Using murine erythroleukemia (MEL) cells, we have obtained evidence that the mRNA for a protein synthesis elongation factor, namely eucaryotic elongation factor Tu (eEF-Tu; also known as EF-la), accumulates in messenger ribonucleoprotein particles (mRNPs) as MEL cell growth slows down. When MEL cells reach stationary phase approximately 60% of the eEF-Tu mRNA is sequestered in mRNP. Similar observations have been made in both mammalian and invertebrate cells by other investigators. More recently, we have shown that when MEL cell growth resumes after addition of fresh medium to stationary cultures, the eEF-Tu mRNP disappears and the mRNA resident in that structure is quantitatively degraded. We have also constructed an in vitro system derived from MEL cells, consisting primarily of nontranslating mRNPs, in which eEF-Tu mRNA, as well as certain other mRNAs, are selectively degraded. This proposal attempts to build on these observations with the aim of providing more detailed molecular mechanisms for these observations. (1) We will construct, transfect, and express a chimeric gene for eEF-Tu in MEL cells which will be under the control of a dexamethasone response promoter. Using this system will determine the cis-acting elements in eEF-Tu mRNA responsible for translational repression. (2) This cis-acting element will be synthesized in vitro and used to isolate the trans-acting cellular factor(s) which repress eEF-Tu synthesis. (3) We will purify and characterize the nuclease associated with MEL cell mRNPs and produce anti-nuclease antibodies. These antibodies will be used to investigate the relationship, if any, between nuclease activity and MEL cell growth and differentiation. (4) We will employ the nuclease to characterize the nuclease sensitive sites in eEF-Tu mRNA and eEF-Tu mRNP with the aim of footprinting the regions of eEF-Tu mRNA that associate with protein. (5) Finally, we will employ the anti-nuclease antibodies to reconstitute mRNPs in vitro by combining mRNP proteins which wash off the particles at 0.5 M NaC1 (the proteins contain the RNAse activity as well as specific mRNA binding proteins) with salt-washed mRNPs. This approach, if successful, will provide a way of examining the structure and function of native mRNPs.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM025434-12
Application #
3273016
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1978-07-01
Project End
1993-11-30
Budget Start
1991-12-01
Budget End
1992-11-30
Support Year
12
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of Mississippi Medical Center
Department
Type
Schools of Medicine
DUNS #
928824473
City
Jackson
State
MS
Country
United States
Zip Code
39216