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. Author manuscript; available in PMC: 2026 Oct 4.
Published in final edited form as: Biochem Biophys Res Commun. 2026 Jun 2;828:154079. doi: 10.1016/j.bbrc.2026.154079

In vitro Cleavage Requirements and Specificities of Mycobacterial RNase E

Abigail R Rapiejko 1, Manchi Reddy 2, James C Sacchettini 2, Scarlet S Shell 1,*
PMCID: PMC13633751  NIHMSID: NIHMS2212609  PMID: 42263604

Abstract

Regulation of RNA pools allows for adaptation to stress, which is important in pathogenic bacteria such as Mycobacterium tuberculosis (Mtb). RNA degradation is a significant contributor to RNA abundance, and Ribonuclease (RNase) E has a rate-limiting role in degradation of most mycobacterial transcripts. Many open questions remain about the RNA substrate requirements and specificities for efficient cleavage by mycobacterial RNase E. Here, we demonstrate that RNase E is only active on substrates with a minimum length of approximately 27 nt using both Mtb and Mycolicibacterium smegmatis (Msm) enzyme variants. Furthermore, we show that mycobacterial RNase E prefers substrates with 5’ monophosphates to 5’ triphosphates. We present that the positions of cleavage events by RNase E within substrates are dictated by both sequence and distance from the RNA ends. Finally, we show that Msm RNase E behaves similarly to Mtb RNase E, validating the use of this model organism for RNA degradation studies.

Keywords: RNase E, RNA degradation, cleavage specificity

Introduction

Mycobacterium tuberculosis (Mtb) remains a global public health threat [1]. An important enzyme likely regulated during physiologically relevant stress is RNase E. RNase E is an endonuclease with a rate-limiting role in bulk mRNA degradation in the nonpathogenic model Mycolicibacterium smegmatis (Msm) [2] and is essential for growth in both Msm and Mtb [3, 4].

Sequence alignments of E. coli and Mtb RNase E suggest that the catalytic domain architecture is conserved [5]. Similarities exist between E. coli and mycobacterial RNase E such as the requirement for magnesium in the active site [6, 7] and the use of zinc for the formation of tetramers [6–8]. However, mycobacterial RNase E also displays many differences from its better-studied E. coli counterpart. Mycobacterial RNase E has two intrinsically disordered regions (IDRs) flanking the catalytic domain, unlike E. coli RNase E which has a single IDR [5] that scaffolds a multiprotein complex called the degradosome. E. coli RNase E has a membrane-binding domain for localization to the inner membrane [9–12], while mycobacterial RNase E is cytoplasmic [13]. Finally, mycobacterial RNase E preferentially cleaves preceding cytidine residues, consistent with mycobacteria being GC-rich organisms [2], whereas E. coli RNase E preferentially cleaves preceding uracil residues, consistent with it being a GC-balanced organism [14, 15]. These important differences motivate studying mycobacterial RNase E, as it is fundamentally different from the better studied E. coli version.

Unanswered questions remain surrounding RNA substrate preferences and specificities of cleavage site position for mycobacterial RNases. Here, we used in vitro RNA cleavage assays with substrates containing exactly one known RNase E cleavage site to investigate these questions using both Mtb and Msm RNase E. We found that RNase E has a minimum substrate length requirement of approximately 27 nt, in contrast to a previous report [7], and we suggest that RNase preparation purity may have contributed to these discrepancies. Additionally, we demonstrate that RNase E prefers to cleave RNAs with 5’ monophosphates to those with 5’ triphosphates. We show that cleavage site proximity to the RNA ends is just as important as cleavage site sequence for dictating RNase E cleavage position. These findings provide a framework for future investigations using RNase E in vitro and have important in vivo implications towards understanding how RNase E selects and degrades its substrates to control RNA metabolism in a globally important pathogen.

Materials and methods

Bacterial strains and culture conditions

LB broth and agar were used to grow E. coli with 50 μg/mL kanamycin and 20 μg/mL chloramphenicol. E. coli NEB-5-alpha (New England Biolabs) was used for cloning. HiFi Assembly (New England Biolabs) was used to make plasmids. Gene IDs are listed in Supplemental Table 1 and expression plasmids are described in Supplemental Table 2.

Overexpression and purification of recombinant proteins

Msm RNase E variants were recombinantly expressed and purified using E. coli BL21(DE3) pLysS transformed with pET42-derived plasmids as described in [2] using 1 M NaCl washing. A truncated version of Msm RNase E containing amino acids 145–823 (deletion of part of the N-terminal IDR and deletion of the full C-terminal IDR) was used in this study as reported [2]. All RNase E variants had N-terminal 6Xhis, 3X FLAG, TEV protease cleavage site, and 4Xgly linker tags. A catalytically inactive version containing D694R and D738R mutations [2, 16] was used as a control.

Mtb enzymes with C-terminal 6xhis tags were overexpressed from pET plasmids in E. coli. Pellets were lysed in 20 mM Tris-HCl (pH 7.5), 500 mM NaCl, and 5% glycerol and loaded onto Ni-NTA resin using an AKTA purification system (Cytiva). Initial preps were washed with this buffer additionally containing 100 mM imidazole and 500 mM NaCl. High salt wash preps used 100 mM imidazole and 1 M NaCl. Proteins were eluted in a 75–500 mM imidazole gradient, dialyzed in 50 mM Tris-HCl, pH 7.5, 100 mM NaCl, 5% glycerol, and 2 mM DTT, run on a Superdex size exclusion column, and concentrated.

Preparation of RNA substrates

RNA oligos used for this study are listed in Supplemental Table 3. Those used in Figure 1A–B and Supplemental Figure 1C were purchased from Integrated DNA Technologies. Sometimes, T4 polynucleotide kinase (New England Biolabs) was used according to the manufacturer’s instructions to monophosphorylate 5’ ends of RNAs purchased with 5’ hydroxyls.

Figure 1. 27 nt is the minimum length required for RNase E cleavage of an atpB-derived RNA.

Figure 1.

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A) Cleavage of 20 nM 22-nt 3’ FAM labeled oligo with 100 nM enzyme. OH represents a 5’ hydroxyl, and P represents a 5’ monophosphate. B) Cleavage of 1.5 μM 22-nt unlabeled oligo with 200 nM enzyme. C-E) Cleavage assays with 100 nM enzyme and RNAs made by IVT: C) 0.5 μM 25 nt RNA; D) 3.4 μM 27 nt RNA; E) 3.2 μM 29 nt RNA. Each panel shows a single gel, with irrelevant lanes cropped out for clarity.

RNA substrates in Figure 1C–E, Figure 2, Figure 3, and Figure 4 were synthesized by in vitro transcription (IVT). dsDNA templates were made by annealing 25 μM of each primer in 10 mM tris-HCl pH 7.9, 50 mM NaCl, and 1 mM EDTA in 20 μL reactions, heating at 95°C for 2 minutes, then cooling 1.5°C per 1.5-minute cycle to 24.5°C.

Figure 2. RNase E cleaves a 29 nt but not 21 nt RNA derived from the ESX-1 locus.

Figure 2.

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RNAs with 5’ monophosphates were derived from the intergenic region between PPE68 and esxB in the ESX-1 locus of Msm. Reactions contained 4.4 μM RNA and 100 nM enzymes. A-C) Cleavage of a 21 nt oligo made by IVT with the cleavage site A) near the 5’ end and B) in the middle of the oligo. C) Cleavage assay with a 29 nt oligo containing a central RNase E cleavage site. Each panel shows a single gel, with irrelevant lanes cropped out for clarity.

Figure 3. RNase E cleaves 5’ monophosphorylated substrates better than 5’ triphosphosphorylated substrates.

Figure 3.

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All reactions contained 1.6 μM RNA with 100 nM enzymes. A) Cleavage assay of the 29 nt atpB RNA with a 5’ monophosphate or 5’ triphosphate. B) Quantification of three replicates of A). The amount of substrate remaining was reported as a percentage of the mock reaction. P: rmonophosphorylated substrate; PPP: triphosphorylated substrate; (−):catalytically inactive enzyme; (+): catalytically active enzyme; S: Msm RNase E; T: Mtb RNase E. Bars represent averages. Error bars represent standard error of the mean. An ordinary one-way ANOVA with Sidak’s multiple comparisons test was used for significance testing (GraphPad Prism). Each panel shows a single gel, with irrelevant lanes cropped out for clarity.

Figure 4. Both sequence and proximity to RNA ends influence RNase E cleavage positions.

Figure 4.

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Reactions contained 3.2 μM RNA with 100 nM enzymes. Cleavage of a 29 nt oligo made by IVT with the cleavage site motif from Figure 1 located 6 nt from the 5’ end A) and B) and 6 nt from the 3’ end in C) and D). The original RNase E cleavage sites are indicated with blue scissors (blue arrowheads on gels). Gray scissors indicate the approximate positions of unknown observed cleavage events leading to two observed cleavage products with sizes in the range of 12–19 nt (red arrowheads on gels). Each panel shows a single gel, with irrelevant lanes cropped out for clarity.

For generating RNAs with 5’ triphosphates (Figure 3), HiScribe® T7 High Yield RNA Synthesis Kit (New England Biolabs) was used with a 20 μL reaction containing 7.5 μL NTP + Buffer mix, 1.5 μL T7 RNA Polymerase, and 250 ng dsDNA template, incubated overnight at 37°C. 2 μL DNase I (New England Biolabs) and 30 μL water were added followed by incubation at 37°C for 15 minutes.

For Figure 3, where we compared matched RNAs with 5’ triphosphates and 5’ monophosphates, we synthesized RNAs by IVT as described above and treated them with RNA 5’ Pyrophosphohydrolase (RppH) (New England Biolabs) according to the manufacturer’s instructions. Mock reactions with no RppH set up in parallel were used for direct comparison of cleavage of monophosphorylated and triphosphorylated substrates.

The 5’ monophosphorylated RNAs used in Figure 1C–E, Figure 2, and Figure 4, which were not directly compared to matched 5’ triphosphorylated RNAs, were synthesized with an excess of AMP to directly produce RNAs with largely monophosphorylated 5’ ends. HiScribe® T7 High Yield RNA Synthesis Kit (New England Biolabs) was used with a 20 μL reaction containing 7.5 μL NTP + Buffer mix, 1.5 μL T7 RNA Polymerase, 250 ng dsDNA template, 20 U murine RNase inhibitor (New England Biolabs), 5 mM DTT, and 25 mM AMP, incubated overnight at 37°C. 2.5 U Turbo DNase (Invitrogen), 1X Turbo DNase buffer, and 80 U murine RNase inhibitor (New England Biolabs) were added, and reactions were brought to a final volume of 100 μL and incubated at 37°C with 200 rpm shaking for 1 hour.

RNAs made by IVT were purified by RNA Clean and Concentrator-5 kit (Zymo) according to the manufacturer’s instructions with an 11 μL elution volume.

RNA cleavage assays

Cleavage assays were done in 10 μL reactions in buffer composed of 20 mM tris-HCl pH 7.9, 100 mM NaCl, 10 mM MgCl2, 10 μM ZnCl2, 0.5% glycerol, 0.01% IGEPAL, and 1 mM DTT. Reactions were incubated at 37°C for 1 hour and stopped by adding an equal volume of RNA loading dye (ThermoScientific) for unlabeled RNAs, or for FAM-labeled RNAs, an equal volume of 12.5 mM EDTA pH 8.0 in formamide. Reactions were heated for 10 minutes at 70°C and run on 20% TBE-urea PAGE gels. Those with unlabeled RNAs were stained with Sybr™ Gold (Invitrogen) then imaged with an Azure 600 imager at 302 nm excitation. FAM-labeled RNAs were imaged using 524 nm excitation. ImageJ (FIJI plugin) was used to quantify band intensities, which were normalized to a mock reaction with no enzymes where indicated. Concentrations of RNA and enzymes are noted in figure legends.

When there were detectable cleavage products, representative images of duplicate assays are shown in the figures. Gels were cropped to show only relevant lanes. Expected RNase E cleavage sites are indicated with scissors, and the sizes of the resulting expected cleavage products are shown. “Nonspecific” refers to unidentified byproducts of IVT synthesis reactions.

Results

27 nt is the minimum length required for RNA cleavage by mycobacterial RNase E

We investigated the cleavage preferences of mycobacterial RNase E using a simple in vitro system. We purchased a 22 nt 3’−6-carboxyfluorescein (FAM)-labeled RNA oligo composed of sequence from the atpB gene from Msm, which contained exactly one RNase E cleavage site [2]. This oligo bore a 5’ hydroxyl, and we used T4 polynucleotide kinase (PNK) to monophosphorylate a portion of it, as we expected RNase E to show a preference for monophosphorylated substrates [6, 7]. Reactions were performed with this oligo and full-length Mtb and Msm RNase E with truncations of portions of its IDRs, as previously reported [2, 16]. Surprisingly, we did not observe any cleavage of the 22 nt substrate by either version of RNase E, regardless of the 5’ end chemistry (Figure 1A). The substrate was cleaved by Mtb RNase J, which we included as a positive control given its 5’ to 3’ exonuclease capability (Figure 1A). To test if the FAM label was interfering with the cleavage activity of RNase E, we repeated the assay with an unlabeled version of the same RNA, but we did not observe any cleavage by RNase E (Figure 1B). As we previously found that a 50 nt RNA including this 22 nt sequence was cleaved by RNase E [2], we suspected that RNase E may have a minimum substrate length requirement. We used in vitro transcription (IVT) to synthesize variants of the substrate with progressively longer lengths. A 25 nt version was not cleaved by RNase E cleavage, but 27 nt and 29 nt versions showed cleavage (Figure 1C–E).

To test if this minimum length requirement for RNase E cleavage was specific to the particular atpB-derived sequence tested above or applicable to other sequences, we tested another known Msm sequence containing one RNase E cleavage site from the intergenic region between PPE68 and esxB in the ESX-1 locus [17]. We found that neither of two different 22 nt versions of the ESX-1 substrate with the cleavage site in different positions were cleaved by RNase E (Figure 2A–B). Increasing the length to 29 nt allowed for cleavage of the substrate by RNase E (Figure 2C). These results suggest that RNase E may have a minimum substrate length requirement unrelated to sequence. This result surprised us, as a previous report had shown that Mtb RNase E cleaved a 13 nt substrate [7].

Recombinant mycobacterial RNase E and RNA helicase preparations can be contaminated with small amounts of highly active E. coli RNases

Preliminary experiments with an Msm RNase E mutant that was predicted to be catalytically inactive led us to suspect that some of our recombinant RNase E preparations were contaminated with E. coli RNases. To mitigate this, we increased the concentration of salt in the wash buffer in our Ni-NTA affinity purification procedure (1 M NaCl rather than 500 mM NaCl). Although Coomassie-stained gels did not reveal major observable differences in purity of preparations done with the two wash protocols (Supplemental Figure 1A–B), we found that RNase E purified using the lower-salt wash protocol produced more cleavage products from a 29 nt RNA substrate (Supplemental Figure 1C). Furthermore, a preparation of the Mtb RNA helicase RhlE done with the lower-salt protocol produced cleavage products of the same sizes as the extra bands observed in the RNase E preps (Supplemental Figure 1C). As a helicase, RhlE is not expected to have any cleavage activity, and high salt washing removed these contaminating bands. With the exception of Supplemental Figure 1, all data shown in this work were generated using RNase E purified with the high-salt protocol.

RNase E prefers to cleave 5’ monophosphorylated transcripts compared to 5’ triphosphosphorylated transcripts

The catalytic domain of RNase E in E. coli contains a pocket that can bind 5’ monophosphates of RNA substrates [6] which is required for efficient cleavage of many RNAs [18–20]. However, some RNA substrates are cleaved by RNase E regardless of 5’ end status suggesting that alternative 5’-end-independent mechanisms for cleavage also exist [21, 22]. To assess the impact of 5’ end chemistry on mycobacterial RNase E cleavage, we compared cleavage of substrates with 5’ monophosphates and triphosphates. We synthesized a 29 nt atpB RNA by IVT, which we expected to be uniformly triphosphorylated. We monophosphorylated a portion of it using E. coli RppH and compared cleavage of the two substrates with RNase E from both Msm and Mtb. Both enzymes cleaved a greater proportion of the 5’ monophosphorylated substrate than the triphosphorylated substrate (Figure 3A–B). This is consistent with our recent report on the impact of the endogenous Mtb RppH on cleavage by Mtb RNase E [13].

Both sequence and proximity to ends appear to influence choice of cleavage position by RNase E

Mycobacterial RNase E prefers to cleave immediately upstream of cytidines, in contrast to proteobacterial RNase Es which cleave in AU-rich regions [14, 15, 23]. However, we have consistently detected cleavage at some purine-cytidine dinucleotides in mycobacterial transcriptomes and not others [2, 24], indicating that other transcript features influence RNase E cleavage. We assessed the impact of moving a verified RNase E cleavage site to different positions within a 29 nt substrate. We shifted the cleavage site assayed in Figure 1 to be 6 nt from either the 5’ end or the 3’ end to determine if RNase E could recognize the sequence of its original cleavage site in a different position, or if proximity to transcript ends also plays a role in cleavage site determination (Figure 4). We expected cleavage products of 6 nt and 23 nt from these new oligos if RNase E cut at the same sequence as in Figure 1. Unexpectedly, we were unable to detect the 6 nt product in any of our assays. However, we observed a band consistent with the expected 23 nt product when the cleavage site was located near the 5’ end of the RNA (Figure 4A–B) or 3’ end (Figures 4C–D). We saw additional products that appeared to be between 12–19 nt long based on our molecular weight standards, indicating that RNase E cut at one or more additional positions besides the original cleavage site (Figure 4A–B). Both the sequence at the cleavage site and the position of the cleavage site are therefore important for predicting cleavage, and the failure of RNase E to cleave 22 nt and 25 nt substrates (Figure 1A–C) was not due to an absolute requirement for a minimum distance between the cleavage site and the 5’ or 3’ ends.

Discussion

Here, we have identified RNA features that impact mycobacterial RNase E cleavage in vitro. Consistent with expectations, we showed that Mtb and Msm RNase E prefer 5’ monophosphorylated substrates to 5’ triphosphorylated ones. The catalytic domain of E. coli RNase E has an allosterically activating pocket that recognizes the 5’ end of the RNA [6]. While no structure of mycobacterial RNase E has been reported, there is a high degree of sequence conservation of the catalytic domain with E. coli [5], so a similar 5’ end allosteric activation mechanism is possible.

We demonstrated that approximately 27 nt is the minimum RNA substrate length required for cleavage by mycobacterial RNase E. This contrasts with E. coli RNase E, which can cleave 10 nt RNAs [18, 25]. RNase E from E. coli [8, 26] and Mtb [7] has been shown to form tetramers. In E. coli, 10 nt RNAs are bound by one tetramer whereas a 15 nt RNA is long enough to be shared by two tetramers, being bound by the 5’ end sensing pocket on one tetramer while simultaneously binding the active site on the second tetramer [6]. RNA length may influence the mode of binding by RNase E. It is unclear why the minimum length requirement for mycobacterial RNase E differs from that of E. coli; a structure of mycobacterial RNase E bound to a substrate will be required to answer this question.

Our finding that substrates shorter than 27 nt were not cleaved by mycobacterial RNase E stands in contrast to a previous report with Mtb RNase E in which a 13 nt oligo was cleaved [7]. Since we did not test the specific RNA sequence used in that report, it is possible that the discrepancies are due to the sequence itself. However, it is also possible that the RNase E preparations used in [7] were contaminated with co-purifying E. coli RNases. We were surprised to find contamination with E. coli RNases in our recombinant protein preparations despite them appearing pure on Coomassie-stained gels. Including 1 M NaCl in the Ni-NTA affinity resin wash buffer effectively removed the contaminating activity from both RNase E and RhlE preparations. We highly recommend using high salt washing and purifying catalytically inactive enzymes in parallel with active protein preparations when purifying recombinant mycobacterial RNase E and RhlE from E. coli to ensure high purity.

In GC balanced E. coli and Salmonella typhimurium, RNase E preferentially cleaves when an uracil is in the +2 position [14, 15, 23]. In GC-rich Msm (~67% G+C), RNase E preferentially cleaves 5’ of cytidines [2]. We show here that a known cleavage site sequence was still cut when moved to positions near the 5’ or 3’ end of a short substrate, but additional products were also made nearer to the center of the substrate. This adds complexity to predicting cleavage positions for RNase E because both sequence and context matter. This result is consistent with a recent study in which we showed that mutating the cytidine at a major RNase E cleavage site in the ESX-1 locus (studied here in Figure 2) did not prevent cleavage at or near that position in Msm cells, indicating that other sequence, secondary structure, or positional features were more important for specifying cleavage at that location [17].

Finally, there is the remarkable functional similarity between Msm and Mtb RNase E in terms of cleavage site positions, 5’ end preferences, length requirements, and sequence context importance. This supports the value of Msm as a comparable nonpathogenic model for studying Mtb RNA metabolism and the roles of RNase E.

Supplementary Material

Supplementary Table

Supplemental Table 1. Msm and Mtb genes and proteins.

Supplemental Table 2. Plasmids used for protein overexpression in BL21 E. coli. All plasmid components are listed from N-terminus to C-terminus.

Supplemental Table 3. RNA oligos.

Supplementary Figure

Supplemental Figure 1. Mtb RNase E and RhlE preparations can be contaminated with small amounts of E. coli RNases.

Acknowledgements

We thank members of the Shell lab for thoughtful discussions and suggestions.

Funding

This work was supported in part by NIH-NIAID award 5TP01AI143575-02 (SSS and JCS), NIH-NIAID award R21 AI156415-01A1 (SSS), NSF-CAREER award 1652756 (SSS), and DoEd GAANN training grant P200A240115 (ARR). The funders had no role in study design.

Footnotes

CRediT authorship contribution statement

Abigail R. Rapiejko: conceptualization, methodology, formal analysis, investigation, writing – original draft, data visualization. Manchi Reddy: investigation. James C. Sacchettini: methodology, supervision, funding acquisition. Scarlet S. Shell: conceptualization, writing – review & editing, supervision, project administration, funding acquisition.

Declaration of competing interest

The authors declare no conflict of interest for this publication.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Table

Supplemental Table 1. Msm and Mtb genes and proteins.

Supplemental Table 2. Plasmids used for protein overexpression in BL21 E. coli. All plasmid components are listed from N-terminus to C-terminus.

Supplemental Table 3. RNA oligos.

NIHMS2212609-supplement-Supplementary_Table.xlsx (15.5KB, xlsx)
Supplementary Figure

Supplemental Figure 1. Mtb RNase E and RhlE preparations can be contaminated with small amounts of E. coli RNases.

NIHMS2212609-supplement-Supplementary_Figure.pdf (600.8KB, pdf)

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