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The start sites of sequences of 18S and 25S rRNA found to be exonuclease resistant as indicated by oligo attachment

Citation

Fleischmann, Jacob et al. (2020), The start sites of sequences of 18S and 25S rRNA found to be exonuclease resistant as indicated by oligo attachment , Dryad, Dataset, https://doi.org/10.5068/D16H37

Abstract

Abstract

 

Background

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. 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.

Results

Oligo-ligation and primer extension again showed the presence of extra phosphate at 5’-end at or near 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.

Conclusions

These 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.

 

Methods

Sequences were obtained from various RNA samples (extracted from C. albicans at stationary growth phase) using distinct primers specifically designed for either 18S or 25S rRNAs.  Several sequences are included and each one is highlighted where the oligo has been attached, 

Funding

Elias, Genevieve, Georgianna/ Atol Charitable Trust

The Monica Lester Charitable Trust