323-54
Dresios, John; Chappell, Stephen A; Zhou, Wei et al. (2006) An mRNA-rRNA base-pairing mechanism for translation initiation in eukaryotes. Nat Struct Mol Biol 13:30-4
Chappell, Stephen A; Edelman, Gerald M; Mauro, Vincent P (2006) Ribosomal tethering and clustering as mechanisms for translation initiation. Proc Natl Acad Sci U S A 103:18077-82
Chappell, Stephen A; Dresios, John; Edelman, Gerald M et al. (2006) Ribosomal shunting mediated by a translational enhancer element that base pairs to 18S rRNA. Proc Natl Acad Sci U S A 103:9488-93
Zhou, Wei; Edelman, Gerald M; Mauro, Vincent P (2005) A positive feedback vector for identification of nucleotide sequences that enhance translation. Proc Natl Acad Sci U S A 102:6273-8
Dresios, John; Aschrafi, Armaz; Owens, Geoffrey C et al. (2005) Cold stress-induced protein Rbm3 binds 60S ribosomal subunits, alters microRNA levels, and enhances global protein synthesis. Proc Natl Acad Sci U S A 102:1865-70
Chappell, Stephen A; Edelman, Gerald M; Mauro, Vincent P (2004) Biochemical and functional analysis of a 9-nt RNA sequence that affects translation efficiency in eukaryotic cells. Proc Natl Acad Sci U S A 101:9590-4
Rogers Jr, George W; Edelman, Gerald M; Mauro, Vincent P (2004) Differential utilization of upstream AUGs in the beta-secretase mRNA suggests that a shunting mechanism regulates translation. Proc Natl Acad Sci U S A 101:2794-9
Chappell, Stephen A; Mauro, Vincent P (2003) The internal ribosome entry site (IRES) contained within the RNA-binding motif protein 3 (Rbm3) mRNA is composed of functionally distinct elements. J Biol Chem 278:33793-800

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