Abstract
BackgroundWe have previously reported 18S and 25S ribosomal RNA molecules in Candida albicans resistant to processive 5′ → 3′ exonuclease, appearing as cells approached stationary growth phase. Initial analysis pointed to extra phosphate(s) at their 5′- end raising the possibility that they were newly transcribed. Here we report on additional experiments exploring this possibility and try to establish which of the RNA polymerases may be transcribing them.ResultsOligo-ligation and primer extension again showed the presence of extra phosphate at the 5′-end of the reported processing sites for both 18S and 25S ribosomal RNA components. Inhibition of Pol I with BMH-21 increased the presence of the molecules. Quantitation with an Agilent Bioanalyzer showed that resistant 18S and 25S molecules are primarily produced in the nucleus. Utilizing an RNA cap specific antibody, a signal could be detected on these molecules via immunoblotting; such signal could be eliminated by decapping reaction. Both the cap specific antibody and eIF4E cap-binding protein, increased fold enrichment upon quantitative amplification. Antibodies specific for the RNA Polymerase II c-terminal domain and TFIIB initiator factor showed the presence of Pol II on DNA sequences for both 18S and 25S molecules in chromatin precipitation and qPCR assays. Rapamycin inhibition of TOR complex also resulted in an increase of resistant 18S and 25S molecules.ConclusionsThese data raise the possibility of a role for RNA Polymerase II in the production of 18S and 25S molecules and indicate that efforts for more direct proof may be worthwhile. If definitively proven it will establish an additional role for RNA Polymerase II in ribosomal production.
Highlights
We have previously reported 18S and 25S ribosomal RNA molecules in Candida albicans resistant to processive 5′ → 3′ exonuclease, appearing as cells approached stationary growth phase
Resistant ribosomal 18S and 25S behaved like 5S molecules, products of Pol RNA Polymerase III (III) with triphosphates at their 5′ end, which we found to be resistant to Terminator digestion
We feel confident that in this yeast there can be two types of 18S and 25S ribosomal RNA components as they have been consistently found on hundreds of Terminator digestions
Summary
We have previously reported 18S and 25S ribosomal RNA molecules in Candida albicans resistant to processive 5′ → 3′ exonuclease, appearing as cells approached stationary growth phase. While three RNA polymerases are involved in ribosome production, the 18S, 5.8S and 25S ribosomal RNA (rRNA) components are thought to be products of polycistronic transcription by RNA polymerase I (Pol I) followed by processing [1,2,3]. A role for Pol II in ribosomal RNA production in Saccharomyces cerevisiae has been described [7]. Transcription was initiated from multiple start sites upstream or downstream from the normal Pol I’s promoter site, still in a polycistronic fashion. It has been seen in a petite strain of S. cerevisiae, involving the selective activation of cryptic Pol II promoters from episomal rDNA elements [8]
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