Background

We have previously found that, in the pathogenic yeast Candida albicans, 18S and 25S ribosomal RNA components, containing more than one phosphate on their 5'-end were resistant to 5'-monophosphate requiring 5' → 3″ exonuclease. Several lines of evidence pointed to RNAP II as the enzyme producing them.

Results

We now show the production of such 18S and 25S rRNAs in Saccharomyces cerevisiae that have been permanently switched to RNAP II (due to deletion of part of RNAP I upstream activator alone, or in combination with deletion of one component of RNAP I itself). They contain more than one phosphate at their 5'-end and an anti-cap specific antibody binds to them indicating capping of these molecules. These molecules are found in RNA isolated from nuclei, therefore are unlikely to have been modified in the cytoplasm.

Conclusions

Our data confirm the existence of such molecules and firmly establish RNAP II playing a role in their production. The fact that we see these molecules in wild type Saccharomyces cerevisiae indicates that they are not only a result of mutations but are part of the cells physiology. This adds another way RNAP II is involved in ribosome production in addition to their role in the production of ribosome associated proteins.

Objectives:

Cell therapies using mesenchymal stromal cells (MSCs) have been proposed as a promising new tool for the treatment of vocal fold scarring. However, the mechanisms by which MSCs promote healing as well as their duration of survival within the host vocal fold have yet to be defined. The aim of this work was to assess the persistence of embedded MSCs within a tissue-engineered vocal fold mucosal replacement in a rabbit model of vocal fold injury.

Methods:

Male rabbit adipose-derived MSCs were embedded within a 3-dimensional fibrin gel, forming the cell-based outer vocal fold replacement. Four female rabbits underwent unilateral resection of vocal fold epithelium and lamina propria and reconstruction with cell-based outer vocal fold replacement implantation. Polymerase chain reaction and fluorescent in situ hybridization for the sex-determining region of the Y chromosome (SRY-II) in the sex-mismatched donor-recipient pairs sought persistent cells after 4 weeks.

Results:

A subset of implanted male cells was detected in the implant site at 4 weeks. Many SRY-II-negative cells were also detected at the implant site, presumably representing native female cells that migrated to the area. No SRY-II signal was detected in contralateral control vocal folds.

Conclusions:

The emergent tissue after implantation of a tissue-engineered outer vocal fold replacement is derived both from initially embedded adipose-derived stromal cells and infiltrating native cells. Our results suggest this tissue-engineering approach can provide a well-integrated tissue graft with prolonged cell activity for repair of severe vocal fold scars.

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 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.

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.