Mechanism of stringent translation initiation: a probe for its biological relevance

Eukaryotic translation initiation is a complex process involving the ribosome, mRNA, Met-tRNAiMet
and numerous eukaryotic initiation factors (eIFs). Decades of studies driven by those using the
model eukaryote yeast Saccharomyces cerevisiae revealed that the key process is the formation
of codon-anticodon base pairing in the small ribosome P-site. Stringent initiation is enabled by
formation of the 48S ribosomal pre-initiation complex (PIC) strictly at the AUG start codon while
excluding initiation at other sites. Intriguingly, however, many non-canonical start sites are utilized
in some biological contexts and diseases such as cancer and neurodegenerative disorders. The
list of non-AUG start sites within the human genome is far from being complete and how the use
of these sites and hence protein production from these sites are regulated remains an open
question.
Thus, Aim 1 of this grant is to make such a list of non-AUG start sites through genome-wide
translation profiling of well characterized cancer model systems, verify some of these sites
and determine the mechanism driving the observed non-AUG translational regulation in cancer.
The Aim 2 is to study the mechanistic role of 5MP and Met-tRNAiMet adenosine N6-
threonylcarbamoylation (t6A) in controlling non-AUG translation. It will be tested if 5MP
mutations found in many types of cancer can alter initiation accuracy, thereby affecting patients'
prognosis. Our preliminary studies suggested that t6A located 3' of Met-tRNAiMet anticodon can
discriminate specifically against GUG and UUG start codons, in contrast to eIF1 being more
universal non-AUG discriminator. Combining molecular dynamics simulation methods, we will test
if the recently discovered cyclic t6A serves the discriminating role and determine how the
cooperation or competition between t6A and eIF1 promotes stringent initiation and leaky scanning
crucial for translational regulation.
The Aim 3 is to study yet a distinct mechanism of start codon selection that is exploited
during the heat shock response at the translational level. This mechanism was discovered
through translational profiling of yeast eIF3i mutant defective in its interaction with RNA-binding
eIF3g subunit at a high temperature. The working hypothesis assumes that, at a high temperature,
stable PIC formation at the AUG codon requires additional mRNA elements anchoring the
initiating ribosome at its entry site. These hypothetical mRNA elements are located downstream
of the start codon and include a novel eIF3g-binding motif 5'-GUCG-3' and a downstream stem-
loop that potentially binds the entry site-side of the 40S, thereby stabilizing the PIC formation.
This model will tested using yeast as a model system.

Grant Number: 5R01GM147542-03
NIH Institute/Center: NIH
Principal Investigator: KATSURA ASANO