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Abstract
During the lytic phase of infection, the gamma herpes virus Kaposi's Sarcoma-Associated Herpesvirus (KSHV) expresses a highly abundant, nuclear noncoding RNA of unknown function. Poly adenylated nuclear RNA (PAN RNA) associates with host poly(A)-binding protein C1 (PABPC1), which normally resides in the cytoplasm where it binds and stabilizes the poly(A) tails of mRNAs, increasing mRNA translation efficiency. During the lytic phase of KSHV infection, PABPC1 is re-localized to the nucleus as a consequence of the host shutoff effect mediated by the viral shutoff exonuclease (SOX) protein: host mRNAs are selectively downregulated while viral mRNAs are expressed. We show that whereas PAN RNA does not appear to be required for the host shutoff effect nor for PABPC1 re-localization, it avidly binds PABPC1 in the nucleus. SOX strongly upregulates the levels of PAN RNA in transient transfection experiments by a mechanism that is dependent on the ability of SOX to mediate the host shutoff effect, on the re-localization of PABPC1 into the nucleus and on the presence of a poly(A) tail on PAN RNA. Depletion of PAN RNA from cells lytically infected with KSHV using antisense oligonucleotides indicates that PAN is required for the production of proteins from late viral mRNAs that are themselves polyadenylated. Our results add to the repertoire of functions ascribed to large noncoding RNAs.
During Drosophila embryogenesis, the transcription factor Prospero influences neuronal differentiation and axonal outgrowth. The prospero pre-mRNA undergoes alternative splicing, but is unique in that it harbors a rare twintron—whereby one intron lies embedded within another. The innermost intron is excised by the major U2-type spliceosome and the outermost by the minor U12-type spliceosome. Previously, an intronic purine-rich element (PRE) was identified as an enhancer of both U2- and U12-type splicing, with a greater effect on the U2-type pathway. We find that the PRE binds Drosophila homologs of hnRNP A1, Hrp38 and Hrp36. RNAi-mediated knockdown of these proteins in S2 cells specifically decreases U2-type splicing of the twintron, surprising since hnRNPs are usually repressive. Conversely, tethering Hrp38 to the twintron increases U2-type splicing. Thus, developmentally-regulated alternative splicing of the prospero twintron can be explained by documented changes in the abundance of these hnRNP A1-like proteins during embryogenesis.