Exonuclease resistant 18S and 25S ribosomal RNA components in yeast are possibly newly transcribed by RNA polymerase II
Abstract
Background: We’ve formerly reported 18S and 25S ribosomal RNA molecules in Candidiasis resistant against processive 5′ ? 3′ exonuclease, appearing as cells contacted stationary growth phase. Initial analysis pointed to extra phosphate(s) in their 5′- finish raising the chance that these were recently transcribed. Ideas set of additional experiments exploring this possibility and then try to establish which from the RNA polymerases might be transcribing them.
Results: Oligo-ligation and primer extension again demonstrated the existence of extra phosphate in the 5′-finish from the reported processing sites for 18S and 25S ribosomal RNA components. Inhibition of Pol I with BMH-21 elevated the existence of the molecules. Quantitation by having an Agilent Bioanalyzer demonstrated that resistant 18S and 25S molecules are mainly created within the nucleus. Having an RNA cap specific antibody, an indication might be detected on these molecules via immunoblotting such signal might be eliminated by decapping reaction. Both cap specific antibody and eIF4E cap-binding protein, elevated fold enrichment upon quantitative amplification. Antibodies specific for that RNA Polymerase II c-terminal domain and TFIIB initiator factor demonstrated the existence of Pol II on DNA sequences for 18S and 25S molecules in chromatin precipitation and qPCR assays. Rapamycin inhibition of TOR complex also led to a rise of resistant 18S and 25S molecules.
Conclusions: These data raise the potential of a job for RNA Polymerase II in producing 18S and 25S molecules and indicate that efforts for additional direct proof might be useful. If for sure proven BMH-21 it’ll establish yet another role for RNA Polymerase II in ribosomal production.