Small RNA-induced mRNA degradation achieved through both translation block and activated cleavage

Abstract

Small RNA (sRNA)-induced mRNA degradation occurs through binding of an sRNA to a target mRNA with the concomitant action of the RNA degradosome, which induces an endoribonuclease E (RNase E)-dependent cleavage and degradation of the targeted mRNA. Because many sRNAs bind at the ribosome-binding site (RBS), it is possible that the resulting translation block is sufficient to promote the rapid degradation of the targeted mRNA. Contrary to this mechanism, we report here that the pairing of the sRNA RyhB to the target mRNA sodB initiates mRNA degradation even in the absence of translation on the mRNA target. Remarkably, even though it pairs at the RBS, the sRNA RyhB induces mRNA cleavage in vivo at a distal site located >350 nucleotides (nt) downstream from the RBS, ruling out local cleavage near the pairing site. Both the RNA chaperone Hfq and the RNA degradosome are required for efficient cleavage at the distal site. Thus, beyond translation initiation block, sRNA-induced mRNA cleavage requires several unexpected steps, many of which are determined by structural features of the target mRNA.

Figure 1.

Minimal sequence requirement for sRNA-induced mRNA degradation in vivo. (A) Description of the different constructs used to determine the minimal sodB to generate RyhB-induced mRNA degradation. (B) Northern blots with a lacZ probe showing the effect of RyhB expression (for 10 min) on various transcriptional sodB-lacZ constructs and the endogenous sodB transcript. The expression of the endogenous sodB transcript and RyhB is also shown. 16S ribosomal RNA was used as a loading control. (C) Northern blots using a lacZ probe showing the effect of RyhB expression (for 10 min) on various translational SodB-LacZ constructs. The expression of the endogenous sodB transcript and RyhB is also shown. 16S ribosomal RNA was used as a loading control.

Figure 2.

RyhB-induced mRNA degradation of various target mRNAs. Northern blots with a lacZ probe showing the effect of RyhB expression (for 10 min) on various transcriptional fumA-lacZ constructs (A) and transcriptional iscRS-lacZ constructs (B). RyhB pairs at nucleotides 664–670 of iscRS, which corresponds to the RBS of iscS. Whereas the iscRS₆₈₇ fusion extends 19 nt into the iscS ORF, the iscRS₁₂₆₈ fusion extend 600 nt into iscS ORF. Expression of endogenous sodB transcript and RyhB is also shown. 16S ribosomal RNA was used as loading control.

Figure 3.

Both the RNA degradosome and RNA chaperone Hfq are essential for the RyhB-induced degradation of the minimal target sodB₄₃₀-lacZ. (A) Northern blots using a lacZ probe showing the effect of RyhB expression (for 10 min) on transcriptional sodB₄₃₀-lacZ fusions (left panel) in wild-type cells (lanes 1,2) and rne131 cells (lanes 3,4), and translational SodB₄₃₀-LacZ fusions (right panel) in wild-type cells (lanes 5,6) and rne131 cells (lanes 7,8). (B) Effect of RyhB expression on β-galactosidase activity of transcriptional sodB₄₃₀-lacZ fusions in wild-type and rne131 cells, and translational SodB₄₃₀-LacZ fusions in wild-type and rne131 cells. (C) Northern blots using a lacZ probe showing the effect of RyhB expression (for 10 min) on transcriptional sodB₄₃₀-lacZ fusions (left panel) in wild-type cells (lanes 1,2) and hfq cells (lanes 3,4), and translational SodB₄₃₀-LacZ fusions (right panel) in wild-type cells (lanes 5,6) and rne131 cells (lanes 7,8). (D) Effect of RyhB expression on β-galactosidase activity of transcriptional sodB₄₃₀-lacZ fusions (left panel) in wild-type and hfq cells, and translational SodB₄₃₀-LacZ fusions (right panel) in wild-type cells and hfq cells.

Figure 4.

The putative cleavage site of sodB₄₃₀ promotes the degradation of a RyhB-resistant sodB₁₃₀ construct. (A) Schematic description of sodB₄₃₀ and sodB_{130 + 30}, with the putative cleavage site represented by the black triangle. (B) Northern blots using a lacZ probe showing the effect of RyhB expression (for 10 min) on sodB₁₃₀-lacZ and sodB_{130 + 30}-lacZ transcriptional fusions. (C) Northern blots using a lacZ probe showing the effect of RyhB during a time-course expression on sodB₁₃₀-lacZ, sodB_{130 + 30}-lacZ, sodB₄₃₀-lacZ, sodB₄₅₈-lacZ, and sodB₆₀₇-lacZ.

Figure 5.

In vitro mapping and mutagenesis of the minimal sodB₄₃₀ cleavage site. (A) Effect of increasing amounts of purified RNA degradosomes (0.5, 0.76, 1.0, and 1.5 ng/μL final) on ³²P-pCp 3′-end radiolabeled sodB₄₅₈ (left panel, lanes 4–8) and sodB_458GCC (right panel, lanes 12–16). (Lanes 1,9) RNase TA ladder. (Lanes 2,10) RNase T1 ladder. (Lanes 3,11) NaOH ladder. (Lanes 4,12) Radiolabeled RNA alone. (B) Local nucleotide sequence (between G400 and G413) of the cleavage site from sodB₄₃₀ (top line) and the mutated sodB_430GCC (bottom). (C) Northern blots using a lacZ probe on sodB₄₃₀-lacZ and sodB_430GCC-lacZ transcriptional fusions in the absence or presence of RyhB expression (for 10 min).

Figure 6.

Translation block alone in the absence of RyhB is not sufficient to cause full degradation of sodB mRNA. (A) Schematic description of the different sodB constructs used to monitor the effect of translation on mRNA steady-state level and stability. The gray bar represents sodB ORF. The arrowhead is the cleavage site at position 407. UAA30 is the stop codon introduced to prematurely terminate translation. GCC represents the mutation introduced to inactivate the cleavage site at position 407. (B) Northern blots using a lacZ probe showing the steady-state levels of sodB₄₃₀, sodB_430UAA30, sodB_430GCC, and sodB_430UAA30GCC constructs. (C) Northern blots using a lacZ probe showing the steady-state level of fumA₃₉₉, fumA_399UAA30, fumA_399GCC, and fumA_399UAA30GCC constructs. (D) Northern blots using a lacZ probe showing the stability of sodB₄₃₀, sodB_430UAA30, sodB_430GCC, and sodB_430UAA30GCC constructs. Rifampicin (250 μg/mL) was added at time 0 before total RNAs were extracted at the indicated time points. Control 16S are shown in Supplemental Figure S8.

Figure 7.

RyhB-induced mRNA cleavage in prior absence of translation on the target mRNA sodB₄₃₀-lacZ. Northern blots using a lacZ probe showing the effect of RyhB expression in wild-type cells on sodB₄₃₀ (A), sodB_430GCC (B), sodB_430UAA30 (C), and sodB_430UAA30GCC (D). (E) Northern blots using a lacZ probe showing the effect of RyhB expression on sodB_430UAA30 in rne131 cells.

Figure 8.

Model for sRNA-induced mRNA degradation (see the text for details).