5' End-independent RNase J1 endonuclease cleavage of Bacillus subtilis model RNA

Abstract

Bacillus subtilis trp leader RNA is a small (140-nucleotide) RNA that results from attenuation of trp operon transcription upon binding of the regulatory TRAP complex. Previously, endonucleolytic cleavage by ribonuclease RNase J1 in a 3'-proximal, single-stranded region was shown to be critical for initiation of trp leader RNA decay. RNase J1 is a dual-specificity enzyme, with both 5' exonucleolytic and endonucleolytic activities. Here, we provide in vivo and in vitro evidence that RNase J1 accesses its internal target site on trp leader RNA in a 5' end-independent manner. This has important implications for the role of RNase J1 in RNA decay. We also tested the involvement in trp leader RNA decay of the more recently discovered endonuclease RNase Y. Half-lives of several trp leader RNA constructs, which were designed to probe pathways of endonucleolytic versus exonucleolytic decay, were measured in an RNase Y-deficient mutant. Remarkably, the half-lives of these constructs were indistinguishable from their half-lives in an RNase J1-deficient mutant. These results suggest that lowering RNase Y concentration may affect RNA decay indirectly via an effect on RNase J1, which is thought to exist with RNase Y in a degradosome complex. To generalize our findings with trp leader RNA to other RNAs, we show that the mechanism of trp leader RNA decay is not dependent on TRAP binding.

FIGURE 1.

A, nucleotide sequence and secondary structure of trp leader RNA bound by 11-mer TRAP complex. Numbering is from the natural start site of trp transcription. GAG and UAG triplet repeats at which TRAP binds are in boldface. Shaded nucleotides in the TBS region (GGGU) and in the 3′-TT region (ACCC) are paired in the alternative structure that forms in the absence of TRAP. Boxed nucleotides in the 5′-SL region were mutated to give a trp leader RNA with an unstructured 5′-terminal region. Nt 96–103 that were replaced by the GC-containing NotI sequence are indicated. The arrow points to nt 34, which was replaced with the NotI sequence (see Fig. 5A). B, predicted secondary structure of the 5′-terminal nucleotides in 5′-SSS RNA.

FIGURE 2.

A, schematic diagrams of various trp leader RNA constructs. RNase J1 target site indicated by open box with downward arrow at cleavage site; the mutated target site in NotI RNAs is indicated by a solid box. Half-life data obtained from Northern blot analysis are shown below each schematic for the wild-type strain and for RNase J1 and RNase Y mutant strains. The half-life of trp leader RNA in the RNase J2 deletion strain is also shown. Values are average of at least three experiments, with S.D. shown. For the RNase Y mutant strains, p values of RNA half-lives compared with the corresponding half-lives in RNase J1 mutant strains are shown in parentheses. For the RNase J2 deletion strain, the p value of trp leader RNA half-life compared with the half-life in the RNase Y strain is in parentheses. B, in vitro assay of RNase J1 endonuclease cleavage of trp leader RNA and NotI RNA. Major cleavage product of ∼100 nt indicated by asterisk at the left. Numbers on top are minutes after addition of RNase J1. Additional experiments with longer incubation times also showed no detectable RNase J1 cleavage of NotI RNA. Control lane (C) contained RNA substrate incubated with the inactive H76A mutant RNase J1. Marker lane (M) contained 5′ end-labeled TaqI fragments of plasmid pSE420 DNA (34), with the sizes of these fragments indicated on the left.

FIGURE 3.

Analysis of trp leader RNA with 5′-terminal strong stem structure (5′-SSS). A, Northern blot analysis of RNase J1 cleavage in vivo, probed with a 3′-proximal probe. Migration of the RNase J1 3′ cleavage product is indicated by the asterisk at right. Marker lane (M) is as described in the legend to Fig. 2. B, RNase J1 endonuclease cleavage of trp leader RNA and 5′-SSS RNA in vitro. Full-length (FL) RNAs are indicated, and major cleavage products are marked by asterisks. Marker lane and control lane (C) are as described in the legend to Fig. 2. Above the gel are the times after addition of RNase J1. The sample in the control lane (−) had no enzyme added and was also incubated for 15 min. C, rate of cleavage of full-length RNA in vitro (average of three experiments). Filled squares, trp leader RNA; open squares, 5′-SSS RNA.

FIGURE 4.

RNase J1 5′-exonuclease activity on 5′-monophosphorylated RNA substrates. Release of ³²P-labeled GMP was assayed by thin-layer chromatography. Solid squares, trp leader RNA; open circles, trp leader RNA with unstructured 5′-terminal region; open triangles, 5′-SSS RNA.

FIGURE 5.

Characteristics of trp:rpsO-TT RNA decay. A, nucleotide sequence of 3′-TT for trp leader RNA and trp:rpsO-TT RNA. Predicted structures are shown, with the calculated ΔG₀ indicated below. Uncertainty about the actual 3′ end of trp:rpsO-TT RNA is indicated by the parentheses. B, Northern blot analysis (5′-proximal probe) of RNase J1 5′ cleavage product, indicated by asterisk at right. Migration of full-length (FL) and read-through (RT) RNA is indicated. The blot is overexposed to show the rapidly degraded 5′ cleavage product. Marker lane (M) is as described in the legend to Fig. 2. C, Northern blot analysis of RNase J1 3′ cleavage product, using trp- and rpsO-specific 3′ probes. Major 3′-terminal fragments are marked with an asterisk. Marker lanes (M) are as described in the legend to Fig. 2. D, accumulation of trp:rpsO-TT RNA 3′ end-containing decay intermediates in wild-type and RNase J1-depleted strains, with or without TRAP present. Below the blot are the amount of 3′-terminal fragments, relative to the amount of full-length RNA in each lane. The marker lane is as described in the legend to Fig. 2.

FIGURE 6.

Analysis of a block to 3′-to-5′ exonucleolytic decay. A, schematic diagram of NotI_34 RNA. B, Northern blot analysis of accumulation of the 50-nt fragment (indicated by the arrow at the right), using a 5′-proximal probe. A fragment predicted to be ∼108 nt is observed in lane 2 (marked with a caret) and faintly in lane 4. Below each lane is the amount of 50-nt fragment, relative to the amount of this fragment in lane 1. The marker lane (M) is as described in the legend to Fig. 2.