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The 11 th International Workshop on KSHV & Related Agents, Birmingham, UK Gene Expression I Abstract 32 PROFILING OF CELLULAR AND VIRAL MICRORNAS IN KAPOSI SARCOMA AND VIRAL-ASSOCIATED LYMPHOMA Andrea J. O’Hara*, Bruce J. Dezube # , William Harrington Jr.^, Blossom Damania*, and Dirk P. Dittmer^* *Department of Microbiology and Immunology, Lineberger Comprehensive Cancer Center, and Curriculum in Genetics and Molecular Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599; # Beth Israel Deaconess Medical Center; ^Sylvester Comprehensive Cancer Center, University of Miami. Abstract MicroRNAs are regulated by gene alteration at the DNA level, transcriptional regulation, and mature miRNA processing via Dicer. Thus far, few studies have simultaneously assessed all three levels of regulation. Using real-time quantitative polymerase chain reaction (QPCR)-based arrays, changes in gene copy number, pre-miRNA and mature miRNA levels for a large set of primary effusion lymphomas (PELs) and primary Kaposi Sarcoma biopsies (KS) has been determined; this includes miRNA gene alterations and concordant changes in pre-miRNA and mature miRNA expression levels. The real-time QPCR based approach confirmed many of the KSHV viral and cellular miRNAs previously cloned from PEL. However, array-based profiling also uncovered many novel PEL–specific miRNAs, since cloning-based approaches are not always saturating. The miRNA expression pattern for neither viral nor cellular miRNAs has hitherto been determined for KS. Furthermore, comprehensive SNP analysis of the viral miRNAs has been profiled to further examine the effects of sequence and processing. This defines the miRNA signature of PEL, KS, KSHV-infected cancers and experimental models. It shows that the transcriptional regulation of pre-miRNA as well as mature miRNA levels contribute nonredundant information that can be used in the classification of human tumors. Presenting author Email: ohara@unc.edu 56
The 11 th International Workshop on KSHV & Related Agents, Birmingham, UK Gene Expression I Abstract 33 INEFFICIENT CODON USAGE IN V-FLIP MRNA LEADS TO TRANSCRIPT INSTABILITY Priya Bellare, Andrew T Dufresne and Don Ganem HHMI and G.W. Hooper Foundation, University of California, San Francisco, California 94143-0552, USA Abstract KSHV v-FLIP is a latent gene product that activates the NF-κB pathway, prolonging the survival of PEL cells and causing the characteristic spindled morphology of latently infected endothelial cells. The expression of v-FLIP is unusual in two regards. (i) The gene is expressed from a bi or tricistronic message that also encodes v-cyclin and LANA genes; to date, no monocistronic v-FLIP mRNA has been consistently detected in infected cells. (ii) The level of v-FLIP protein in infected or transfected cells is extremely low, even when a monocistronic mRNA is artificially engineered. Here we show that the low abundance of v-FLIP mRNA can be attributed to instability of the v-FLIP message, which in turn is a consequence of its sub-optimal codon usage. We transfected 293 cells with a vector designed to produce a monocistronic v-FLIP mRNA expressing Flag-tagged v-FLIP. As expected, v-FLIP protein levels were nearly undetectable; surprisingly, however, v- FLIP mRNA levels were also extremely low. Examination of codon usage in v-FLIP showed it to be dominated by poorly used codons throughout the length of the ORF. Expression of a re-engineered v-FLIP gene with efficient codon usage not only generated abundant v-FLIP protein but also restored mRNA accumulation to levels readily detected by Northern blotting. Analysis of chimeras of optimal and sub-optimal v-FLIP coding sequences reveals that mRNA stability cannot be mapped to a unique region of the mRNA; however, when an unstable v-FLIP construct is placed 3’ to a well expressed ORF, the resulting bicistronic mRNA is stable. These results explain many, but perhaps not all, of the prior conundrums of v-FLIP expression, and provide a striking example of translational regulation of RNA accumulation, the mechanism of which is now under study. Presenting author Email: Priya.Bellare@ucsf.edu 57
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