Kinetics and efficiency of polyadenylation of late polyomavirus nuclear RNA: generation of oligomeric polyadenylated RNAs and their processing into mRNA.

Abstract

The rate and efficiency of polyadenylation of late polyomavirus RNA in the nucleus of productively infected mouse kidney cells were determined by measuring incorporation of [3H]uridine into total and polyadenylated viral RNAs fractionated by oligodeoxythymidylic acid-cellulose chromatography. Polyadenylation is rapid: the average delay between synthesis and polyadenylation of viral RNA in the nucleus is 1 to 2 min. However, only 10 to 25% of viral RNA molecules become polyadenylated. Polyadenylated RNAs in the nucleus are a family of molecules which differ in size by an integral number of viral genome lengths (5.3 kilobases). These RNAs are generated by repeated passage of RNA polymerase around the circular viral DNA, accompanied by addition of polyadenylic acid to a unique 3' end situated 2.2 + n(5.3) kilobases from the 5' end of the RNAs (n can be an integer from 0 to at least 3). Between 30 and 50% of the sequences in nuclear polyadenylated RNA are conserved during processing and transport to the cytoplasm as mRNA. This is consistent with the molar ratios of nuclear polyadenylated RNAs in the different size classes, and it suggests that most polyadenylated nuclear RNA is efficiently processed to mRNA. Thus, the low overall conservation of viral RNA sequences between nucleus and cytoplasm is explained by (i) low efficiency of polyadenylation of nuclear RNA and (ii) removal of substantial parts of polyadenylated RNAs during splicing. The correlation between inefficient termination of transcription and inefficient polyadenylation of transcripts suggests that these two events may be causally linked.During the late phase of infection of mouse cells by polyomavirus, only about 5% of the viral RNA synthesized in the nucleus is successfully processed and transported to the cytoplasm as mRNA (2). This low efficiency of conservation of viral RNA during processing can be explained partly by the fact that RNA polymerase II may traverse the circular viral DNA genome (5,292 nucleotide pairs [11,35]) several times before terminating transcription (1,3). This leads to production of giant RNAs containing several tandemly repeated copies of the entire nucleotide sequence of one of the strands of the viral DNA. Only a single mRNA body sequence per precursor RNA molecule is conserved during RNA splicing (8,15,21) I have confirmed that only a small fraction (10 to 25%) of nuclear viral RNA is polyadenylated, that it is polyadenylated within less than 2 min of its synthesis, and that the remaining 75 to 90% of nuclear viral RNA is apparently never polyadenylated and cannot therefore serve as a precursor to viral mRNA in the cytoplasm. Nuclear viral RNA which is polyadenylated exists as molecules of discrete lengths of about 2.2 + n(5.3) kilobases (kb). These RNAs represent a series which have the same 5' and 3' ends but differ in the number of genome traverses made by RNA polymerase II before terminating. These results are consis-722 tent with the idea that most polyadenylated nuclear RNAs are successfully processed into mature mR...

The length but not the sequence of the polyoma virus late leader exon is important for both late RNA splicing and stability

“…Cleavage at this site in polyoma has been shown to be tightly coupled to polyadenylation (37). Full-length probe is indicative of readthrough transcripts not polyadenylated at the expected site and perhaps due to multi-genomic transcripts.…”

Use of a novel S1 nuclease RNA-mapping technique to measure efficiency of transcription termination on polyomavirus DNA.

“…If the bands in lane 4 were generated by RNAs whose 3' ends lie within HpaII fragment 3, they would have become smaller, not larger, when these RNAs were hybridized to PstI fragment 2 (Fig. 1).…”

“…RNA was extracted with phenol-CHCl3 and treated with RNasefree DNase (DPRF; Worthington Diagnostics, Freehold, N.J.) as described previously (6). Oligo(dT)-cellulose chromatography was carried out as described previously (4), except that the RNA load was passed three times in succession through the column before washing and elution.…”

“…As a result, some RNA polymerase molecules traverse the circular, 5.3-kilobase (kb), double-stranded polyomavirus genome several times before dissociating and releasing their RNA chains. RNA molecules up to four times genome length have been detected in the nuclei of productively infected cells (4,5). These multigenome-length RNAs undergo splicing within the nucleus to form 19S, 18S, and 16S viral mRNAs (18,27).…”

“…It has been postulated that the presence of multiple leaders may increase the ability of polyomavirus L-strand mRNAs to be translated (27), thereby conferring a selective advantage to virus replication. In this light, the low termination efficiency which leads to production of multigenome-length mRNA precursors can be seen as a feature designed ultimately to enhance viral replication, although paradoxically it leads to removal by splicing and degradation in the nucleus of most of the viral RNA synthesized (2,4).…”