Contents - College of Medical and Dental Sciences - University of ...
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The 11 th International Workshop on KSHV & Related Agents, Birmingham, UK<br />
Gene Expression I Abstract 33<br />
INEFFICIENT CODON USAGE IN V-FLIP MRNA LEADS TO TRANSCRIPT<br />
INSTABILITY<br />
Priya Bellare, Andrew T Dufresne <strong>and</strong> Don Ganem<br />
HHMI <strong>and</strong> G.W. Hooper Foundation, <strong>University</strong> <strong>of</strong> California, San Francisco, California<br />
94143-0552, USA<br />
Abstract<br />
KSHV v-FLIP is a latent gene product that activates the NF-κB pathway, prolonging the<br />
survival <strong>of</strong> PEL cells <strong>and</strong> causing the characteristic spindled morphology <strong>of</strong> latently<br />
infected endothelial cells. The expression <strong>of</strong> v-FLIP is unusual in two regards. (i) The<br />
gene is expressed from a bi or tricistronic message that also encodes v-cyclin <strong>and</strong> LANA<br />
genes; to date, no monocistronic v-FLIP mRNA has been consistently detected in infected<br />
cells. (ii) The level <strong>of</strong> v-FLIP protein in infected or transfected cells is extremely low, even<br />
when a monocistronic mRNA is artificially engineered. Here we show that the low<br />
abundance <strong>of</strong> v-FLIP mRNA can be attributed to instability <strong>of</strong> the v-FLIP message, which<br />
in turn is a consequence <strong>of</strong> its sub-optimal codon usage. We transfected 293 cells with a<br />
vector designed to produce a monocistronic v-FLIP mRNA expressing Flag-tagged v-FLIP.<br />
As expected, v-FLIP protein levels were nearly undetectable; surprisingly, however, v-<br />
FLIP mRNA levels were also extremely low. Examination <strong>of</strong> codon usage in v-FLIP showed<br />
it to be dominated by poorly used codons throughout the length <strong>of</strong> the ORF. Expression<br />
<strong>of</strong> a re-engineered v-FLIP gene with efficient codon usage not only generated abundant<br />
v-FLIP protein but also restored mRNA accumulation to levels readily detected by<br />
Northern blotting. Analysis <strong>of</strong> chimeras <strong>of</strong> optimal <strong>and</strong> sub-optimal v-FLIP coding<br />
sequences reveals that mRNA stability cannot be mapped to a unique region <strong>of</strong> the<br />
mRNA; however, when an unstable v-FLIP construct is placed 3’ to a well expressed ORF,<br />
the resulting bicistronic mRNA is stable. These results explain many, but perhaps not all,<br />
<strong>of</strong> the prior conundrums <strong>of</strong> v-FLIP expression, <strong>and</strong> provide a striking example <strong>of</strong><br />
translational regulation <strong>of</strong> RNA accumulation, the mechanism <strong>of</strong> which is now under<br />
study.<br />
Presenting author Email: Priya.Bellare@ucsf.edu<br />
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