While in eukaryotes three RNA polymerases are involved in ribosome production  under usual growth conditions, the 18S and 25S ribosomal RNA (rRNA) components are thought to be exclusively the products of transcription by RNA polymerase I (Pol I) followed by processing . We have observed recently, in Candida albicans during nutritional depletion and with TOR inhibition, the appearance of 18S and 25S rRNA molecules, resisting digestion by a 5′-phosphate-dependent exonuclease, indicating that they were different from the usual processed rRNA transcripts . Candida albicans, a eukaryotic yeast, is a major cause of invasive fungal disease especially in immune compromised patients . Ribosomes of eukaryotic cells are assembled from four individual rRNAs and 79 proteins . As in Saccharomyces cerevisiae, genes coding for rRNA (rDNA) in C. albicans are repeated multiple times in tandem , allowing for efficient transcription by Pol I. Like other eukaryotes, the current accepted mechanism of the production of the 18S and 25S components of the ribosome in this yeast, is transcription of a 35S copy of the rDNA, followed by post and co-transcriptional processing of the nascent RNA . Typically, processed RNA molecules will have a single phosphate on their 5’-end making them vulnerable to processive 5′→3′ exonucleases (P53E) that digests only RNA that has a 5′-monophosphate end . Therefore, after digestion by such an exonuclease, it was unexpected to find 18S and 25S rRNA molecules in total RNA isolated from C. albicans entering its stationary phase . Similar molecules were appearing also in yeast, whose TOR was inhibited by rapamycin . This background information is illustrated in Fig 1A. Pyrophosphatase digestion which separates linked phosphates, made these resistant 18S and 25S molecules vulnerable again to 5’-exonulease digestion . This indicated that these molecules contained more than a single phosphate at their 5’-end. This in turn raised the possibility that they were newly transcribed rather than processed, as polymerases use triphosphate nucleotides when they initiate transcription. Another serial enzyme digestion included alkaline phosphatase (AP) followed by P53E digestion, in this case the rRNAs remained protected . This could have been due to either more than one phosphate being digested by AP resulting in 5′-OH, or further modification in rRNA, such as a 5’-cap protecting against both AP and P53E, again preventing exonuclease digestion. Additionally, we have previously found that C. albicans grown overnight, polyadenylates some of its 18S  and 25S  rRNA molecules, a feature associated with Pol II transcription . These features prompted us to see whether Pol II is involved with ribosomal rRNA transcription.
Features Poly(A) RNA Selection Kit