REGULATION OF PLANT RNA VIRUS PROTEIN SYNTHESIS

Overview: When an RNA virus infects a cell, it must out-compete host cell mRNA to produce viral proteins. Luteovirus barley yellow dwarf virus (BYDV) and tombavirus maize necrotic streak virus (MNeSV) are aphid-borne threats to cereal crops world-wide. These positive strand RNA viruses are some of the most efficiently
translated mRNA known. These viruses use a number of strategies for efficient translation, often involving unique RNA structures. This efficient translation allows these plant viruses to serve as low cost means to produce proteins of therapeutic value, but emerging viruses remain a problem both for world health and food supply.

 

Research: We are investigating three critical aspects in selection of mRNA for translation and assembly of an initiation complex. These are 1) identify the role of eIF3 in ribosome recruitment; 2) analyze the novel ribosome transfer from the 3’ UTR and recruitment to the AUG and 3) determine structural elements necessary for interactions of the 40S initiation complex. We are using ensemble fluorescence spectroscopy, structural probing utilizing SHAPE and capillary electrophoresis, and FRET techniques. In collaboration with Prof. Ruben Gonzalez, Columbia University, we are employing a novel application of single molecule fluorescence, to determine the rate of ribosome transfer from the 3’ UTR to the 5’ UTR. In collaboration with Prof. Amedee des Georges, ASRC, structural information to further understand these interactions and potentially
design therapeutic agents are being obtained from Cryo-EM experiments.

NOVEL MAMMALIAN PROTEIN SYNTHESIS MECHANISM

Translation of mRNAs into proteins plays a major role in gene expression. Increasingly, it is
recognized that many diseases are the result of mis-regulation of this process (e.g. cancer, diabetes and
neurological diseases). Our long term goal is to understand and modulate non-canonical translation initiation.
For most cellular mRNA, translation initiation involves recognition of the mRNA 5’ m7G triphosphate (the cap)
by eIF4E, the cap binding protein. However, under cellular stress conditions, e.g. tumor hypoxia, where cap-
dependent translation is compromised, a subset of mRNAs is still translated efficiently. These mRNAs switch
from a cap-dependent to a cap-independent mechanism for expression. The mechanisms that regulate
switching between cap-dependent and cap-independent translation of specific mRNAs remain unknown,
impeding our understanding and ability to modulate this aspect of health and disease.

 

Previous studies have shown that switching of a prototypical subset of mRNAs, HIF-1α, FGF-9, VEGF
and p53, from cap-dependent to cap-independent translation initiation as a result of eIF4E inhibition,
correlates with increased levels of eIF4G or it’s homolog, DAP5, proteins responsible for recruitment of the
small ribosomal subunit. In addition, these mRNAs have been shown to contain highly structured 5’
untranslated regions (UTRs) that act as translational enhancer (TE) elements. Utilizing ensemble and single
molecule fluorescence, and complementary cellular and molecular biology approaches, we are characterizing
the mechanism of switching between cap-dependent and cap-independent modes of translational initiation.
Our research will broadly impact the field by characterizing the essential mechanism of translational initiation
switching and elucidate a physiologically relevant model for how TE-mRNAs are regulated for translation.
These studies have the potential to uncover novel mechanisms of translational regulation applicable to other
mRNAs and give insight into cap-dependent mechanisms as well.