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. 2020 Jun 19;17(10):1427–1441. doi: 10.1080/15476286.2020.1771946

Figure 3.

Figure 3.

Determination of the presence and percentages of circular pre-rRNA intermediates in total cellular rRNA pool. (a) Schematic shows the predicted circularization coupled with EndA cleaving at BHB motifs (grey shaded boxes, upper panel) to separate pre-16S and pre-23S rRNA from the polycistronic transcript; the spliced products are presumably ligated by RtcB as circular pre-rRNA intermediates (lower panel). Thick black lines indicate mature rRNAs. Thin dark cyan and red lines indicate the precursor sequences from BHB 5′ splice site to the mature 5′ end of 16S or 23S rRNA and that from the mature 3′ end of 16S or 23S rRNA to BHB 3′ splice site, respectively. Same labels are also used in the following figures. (b) Detection of the circular pre-16S and pre-23S rRNA intermediates using ligation-omitted cRT-PCR as described in Materials and methods. The PCR primer pairs are shown in (A), the nested PCR products on cDNA templates that are reverse-transcribed from total RNA with (+) or without (−) T4 RNA ligase pretreatment were detected on a 2% agarose gel (upper panel). Longer and shorter arrows specify the products of the first and second round of PCR, respectively. The lower panels show the DNA sequencing results of the PCR products with an inverted ligation between the 3′ half and 5′ half of 16S and 23S rRNA BHBs. (c–d) Northern blot detected the circular pre-16S (c) and pre-23S rRNA intermediate (d) (left panel) and total cellular 16S or 23S rRNA (right panel) using different probes that spanned the junction region or targeted the mature ends of rRNAs. Locations of the probes are shown in (A). Total RNA contents used are indicated at the top of panel. M1 and M2 indicated ssRNA size markers. (E–F) qRT-PCR determined the percentages of circular pre-rRNA intermediates in the total pool using primers matching the precursor and mature sequences. Percentages of the circular pre-rRNA intermediates in the total were determined at the early, middle, late and stationary cultures. All of the data were calculated from three independent measurements and standard deviations are shown.