RNase J mutant phenotypes.

<p>Cultures grown over night, were diluted 10⁵ and 10⁶ times, whereupon 5 µl were spotted on either MH, RH, LB, or LB+Mg agar plates with 10 mg/l uracil, and incubated at the indicated temperatures until the WT colonies reached an approximately similar size. LB+Mg denotes LB plates supplemented with 5 mM MgCl₂. Growth of a strain was then scored by the size (or absence) of visible colonies compared to the WT colonies. A) Previously published strains. B) RNase J mutants generated in this study. A and B were spotted on the same plates.</p

Both maturation and inactivation of RNase P RNA is carried out by RNase J.

<p>A) Histogram showing the percentage of reads mapping to a given position, out of the total number of reads mapping to the putative SA1279-<i>rnpB</i> operon in each strain (shown in parentheses). Only positions of interest are included, but the full data-set can be found in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004207#pgen.1004207.s008" target="_blank">Table S6</a>. +1: The putative transcription start site of SA1279. +452 to +477: The RNase J mutants accumulate RNA with 5′-ends in this region. +485: The putative transcription start site of <i>rnpB</i>, a major detected RNA species in the WT, ΔY and ΔcshA, but very reduced in the RNase J mutants. +499: A major detected RNA species in the WT, ΔY and ΔcshA, however it is absent from the RNase J1 mutants and reduced in the ΔJ2 strain. B) The layout of the region around SA1279 and <i>rnpB</i>. DNA is represented as a wavy line, and RNA transcripts as straight black lines. (PP)P indicates a mix of tri- and mono-phosphorylated RNA, generated by pyrophosphohydrolases. Small blue arrows indicate the PCR-primers used to amplify circularised RnpB and SA1279-RnpB for mapping the 5′ and 3′-ends. R1 indicates the probe used for the Northern blot shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004207#pgen.1004207.s002" target="_blank">Figure S2</a>. C) A blow-up of the region from +420 to +540, showing the proposed model for converting the +1 transcript into mature RnpB. P indicates mono-phosphorylation. D) Predicted secondary structures of RnpB, generated using mfold with default settings <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004207#pgen.1004207-Zuker1" target="_blank">[30]</a>, and based on the crystal structures of RNase P RNA <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004207#pgen.1004207-Reiter1" target="_blank">[27]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004207#pgen.1004207-Kazantsev1" target="_blank">[31]</a>. Within the RNase P structure, the thin dotted arrows indicate the path of the RNA through the secondary and tertiary structure of RNase P, the RBS and start codon of SA1278 are in bold, and the region where the anti-sense RNA can hybridise is indicated with a thick black line. E) The difference in average length of RnpB in WT, ΔJ1, and ΔJ1ΔJ2 strains, revealed by the length of the PCR-product generated across the 5′/3′ junction. Results of the cloned and sequenced PCR-products are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004207#pgen-1004207-t006" target="_blank">Table 6</a>. M: Marker.</p

In ΔJ1ΔJ2, RnpB begins upstream of the +485 putative transcription start site, but 3′-ends are unaffected.

<p>*) If the 5′ base and the 3′ base are identical, thus preventing exact mapping of the ends, then a range of possible 5′ and 3′ positions are given.</p

mRNA maturation by RNase J reveals a potential regulation of translation.

<p>A) Histogram showing the percentage of reads mapping to a given position, out of the total number of reads mapping to the SA2322 transcript in each strain (shown in parentheses). Only positions of interest are included, but the full data-set can be found in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004207#pgen.1004207.s009" target="_blank">Table S7</a>. +1 and +2: The putative transcription start sites. +52: A major detected RNA species in the WT, ΔY and ΔcshA, however it is absent from the RNase J1 mutants and strongly reduced in the ΔJ2 strain. B) The SA2322 locus with important positions indicated. C) A schematic view of the fate of SA2322 transcripts. A newly formed transcript can form a secondary structure, shown in panel D, which partially sequesters the ribosome binding site (RBS). RNase J can shorten the transcript by 51 nt, and is presumably blocked from further exonucleolytic digestion by ribosomes binding to the RBS. (PP)P indicates a mix of tri- and mono-phosphorylated RNA, generated by pyrophosphohydrolases. D) Predicted secondary structure at the 5′-end of the SA2322 transcript. ΔG values predicted by the mfold algorithm are in kcal/mol. RBS and start codon are indicated in bold.</p

Enrichment of immature 16S rRNA in RNase J mutants.

<p>A) The curves show the number of reads mapping to each position of the immature 16S rRNA (mature 16S starts at position M1) in the mutant strains, divided by the number of reads mapping to the same position in the WT data. Data from RNase Y and cshA deletion mutants <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004207#pgen.1004207-Redder1" target="_blank">[21]</a> have been included as controls. The number of reads for each strain has been normalised to the sum of reads mapping to positions between the 16S rRNA RNase III processing site <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004207#pgen.1004207-Lioliou1" target="_blank">[25]</a> and the 3′-end of the mature 16S rRNA. Blue bars indicate how many thousands of reads mapped to each position in the WT data-set. B) Overview of the region important for the maturation steps after RNase III has cleaved the processing stem. Positions of 5′-ends that accumulate in the RNase J mutants are indicated in green, and red positions are much less abundant in the RNase J mutants compared to the WT strain. No significant changes are observed at the yellow positions. M1 denotes the first nucleotide of the mature 16S rRNA. The Anti-Shine-Dalgarno (Anti-SD) and the 3′-end of the mature 16S rRNA are indicated. 5′-ends at positions 28+M and 27+M are relatively frequently observed (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004207#pgen.1004207.s005" target="_blank">Table S3</a>), and might be a hotspot for the ribonuclease that cleaves between 93+M and M1, prior to trimming by RNase J (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004207#s3" target="_blank">discussion</a> for details). The asterisk indicates where two of the five 16S rRNA genes in <i>S. aureus</i> have one base less.</p

Complementation of the RNase J deletions.

<p>Spotting and interpretation was carried out as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004207#pgen-1004207-g001" target="_blank">Figure 1</a>, but with 10 µg/ml chloramphenicol added to both the overnight culture and in the agar plates, to ensure against loss of the complementing plasmids. pEB01 is the empty plasmid. The faint white rings seen randomly distributed on the RH medium are not colonies, but are air bubbles that often form inside the agar matrix during incubation of this medium.</p

SA1075 mRNA inactivation by RNase J competes with translation initiation.

<p>A) Histogram showing the percentage of reads mapping to a given position, out of the total number of reads mapping to the SA1075 transcript in each strain (shown in parentheses). Only positions of interest are included, but the full data-set can be found in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004207#pgen.1004207.s010" target="_blank">Table S8</a>. +1: The putative transcription start site. +16: The major detected RNA species in the WT, ΔY and ΔcshA. +45: Position where ΔJ1ΔJ2 differs from ΔJ1, ΔJ2 and J1^AGA, and appears similar to WT. B) Important positions indicated on the SA1075 gene. +1: Transcription Start Site. RBS: Ribosome Binding Site. R2 indicates the probe used for the Northern blot in panel D. C) Hairpin structure predicted by the mfold algorithm, which sequesters the RBS and start-codon (shown in bold). No secondary structure was predicted for the 50 nucleotides downstream of position +45. D) Northern blot of the SA1075 transcript, using probe R2. The +1 and +16 RNA species are not resolved, and can be seen as a single band, however the +45 species is clearly visible in the ΔJ1, ΔJ2, and J1^AGA strains. The marker was stained with methylene blue and photographed. As a loading control, the Northern blot was stripped and re-probed to detect the 5S rRNA. E) The proposed model for determining the fate of SA1075 mRNA via competition between RNase J, ribosomes and the nuclease that cleaves at position +16. (PP)P indicates a mix of tri- and mono-phosphorylated RNA, generated by pyrophosphohydrolases. Nascent SA1075 mRNA can either be occupied by ribosomes, binding to the RBS, or form Hairpin I which sequesters the RBS. Ribosomes will shield position +45 from RNase J, but the hairpin will not. If cleavage at position +16 occurs before RNase J has cleaved at position +45, then the RBS will be liberated from Hairpin I, and ribosomes can initiate translation. If ever the +45 cut is made by RNase J, then the mRNA, which no longer has RBS or start-codon, is immediately degraded (possibly by the RNase J1+J2 complex). Either RNase J1 or RNase J2 can perform a cleavage at position +45. The loss of both RNases prevents the +45 RNA species from being generated, thus explaining why the WT and the ΔJ1ΔJ2 strains appear similar in panel A (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004207#s3" target="_blank">discussion</a> for details and other potential explanations).</p

Reads mapping to the RNase J1 and RNase J2 genes in the RNase J deletion mutants and in unrelated strains (GHU-12 to 26).

<p>*) The GHU strains have wild-type RNase J loci and are unrelated to this study, except that they originate from the same lineage as strains ΔJ1, ΔJ1 and ΔJ1ΔJ2.</p

Flowchart showing the mapping of 5′ RNA ends with the EMOTE assay.

<p>The 5′-bases of mono-phosphorylated RNA (asterisks) were identified using the following technique. Thin and thick lines denote RNA and DNA, respectively. Open arrows indicate primer elongation. A) The RNA oligo Rp5 (green) is ligated to total RNA from each strain, adding a known sequence to all mono-phosphorylated RNAs (brown), and excluding tri-phosphorylated RNAs (black). The majority of 16S and 23S rRNA are removed by hybridisation to magnetic beads. The two underlined cytidine nucleotides at the 3′-end of Rp5 will be explained below. B) Reverse transcription is performed with a semi-random primer (DROAA), which cannot anneal to the Rp5 sequence. The 5′-ends of the cDNA will all have the sequence of Illumina adaptor B (purple), but only cDNA made from Rp5-ligated RNA will end in a known sequence (D5), which is complementary to Rp5 (green). C) The cDNA from each strain is amplified using one primer, B, that anneals to the Illumina adaptor B (purple), and a second primer, D5xxx, that anneals to D5 (green) and adds the Illumina adaptor A sequence (light green) as well as a short bar-code (light green “XXX”) which is unique to each strain. The D5xxx primers only have the first 16 nt of Rp5, so any PCR product where the D5xxx primers have annealed to non-Rp5 sequence, will not include base 17 and 18 of Rp5. D) The PCR products from all the strains are mixed together and run on an agarose gel, whereupon the appropriate size-range of the smear (P-lanes) is extracted. E) Illumina sequencing of 50 bases from the D5-side then reveals the bar-code (light green, identifying from which strain the original RNA came), the Rp5 sequence (green), the first base in the original mono-phosphorylated RNA (asterisk), and the subsequent 23 bases (brown), allowing an unambiguous mapping of the 5′-end of each detected RNA. F) Bioinformatic analyses to (I) use the bar-code sequence to assign each read to the correct strain. (II) Verify that the two cytosine bases at the 3′-end of the Rp5 sequence are present to remove reads that originate from a mis-priming of the D5xxx primer. (III) align the 24 bases to the genome, to determine the exact position and orientation of each 5′-base. (IV) Tabulate and analyse the data (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004207#s4" target="_blank">materials and methods</a>).</p

Oligos for 5′ EMOTE.

<p>Oligos for 5′ EMOTE.</p