Studies on the processing of polyoma virus RNA
Creators: name: Petric, Martin
Publisher: name: University of British Columbia
Year: 2011

description: The main objective of these studies was to analyze the processing of polyoma virus RNA transcripts. An examination was made of the intracellular distribution of polyoma virus RNA molecules synthesized late during productive infection in mouse kidney cells. The virus specific RNA from whole cells, and their nuclear, cytoplasmic and polyribosomal fractions were compared with respect to sedimentation behaviour (on sucrose gradients), electrophoretic mobility (on polyacrylamide gels) and base sequence homology (by competition hybridization tests). Sedimentation and electrophoretic analysis revealed marked heterogeneity in polyoma RNA from all cell fractions. This heterogeneity and size distribution were essentially the same on dimethylsulfoxide sucrose gradients. The viral RNA resedimented true to its original distribution on a sucrose gradient. The polyoma RNA from different regions of a sucrose gradient contained common sequences as revealed by cross-competition hybridization experiments. However, the polyribosome associated polyoma RNA was devoid of the &gt;28s species found in the other fractions. Thus it appeared that polyoma RNA, including RNA of apparent size in excess of one genome length, was synthesized in the nucleus and cleaved to smaller peices in association with the polyribosome s. The accumulation of the polyoma RNA in the nuclear fraction pro-ceded at a rate similar to that of the cellular RNA, while the corresponding rate of accumulation of viral RNA in the cytoplasmic fraction was similar to the cell RNA for 30 minutes, but did not increase, as did that of the cell RNA, with increased labelling times. The polyoma RNA labelled in the cells during a 15 minute pulse was significantly larger (46% sedimented faster than 28s) than that labelled for 2 hours (25% sedimented faster than 28s). Pulse and chase studies with Actino-mycin D and excess uridine revealed that the majority (up to 75%) of the polyoma RNA labelled in a 40 minute pulse was subsequently degraded within the nucleus within one hour of chase, while the remainder disappeared from the nucleus at a slower rate in the next five hours of chase. In addition, the larger (&gt;28s) polyoma RNA in the nuclear fraction was degraded slightly faster than the small (&lt;18s) RNA. In the cytoplasmic fraction, the quantity of labelled viral RNA decreased slowly during the chase period and all size classes were degraded at approximately the same rate. Polyadenylate sequences were found associated with polyoma RNA. The percentage of the viral RNA molecules containing polyadenylate sequences increased as the RNA was processed from the nuclei through to the polyribosomes, suggesting that only poly (A) containing polyoma RNA molecules can be properly processed. The methodology utilized in the isolation and detection of poly (A) sequences was examined in more detail. The method of isolationof poly (A) sequences, by binding to poly (U) fixed to glass fiber filters, was found to be highly dependent on the salt concentration of the binding buffer, through it did select for non-ribosomal heterogeneous cellular RNA which had ribonuclease resistant poly (A) stretches. The method of extraction of the RNA was found to have a considerable effect on the subsequent binding properties of these molecules.