Scientific Report 2005
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.