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Scientific Report 2005


Neurobiology




Translational Regulation of Gene Expression


V.P. Mauro, S.A. Chappell, G.W. Rogers, Jr., W. Zhou, J. Dresios, D.C.Y. Koh, G.M. Edelman

We focus on understanding the mechanisms that underlie the initiation of translation. In eukaryotic cells, mRNAs recruit the translation machinery at a cap structure, a modified nucleotide found at the 5′ ends of mRNAs, or at a sequence contained within the mRNA termed an internal ribosome entry site (IRES). Recruitment at the cap structure requires various initiation factors, whereas internal initiation can occur via different mechanisms that vary in their requirements for trans-acting factors.

In earlier studies, we showed that some IRESs are composed of shorter functional elements. Currently, we are examining how different IRES elements recruit the translation machinery. To this end, we have developed a powerful new method to facilitate the identification of new IRES elements.

In the new method, a positive feedback mechanism is used to amplify the activities of individual IRES elements. The positive feedback vector encodes a dicistronic mRNA with a reporter gene as the first cistron and the yeast Gal4/VP16 transcription factor as the second cistron. Transcription of this mRNA is driven by a minimal promoter containing 4 copies of the Gal4 upstream activation sequence. In this method, the presence of an IRES in the intercistronic region facilitates the translation of Gal4/VP16, which then binds to the upstream activation sequences and triggers a positive feedback loop that escalates the production of dicistronic mRNA and Gal4/VP16. A corresponding increase in the translation of the first (reporter) cistron is monitored. This reporter also enables isolation of IRES-positive cells. We are using this vector system to identify and analyze new IRES elements.

We are also studying the mechanism by which an IRES module in the 5′ leader of the mRNA encoding the Gtx homeodomain protein affects the initiation of translation. This IRES module is 9 nucleotides long and is complementary to a segment of the 18S rRNA, which is the RNA component of the small (40S) ribosomal subunit. The results of earlier studies, in which we used binding assays, ultraviolet cross-linking, and functional analyses, suggested that the Gtx IRES module enhanced translation by a mechanism that involved base pairing to 18S rRNA. Our most recent results indicated that this IRES element can facilitate the nonlinear movement of ribosomes along the 5′ leader of an mRNA.

In addition, findings from studies in yeast provided direct evidence that the mechanism underlying the activity of the Gtx IRES module requires base pairing to 18S rRNA. These findings indicated that the activity of this IRES element requires a complementary match within the 18S rRNA. We also showed that the activity of this IRES module can be disrupted by point mutations of the IRES sequence and that activity can be restored by mutations of the 18S rRNA that restore complementarity. These studies provide the first direct evidence of an mRNA-rRNA base-pairing mechanism in eukaryotes.

In other studies, by probing RNA accessibility in living cells, we are testing the notion that accessibility is a key factor in determining whether or not the nucleotide triplet AUG is recognized as an initiation codon. We expect that the results of our studies will provide new insights into the basic mechanisms of the initiation of translation.

 

Vincent P. Mauro, Ph.D.
Associate Professor


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