Degradation of the <i>mreBH ykpC</i> mRNA depends on both RNase Y and J1.

<p>(A) Example of trace of the expression data around the <i>mreBH ykpC</i> locus. Lanes are as follows (from top to bottom): (1) Genbank annotation, (2) effect of the depletion of each RNase (log2 ratio −IPTG to wt, calculated on normalized values) on the positive strand, (3) expression signal of wt and RNase depleted strains (normalized log2 values) on the positive strand, (4) summary of the gene-level statistical analysis showing primary (1°) RNase effect (5) expression signal on the negative strand, (6) effect of the depletion of each RNase on the negative strand. The color code for experiments is: wild-type, green; RNase III, violet; RNase J1, blue; RNase Y, red. In the plots of expression signal (lanes 3 and 5), the horizontal black line represents the global median over the whole chromosome and the two horizontal gray lines indicate 5× and 10× this value. In the plots of log2 ratios, the horizontal black line corresponds to base-line (no change) and two horizontal gray lines on either side indicate 2× up and 2× down changes. The summary of the gene-level statistical analysis shows which genes or expression segments were affected by the depletion of at least one of the three RNases: thick green line, gene showing decreased expression in at least one RNase depletion experiment; thick violet, blue or red line, gene showing increased expression in at least one RNase depletion experiment (in this case, the color indicates which depletion was observed to have the greatest effect); thick gray line, gene showing both increased and decreased expression depending on the RNase considered. Color codes for the Genbank annotation are as follows: cyan and magenta, annotated protein coding sequences on the positive and negative strands, respectively (solid symbol when function is known; hollow symbol when function is considered unknown in Genbank); red, ribosomal RNA; dark blue, tRNA; green, Misc_RNA. Traces were plotted using MuGen <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002520#pgen.1002520-Hoebeke1" target="_blank">[43]</a>. Only the signal from unique oligos are shown; gaps are due to non-unique genome sequences. Note: care should be taken when interpreting the log2 ratio signal on the non-coding strand because ratios of values close to background do not have a direct biological interpretation. In particular, these ratios are affected by artifacts such as those caused by reverse transcriptase copying of the cDNA strand, despite the presence of 40 µg/mL actinomycin D in this step. (B) Structure and predicted degradation pathway(s) of the <i>mreBH ykpC</i> transcript. ORFs are shown as large white arrows, transcripts as thin black arrows. Scissors indicate cleavage by RNase Y, ‘Pacman’ symbols represent 5′-3′ degradation by RNase J1. A short thick line indicates the position of the probe used. (C) Northern blot of total mRNA isolated at times after addition of rifampicin (rif) from wild-type and RNase Y depleted (<i>Pspac-rny</i>−IPTG) and RNase Y induced (<i>Pspac-rny</i>+IPTG) cells. The blot was probed with 5′-labeled oligo CCB832 (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002520#pgen.1002520.s015" target="_blank">Table S6</a>) and reprobed with oligo HP246 against 5S rRNA. The half-lives of the different RNA species (A, B, C) from the <i>mreBH ykpC</i> are given under the Northern blot. Migration positions of RNA markers are shown to the right of the blot. (D) Northern blot of total mRNA isolated at times after addition of rifampicin (rif) from wild-type and RNase J1 depleted (<i>Pspac-rnjA</i>−IPTG) and RNase J1 induced (<i>Pspac-rnjA</i>+IPTG) cells. Description as in panel (B); nd is not detected.