NanoRNAs Prime Transcription Initiation In Vivo Seth R. Goldman, Josh S. Sharp, Irina O. Vvedenskaya, Jonathan Livny, Simon L. Dove, Bryce E. Nickels  Molecular Cell  Volume 42, Issue 6, Pages 817-825 (June 2011) DOI: 10.1016/j.molcel.2011.06.005 Copyright © 2011 Elsevier Inc. Terms and Conditions

Molecular Cell 2011 42, 817-825DOI: (10.1016/j.molcel.2011.06.005) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 1 Controlled Depletion of Orn in P. aeruginosa (A) Strategy to enable controlled depletion of Orn. Schematic depicts how the presence of SspB targets Orn-VDAS4 for ClpXP-mediated degradation. (B) Controlled depletion of Orn. JS2 cells (lanes 1–3), which express Orn-VDAS4, or JS3 cells (lanes 4–6), which express Orn-VDAS4 and carry VSV-G-tagged SspB (V-SspB) under the control of an IPTG-inducible promoter, were first grown to mid-log (time 0), then grown for a further 90 min either in the absence (−) or presence of IPTG (+). Cells were harvested at the indicated time points and analyzed for protein content by western blot using an antibody against the VSV-G tag (upper panel) or, to control for sample loading, an antibody against the α subunit of RNAP (bottom panel). (C) Depletion of Orn does not affect the cellular growth rate. Growth of JS3 cells, JS2 cells, and JS1 cells (which express untagged Orn and carry V-SspB under the control of an IPTG-inducible promoter) was monitored over a 24 hr period. Arrow indicates the time point at which IPTG (when present) was added to cells. (D) Accumulation of nanoRNAs in vivo. JS3 cells, JS2 cells, or JS1 cells were grown to mid-log in the presence of 32Pi, then grown for a further 90 min either in the absence (−) or presence of IPTG (+). Cells were either harvested immediately (–) or grown in the presence of rifampicin (+) for an additional 60 min prior to harvesting. The acid soluble fraction obtained from cell extracts was either treated with purified Orn (+) or untreated (–) prior to electrophoresis on a 22.5% denaturing acrylamide gel. Sizes of labeled nanoRNAs were estimated based upon comparison to RNA standards that carry a 5′ triphosphate group and a 3′ hydroxyl group (lane 1). RNA standards carrying a 5′ monophosphate group and a 3′ hydroxyl group migrate within a similar size range as the 5′ triphosphate-carrying standards (Figure S5). The asterisk in lane 2 indicates an Orn-insensitive impurity in the RNA standards that migrates at the same position as the 2 nt transcript. We note that nondepleted JS3 cells carry ∼2-fold less Orn-VDAS4 than JS2 cells (B). Furthermore, nondepleted JS3 cells accumulate 2 nt nanoRNAs upon treatment with rifampicin, whereas JS2 cells do not (compare lanes 9 and 17). Thus, even a modest reduction in the abundance of Orn appears to enable the accumulation of detectable quantities of 2 nt nanoRNAs in rifampicin-treated P. aeruginosa cells. Molecular Cell 2011 42, 817-825DOI: (10.1016/j.molcel.2011.06.005) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 2 Priming of Transcription Initiation with 2–4 nt RNAs Can Alter the TSS In Vitro Top shows galP1/cons promoter sequences extending from position –5 to +3 (the galP1/cons promoter is a derivative of the E. coli galP1 promoter with a consensus extended –10 element). Bottom shows results of primer extension analysis of RNA transcripts produced during in vitro transcription assays performed using a DNA fragment containing the galP1/cons promoter (10 nM). Assays were done using P. aeruginosa RNAP (50 nM) in the presence of 100 μM NTPs in the absence (–) or presence of 100 μM of the indicated 2–4 nt RNA (see the Supplemental Information for details). The positions of the 5′ and 3′ ends of each small RNA are indicated below the gel along with the percentage of transcripts shifted by each RNA (averages of duplicate measurements). Highlighted in red are RNAs that effectively compete with NTPs and shift the TSS of >10% of transcripts initiated from galP1/cons. Molecular Cell 2011 42, 817-825DOI: (10.1016/j.molcel.2011.06.005) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 3 NanoRNAs Prime Transcription Initiation In Vivo (A–C) Accumulation of nanoRNAs leads to TSS shifting as detected by high-throughput sequencing. Graphs show average percentage of all transcripts (A and C) or 5′ triphosphate carrying transcripts (B) initiated at positions –3 to +3 relative to the primary TSS for 148 promoters. Data were derived from the analysis of transcripts isolated from cells of strain JS1.F harboring plasmid pPSV35 (nondepleted), and cells of strain JS3.F harboring plasmids pPSV35 (depleted), pPAOrn (depleted + Orn), or pNrnB (depleted + NrnB). Plotted are the averages and standard deviations for two independent measurements (Table S3). (D) TSS shifting observed in vivo can be recapitulated in vitro using 2 nt RNAs. Shown is the sequence of the rrnP2 promoter, the promoter associated with PA4843, and the promoter associated with PA3978. The primary TSS (position +1) is indicated by the arrow, and putative promoter elements are in red. The box on the bottom left of each panel shows the results of primer extension analysis performed using transcripts isolated from the indicated cells. The box on the bottom right of each panel shows primer extension analysis of RNA transcripts produced during in vitro transcription assays performed in the absence (–) or presence of the indicated 2 nt RNA (see the Supplemental Information for details). Molecular Cell 2011 42, 817-825DOI: (10.1016/j.molcel.2011.06.005) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 4 NanoRNA-Mediated Priming of Transcription Initiation In Vivo Leads to Changes in Gene Expression (A and B) Effect of Orn depletion on gene expression in P. aeruginosa as determined by DNA microarray. Data were derived from the analysis of transcripts isolated from cells of strain JS1.F harboring plasmid pPSV35 (nondepleted), and cells of strain JS3.F harboring plasmids pPSV35 (Orn-depleted), pPAOrn (Orn-depleted + Orn), or pNrnB (Orn-depleted + NrnB). The table in (A) shows the number of genes whose expression changes by a factor of 2 or more upon depletion of Orn-VDAS4, or upon depletion of Orn-VDAS4 in the presence of either wild-type Orn (supplied by plasmid pPAOrn) or NrnB (supplied by plasmid pNrnB). (B) shows a heatmap representation of the 1158 genes whose expression changes by a factor of 2 or more upon depletion of Orn-VDAS4. Also shown are the corresponding effects on expression of these genes when either wild-type Orn or NrnB is supplied ectopically and Orn-VDAS4 is depleted. Molecular Cell 2011 42, 817-825DOI: (10.1016/j.molcel.2011.06.005) Copyright © 2011 Elsevier Inc. Terms and Conditions