Coupled degradation of a small regulatory RNA and its mRNA targets in Escherichia coli

Abstract

RyhB is a small antisense regulatory RNA that is repressed by the Fur repressor and negatively regulates at least six mRNAs encoding Fe-binding or Fe-storage proteins in Escherichia coli. When Fe is limiting, RyhB levels rise, and target mRNAs are rapidly degraded. RyhB is very stable when measured after treatment of cells with the transcription inhibitor rifampicin, but is unstable when overall mRNA transcription continues. We propose that RyhB turnover is coupled to and dependent on pairing with the target mRNAs. Degradation of both mRNA targets and RyhB is dependent on RNase E and is slowed in degradosome mutants. RyhB requires the RNA chaperone Hfq. In the absence of Hfq, RyhB is unstable, even when general transcription is inhibited; degradation is dependent upon RNase E. Hfq and RNase E bind similar sites on the RNA; pairing may allow loss of Hfq and access by RNase E. Two other Hfq-dependent small RNAs, DsrA and OxyS, are also stable when overall transcription is off, and unstable when it is not, suggesting that they, too, are degraded when their target mRNAs are available for pairing. Thus, this large class of regulatory RNAs share an unexpected intrinsic mechanism for shutting off their action.

Figure 1.

Degradation of full-length sodB mRNA. (A) Cells (EM1055, wild-type) were grown at 37°C to an O.D.₆₀₀ of 0.4. 2,2′-dipyridyl (dip) was added at 250 μM and samples taken as indicated and RNA extracted as described in Materials and Methods. Northern blots for sodB and RyhB were done as described previously (Massé and Gottesman 2002). (B) ryhB^- cells (EM1238) were grown and processed as in A. (C) Half-life of sodB message. EM1055 cells were grown as in A, but without the addition of 2,2′-dipyridyl (dip). Rifampicin was added at 250 μg/mL at time 0, and samples taken as indicated.

Figure 2.

RNase III is not required for sodB degradation. Cells were grown in LB at 37°C. At an O.D.₆₀₀ of 0.3, the culture was split and 250 μM 2,2′-dipyridyl was added to one culture and growth was continued for 15 min. The total RNA was then extracted as described in Materials and Methods and sodB mRNA detected by Northern blot. (A) EM1055 (wild-type). (B) EM1321 (rnc-14::Tn10). (C) EM1322 (rnc-14::Tn10ΔryhB::cat).

Figure 3.

RNase E is necessary for degradation of RyhB targets. (A) Strains were grown at 30°C to an O.D.₆₀₀ of 0.4, a portion of the culture was shifted to 43.5°C for 10 min, and 250 μM 2,2′-dipyridyl was added to a portion of the 30°C and a portion of the 43.5°C cultures. After 10 min, samples were harvested, and RNA extracted and analyzed as described in Materials and Methods. The strains used were EM1279 (wild-type), EM1277 (rne-3071), EM1280 (rne-3071 ΔryhB::cat), and EM1282 (ΔryhB::cat). (B) Strains were grown at 37°C to an O.D.₆₀₀ of 0.4, induced for 10 min with 2,2′-dipyridyl, and samples removed and processed as in A. Strains used were EM1055 (wild-type), EM1368 (pnp::tn5), EM1375 (rneΔ10), EM1376 (rneΔ14), and EM1377 (rne-131). RyhB levels were determined by dot blot (Materials and Methods). (C) Experiment as in B. The strain used is EM1377 (rne-131), but samples removed up to 20 min after 2,2′-dipyridyl addition. (D) Cells (EM1377, rne-131) grown and treated with rifampicin as in Figure 1C.

Figure 4.

Northern blot analysis of RyhB stability. (A) Strains were grown at 37°C to an O.D.₆₀₀ of 0.4., and 250 μM 2,2′-dipyridyl was added. After 10 min of incubation, rifampicin was added at 250 μg/mL, incubation continued, and samples were removed and RNA was extracted as decribed in Materials and Methods. The strain used is EM1266 (hfq-2; Hfq⁺ phenotype). (B) Experiment as in A. The strain used is EM1265 (hfq-1:Hfq^- phenotype). (C) Strains were grown at 30°C to an O.D.₆₀₀ of 0.4, then the culture was shifted to 43.5°C for 10 min, and 250 μM 2,2′-dipyridyl was added. After 10 min, samples were harvested and RNA extracted. The strain used is EM1342 (hfq-1rne-3071). (D) Strains were grown at 37°C to an O.D.₆₀₀ of 0.4., and 250 μM 2,2′-dipyridyl was added. After 5 min of incubation, FeSO₄ was added at 100 μM. Samples were removed at the time indicated, and RNA was extracted as decribed in Materials and Methods. The strain used is EM1055 (wild-type). Northern blot analysis for sodB was performed as described (Massé and Gottesman 2002). (E) As in D, except that rifampicin (250 μg/mL) was added simultaneously with FeSO₄. The strain used is EM1055 (wild-type).

Figure 5.

Alternative promoters for small RNA expression. (A) EM1059 + pBAD-ryhB. Cells were grown in LB with ampicillin (50 μg/mL) to an O.D.₆₀₀ of 0.4, and arabinose added (0.01%) for 15 min. The culture was centrifuged briefly (5 min) to wash out the arabinose, and resuspended in an equal volume of LB with glucose (0.2%), either with or without rifampicin (250 μg/mL). Samples were removed at the intervals shown and RNA extracted and probed for RyhB. The -15 (min) panel shows sodB expression right before addition of arabinose. (B) Same as A but with pBAD-dsrA. The strain used is EM1063 + pBAD-dsrA. (C) Same as A but with pBAD-oxyS. The strain used is EM1059 + pBAD-oxyS.

Figure 6.

RNase dependence of RyhB degradation. (A) Strains were grown at 30°C to an O.D.₆₀₀ of 0.4, then the culture was shifted to 43.5°C for 10 min, and 250 μM 2,2′-dipyridyl was added. After 5 min of incubation, FeSO₄ was added at 100 μM. Samples were removed at time indicated and RNA was extracted as decribed in Materials and Methods. The strain used is EM1279 (wild-type). (B) Same as in A. The strain used is EM1277 (rne-3071). (C) Strains were grown at 37°C to an O.D.₆₀₀ of 0.4., and 250 μM 2,2′-dipyridyl was added. After 5 min of incubation, FeSO₄ was added at 100 μM. Samples were removed at times indicated and RNA was extracted as decribed in Materials and Methods. The strain used is EM1377 (rne-131).

Figure 7.

Model for self-limited regulation by a small pairing RNA. Normal growth: Only small amounts of the small RNA (sRNA) may be expressed; these will be used quickly, clearing them from the cell. Hfq may bind to some portion of the target mRNAs. Step 1: In response to a stress or other regulatory signal (Fe limitation for RyhB), synthesis of the sRNA rapidly increases and is bound by Hfq (yellow ring). Step 2: The small RNA pairs with its target. Pairing is frequently but not always near the start of translation; in some cases, two or more short (7-9 nucleotides) regions of complementarity are used (for review, see Gottesman 2002). Both small RNA and target mRNA are degraded rapidly in an RNase E-dependent process (scissors). As long as the small RNA continues to be synthesized at a high rate, it will accumulate, and target messages will be destroyed. Step 3: After the stress signal is lost, synthesis of the small RNA will drop to basal levels. The sRNA that has accumulated will be degraded as it is used, allowing reaccumulation of the target mRNAs (Normal growth).