Processing and Stability of Inducibly ExpressedrpsOmRNA Derivatives inBacillus subtilis

Abstract

The Bacillus subtilis rpsO gene specifies a small (388-nucleotide), monocistronic mRNA that encodes ribosomal protein S15. We showed earlier that rpsO mRNA decay intermediates accumulated to a high level in a strain lacking polynucleotide phosphorylase. Here, we used inducibly expressed derivatives of rpsO, encoding smaller RNAs that had the complex 5 region deleted, to study aspects of mRNA processing in B. subtilis. An IPTG (isopropyl-␤-D-thiogalactopyranoside)-inducible rpsO transcript that contained lac sequences at the 5 end, called lac-rpsO RNA, was shown to undergo processing to result in an RNA that was 24 nucleotides shorter than full length. Such processing was dependent on the presence of an accessible 5 terminus; a lac-rpsO RNA that contained a strong stem-loop at the 5 end was not processed and was extremely stable. Interestingly, this stability depended also on ribosome binding to a nearby Shine-Dalgarno sequence but was independent of downstream translation. Either RNase J1 or RNase J2 was capable of processing lac-rpsO RNA, demonstrating for the first time a particular in vivo processing event that could be catalyzed by both enzymes. Decay intermediates were detected in the pnpA strain only for a lac-rpsO RNA that was untranslated. Analysis of processing of an untranslated lac-rpsO RNA in the pnpA strain shortly after induction of transcription suggested that endonuclease cleavage at 3-proximal sites was an early step in turnover of mRNA.The rate of decay of an mRNA is an important component in determining the level of gene expression. Studies of the mechanism of mRNA decay in Escherichia coli have progressed based on a detailed knowledge of the ribonucleases involved in the process and the construction of RNase mutant strains. A similar level of understanding of the mechanism of mRNA decay has not been achieved for the model grampositive organism Bacillus subtilis. Earlier studies of a number of B. subtilis mRNAs, some of which were constitutively or inducibly stable, revealed that mRNA decay in B. subtilis initiates from the 5Ј end (9) and that polynucleotide phosphorylase (PNPase) (encoded by the pnpA gene) plays a major role in 3Ј-to-5Ј exonucleolytic degradation (15,30,42). More recently, the role of RNase J1 and RNase J2 ribonucleases (18) has become paramount. Of the two, only RNase J1 is essential, and reduced expression of RNase J1 results in an increase in global mRNA half-life, suggesting a general role for RNase J1 in initiation of decay. RNase J1 has been shown to be involved in decay and processing of a number of specific RNAs (3,7,14,18,44), and a recent study demonstrated the effect of reduced RNase J1 and/or lack of RNase J2 on hundreds of B. subtilis mRNAs (26). While the RNase J enzymes were initially purified on the basis of their endoribonuclease activity, it was shown subsequently that RNase J1 also has 5Ј-to-3Ј exoribonuclease activity (27), which is inhibited by a 5Ј triphosphate end (14,25).A model for mRNA turnover in B. subtilis involves RNase J1in two possible pathways...

Regulatory RNAs in Bacillus subtilis: a Gram-Positive Perspective on Bacterial RNA-Mediated Regulation of Gene Expression

“…Nonetheless, when the initiation codon for translation was mutated in an mRNA with a strong 5= secondary structure, the mRNA half-life was still reduced (49). The latter finding is consistent with the results from studies on the 5=UTR S363 preceding aprE and an artificial stemloop preceding rpsO (82), where the stem-loop and ribosome binding were found to be more important than translation (83).…”

What is the role of RNase J in mRNA turnover?

“…However, a model of ribosome-dependent endonucleolytic cleavage by RNase J1 at the upstream boundary of the stalled ribosome is favored by the authors for both ∆ermC and lac-rpsO. 20,22,23 It is not immediately clear to me, however, why these mRNAs would be any different to the cryIIIA and hbs mRNAs. Since the generation of the ribosome-protected species is inhibited by the addition of secondary structures to the 5' end of the ∆ermC and lac-rpsO transcripts, this was interpreted as evidence that the endonucleolytic cleavage by RNase J1 is 5'-end dependent, with the enzyme scanning to the site of ribosome-dependent cutting.…”

“…Two similar examples have been studied by the Bechhofer laboratory: a lac-rpsO fusion in which an inducible lac promoter and a very strong SD (12 nts complementarity to the 3' end of 16S rRNA) was fused to a leaderless rpsO gene 20 and a transcript called ∆ermC, which is stabilized by an erythromycin (Em)-induced stalled ribosome near the 5' end of the transcript. 17 In both cases, a shorter RNA is protected by a ribosome at its 5' end.…”

“…This was interpreted as RNase J1 and J2 having overlapping substrates in the case of the transcriptome analysis and in the case of removal of the 5' end of the lac-rpsO fusion. 11,20 Since RNase J2 alone has much scRNA that is 4 nts shorter on the 3' end. The maturation of this species is thought to involve cleavage by RNase J1 about 12 nts downstream of the major mature 3' end, followed by digestion by 3'-to-5' exonucleases.…”

Bacillus subtilis RNase J1 endonuclease and 5′ exonuclease activities in the turnover of ΔermC mRNA

“…More likely, the regulation of mRNA stability by interference with RNase J1 activity occurs at the binding step at the 59 end, upstream of ribosome flow. We have found that, when the same stem-loop structure that is present in the SSL>11 construct was placed at the 59 end of an RNA that has a 59-proximal RNase J1 target site, cleavage by RNase J1 at this site was virtually abolished (Yao and Bechhofer 2009).…”