Growth phase-dependent control of transcription start site selection and gene expression by nanoRNAs

Abstract

Prokaryotic and eukaryotic RNA polymerases can use 2- to ∼4-nt RNAs, "nanoRNAs," to prime transcription initiation in vitro. It has been proposed that nanoRNA-mediated priming of transcription can likewise occur under physiological conditions in vivo and influence transcription start site selection and gene expression. However, no direct evidence of such regulation has been presented. Here we demonstrate in Escherichia coli that nanoRNAs prime transcription in a growth phase-dependent manner, resulting in alterations in transcription start site selection and changes in gene expression. We further define a sequence element that determines, in part, whether a promoter will be targeted by nanoRNA-mediated priming. By establishing that a significant fraction of transcription initiation is primed in living cells, our findings contradict the conventional model that all cellular transcription is initiated using nucleoside triphosphates (NTPs) only. In addition, our findings identify nanoRNAs as a previously undocumented class of regulatory small RNAs that function by being directly incorporated into a target transcript.

Figure 1.

Ectopic production of a nanoRNase (Orn or NrnB) prevents RNA polymerase (RNAP) from using nanoRNAs as primers for transcription initiation.

Figure 2.

Effects of ectopic production of a nanoRNase on transcription start site selection. (A) Analysis of transcription start sites during the exponential phase of growth. Average percentage of all transcripts initiated at positions −3 to +3 for the 270 promoters listed in Supplemental Table 1 as determined by high-throughput sequencing. Analysis was performed using RNA transcripts isolated from E. coli cells during the exponential phase of growth that harbored an empty plasmid (wt) or a plasmid that specifies production of E. coli Orn, B. subtilis NrnB, or NrnB^DHH. Plotted are the mean + SD derived from two independent measurements. (B,C) Analysis of transcription start sites during the stationary phase of growth. Average percentage of all transcripts (B) or 5′ triphosphate-carrying transcripts (C) initiated at positions −3 to +3 for the seven promoters with start sites that were significantly affected by ectopic production of a nanoRNase (Supplemental Table 3) as determined by high-throughput sequencing. Analysis was performed using RNA transcripts isolated from E. coli cells during the stationary phase of growth that harbored an empty plasmid (wt) or a plasmid that specifies production of E. coli Orn, B. subtilis NrnB, or NrnB^DHH. Plotted are the mean + SD derived from two independent measurements. (D) Average percentage of all transcripts (all), 5′ triphosphate-carrying transcripts (ppp), 5′ monophosphate-carrying transcripts (p), or 5′ hydroxyl-carrying transcripts (OH) initiated at positions −3 to +3 for the four promoters listed in Supplemental Table 4 as determined by high-throughput sequencing. Analysis was performed using RNA transcripts isolated from E. coli cells during the stationary phase of growth that harbored an empty plasmid. Plotted are the mean + SD derived from two independent measurements.

Figure 3.

Growth phase-dependent regulation of transcription start site selection by nanoRNA-mediated priming. (A) Average percentage of all transcripts initiated at positions −3 to +3 for the five promoters listed in Supplemental Table 5 as determined by high-throughput sequencing. Analysis was performed using RNA transcripts isolated from E. coli cells during the exponential phase of growth (exp) or the stationary phase of growth (sta) from cells harboring an empty plasmid (wt) or a plasmid that specifies production of E. coli Orn, B. subtilis NrnB, or NrnB^DHH. Plotted are the mean + SD derived from two independent measurements. (B) Primer extension analysis of transcript 5′ ends generated during transcription initiation from plasmid-borne copies of the promoters associated with tomB and bhsA. Analysis was performed using RNA transcripts isolated from E. coli cells during the exponential phase of growth (exp) or the stationary phase of growth (sta). Cells harbored a plasmid carrying the indicated promoter along with an empty plasmid (wt) or a plasmid that specifies production of E. coli Orn, B. subtilis NrnB, or NrnB^DHH. Putative −10 and −35 elements of each promoter are highlighted in red. Position +1 is indicated by the arrow.

Figure 4.

Growth phase-dependent regulation of gene expression by nanoRNA-mediated priming. (A) Northern blot analysis of tomB and bhsA transcripts during the exponential phase of growth or the stationary phase of growth in cells that harbored an empty plasmid (wt) or a plasmid that specifies production of B. subtilis NrnB. The bottom panel shows an ethidium bromide-stained gel of the RNA samples used for Northern blotting. The two most prominent bands correspond to the 23S and 16S rRNAs. (B) Graphs show the abundance of bhsA transcripts relative to the abundance of rpoD transcripts during the exponential phase of growth or the stationary phase of growth in cells that harbored an empty plasmid (wt) or a plasmid that specifies production of E. coli Orn, B. subtilis NrnB, or NrnB^DHH. Relative transcript abundance was measured by quantitative real-time RT–PCR using the comparative Ct method (ΔΔCt) (Livak and Schmittgen 2001). Plotted are the means derived from three independent measurements. Error bars represent the relative expression values calculated from plus or minus one SD from the mean ΔΔCt.

Figure 5.

−1/+1 TA represents a nanoRNA response element. (A) Sequences of seven promoters with start sites that were significantly affected by ectopic production of a nanoRNase during the stationary phase of growth. Putative −10 and −35 elements of each promoter are highlighted in red. Position +1 is indicated by the arrow. (B) Average percentage of all transcripts initiated at positions −3 to +3 for the 44 promoters listed in Supplemental Table 2 that carry a T at position −1 and an A at position +1 (top) or the 181 promoters listed in Supplemental Table 2 that did not carry a T at position −1 and an A at position +1 (bottom). Analysis was performed using RNA transcripts isolated from E. coli cells during the stationary phase of growth that harbored an empty plasmid (wt) or a plasmid that specifies production of E. coli Orn, B. subtilis NrnB, or NrnB^DHH. Plotted are the mean + SD derived from two independent measurements. (C) Primer extension analysis of transcript 5′ ends generated during transcription initiation from the wild-type tomB and bhsA promoters (−1/+1 TA) or a derivative carrying an A+1G substitution (−1/+1 TG). Analysis was performed using RNA transcripts isolated from E. coli cells during the exponential phase of growth (exp) or the stationary phase of growth (sta). Cells harbored a plasmid carrying the indicated promoter along with an empty plasmid (wt) or a plasmid that specifies production of B. subtilis NrnB. (D) Primer extension analysis of transcript 5′ ends generated during transcription initiation from the indicated derivative of the lacUV5 promoter. Analysis was performed using RNA transcripts isolated from E. coli cells during the exponential phase of growth (exp) or the stationary phase of growth (sta). Cells harbored a plasmid carrying the indicated lacUV5 promoter derivative along with an empty plasmid (wt) or a plasmid that specifies production of E. coli Orn, B. subtilis NrnB, or NrnB^DHH. The −10 and −35 elements of the lacUV5 promoter are highlighted in red. Position +1 is indicated by the arrow.

Figure 6.

Model of growth phase-dependent nanoRNA-mediated priming of transcription initiation. The accumulation of nanoRNAs beginning with the sequence UA and carrying a 5′ hydroxyl during the stationary phase of growth is depicted. We propose that the accumulation of these nanoRNAs occurs through the degradation of full-length transcripts initiated by a cleavage event specifically targeted to the phosphodiester bond 5′ to the sequence UA (in red). Subsequent processing of these fragments by 3′-to-5′ exonucleases results in the generation of 2- to 4-nt nanoRNAs that are either degraded by Orn or used to initiate transcription from promoters carrying a −1/+1 TA sequence element.