Mycobacterium tuberculosis MutT4/RppH is an RNA pyrophosphohydrolase that forms condensate-like bodies and impacts mRNA degradation

Abstract

Bacterial adaptation to stress involves changes in transcription and messenger RNA (mRNA) degradation. In Escherichia coli, the Nudix hydrolase RppH initiates mRNA degradation by removing pyrophosphate from mRNA 5'-ends, converting 5'-triphosphates to 5'-monophosphates. We aimed to identify the RppH homolog in the globally important pathogen Mycobacterium tuberculosis (Mtb). We identified the protein encoded by Rv3908, previously annotated as a nucleotide pool cleanser mutT4, as the predominant mycobacterial RppH. Deletion of rppHMtb increased the relative abundance of 5'-triphosphates on myriad mRNAs across the transcriptome. Purified RppHMtb converted mRNA 5'-triphosphates into monophosphates, and stimulated degradation by RNase E and RNase J in vitro to varying extents. Surprisingly, deletion of rppHMtb had mixed impacts on mRNA degradation in vivo, suggesting that it may not sensitize most transcripts to degradation. RppHMtb has intrinsically disordered regions (IDRs), which often participate in biomolecular condensate formation. Microscopy showed that RppHMtb forms condensate-like bodies that localize with RNases and dissociate upon addition of rifampicin. The N-terminal IDR is sufficient for condensate-like body formation. Deletion of rppHMtb leads to higher outer membrane permeability and resistance to oxidative stress. We conclude that MutT4 is the mycobacterial RppH, assembling in condensate-like bodies with RNases but having unexpectedly complex impacts on mRNA degradation rates.

Graphical Abstract

Figure 1.

MutT4 is an RppH that acts on Mtb mRNAs. (A) Schematic of splinted ligation. The adapter is ligated only if the target mRNA has a 5′ monophosphate. Ligation is performed with and without treatment by E. coli RppH to convert 5′ triphosphates to monophosphates. qPCR reveals the abundance of ligated mRNA (primers 1 and 3) relative to total mRNA (primers 2 and 3). (B, C) Screening of Mtb Nudix gene deletion mutants and complemented strains by splinted ligation of a transcript in the endogenous mRNA pool. ΔΔCt indicates the impact of in vitro treatment with E. coli RppH on the ligatability of the Rv3248c transcript, after normalizing to total transcript abundance. Higher ΔΔCt values indicate a higher proportion of the endogenous Rv3248c mRNA was triphosphorylated. Mean and standard deviation of three biological replicates are shown for panel (C). (D) MutT4/RppH has RppH activity in vitro. Splinted ligation of in vitro transcribed Rv3248c mRNA (5′ triphosphorylated) treated with purified MutT4/RppH, MutT4/RppH E118Q, or no enzyme (mock). Each value represents results from the assay performed with an independent protein preparation. A one-way ANOVA was performed with Dunnett’s test for panel (B) and Tukey’s test for panels (C) and (D). *** P ≤ .001, **** P ≤ .0001.

Figure 2.

RppH_Mtb has a transcriptome-wide role as an RppH, with a greater steady-state impact on transcripts that start with guanosine and have minimal secondary structure near their 5′ ends. (A) Schematic of 5′-end-directed RNA-seq. Total RNA was used to construct 5′-end-directed libraries with or without pretreatment with E. coli RppH to convert 5′ triphosphates to monophosphates. Only transcripts with 5′ monophosphates (whether endogenous or produced by E. coli RppH) can be ligated to adapters and therefore be captured in the libraries. Comparison of the number of reads obtained in libraries made from RppH_{E. coli}-treated versus untreated RNA indicates the extent to which the RNA was endogenously monophosphorylated. The “log₂ coverage ratio” (green) and “rppH_Mtb impact” (blue) are metrics used in subsequent panels. (B) log₂ coverage ratio of ±RppH_{E. coli} libraries for reads derived from RNA 5′ ends previously classified as TSSs (teal curve, n = 1687) or RNase cleavage sites (pink, n = 590) (Zhou and Sun et al. 2023). The RNase cleavage sites produce RNAs with monophosphorylated 5′ ends and are therefore expected to have log₂ coverage ratios of ∼0, while TSSs produce RNAs with triphosphorylated 5′ ends and are therefore expected to have log₂ coverage ratios >0. The median TSS log₂ coverage ratio for each strain is shown in teal. (C–F) For each TSS quantified in panel (B), the impact of rppH_Mtb on 5′ end phosphorylation status was quantified by calculating the difference in log₂ coverage ratio ±RppH_{E. coli} between the deletion strain and the WT strain (see panel A). A higher number indicates a larger impact by rppH_Mtb. (C) Transcripts with 5′ ends mapping to previously published TSSs were grouped according to their first two nt. As most transcripts initiate with A or G, those initiating with C or U are not shown. (D, E) Transcripts with 5′ ends mapping to published TSSs that initiated with G were binned into quartiles according to the impact of rppH_Mtb on their 5′ end phosphorylation status. Secondary structure characteristics of the quartile least affected by rppH_Mtb (Q1) and the quartile most affected by rppH_Mtb (Q4) were compared. The first 20 nt of each transcript were computationally folded and the probability that the first 5 nt were unpaired in these structures (D) as well as the MFE of the structures (E) were determined. (F) The impact of rppH_Mtb on leadered versus leaderless transcripts was compared. * P ≤ .05, ** P ≤ .01, **** P ≤ .0001. A one-way ANOVA was performed with Tukey’s test for panel (C). Mann–Whitney test was performed for panels (D–F).

Figure 3.

RppH_Mtb sensitizes transcripts to cleavage by RNase E and RNase J in vitro. Pre-treatment with RppH_Mtb stimulates degradation of an in vitro transcribed RNA (Rv3248c) by Mtb RNase E (A) and RNase J (B). Values represent the mean and standard deviation of three or four independent replicates. Cleavage was expressed as percentage decrease in substrate band intensity after incubation with RNase E or RNase J compared to the corresponding untreated control. A representative gel is shown in Supplementary Fig. S4. * P ≤ .05, RM one-way ANOVA.

Figure 4.

Deletion of rppH_Mtb does not lead to slower degradation of most transcripts in vivo during log phase growth. Half-lives of ten transcripts were measured by blocking transcription with rifampicin (RIF) and quantifying mRNA abundance by qPCR. (A–C) Transcript abundance over time following RIF treatment is displayed on a log₂ scale by plotting –Ct values from reactions performed with 400 pg of cDNA. Points and error bars represent the means and SDs of biological quadruplicate samples. Slopes were calculated by linear regression (strain lines), and half-life = −1/slope. Because RIF does not block transcription elongation, there is a plateau before the observed decrease in mRNA levels when the qPCR primers are distal from the transcription start site (infB, sigA, plc); in these cases, only the timepoints after the plateau were used for linear regression. The first nt of each transcript is indicated, and the start codon is shown in green. (A) Three transcripts for which deletion of rppH_Mtb had an above-average effect on 5′ end phosphorylation. (B) Three transcripts for which deletion of rppH_Mtb impacted steady-state abundance. (C) Four transcripts for which deletion of rppH_Mtb did not have an above-average impact on 5′ status or impact abundance. (D) Half-lives calculated from the linear regressions shown in panels (A–C). Error bars denote 95% confidence intervals. The upper error bar for esxD was clipped for visualization purposes. P-values were determined by pairwise linear regression comparisons. Those not shown were > .05. * P ≤ .05; ** P ≤ .01; *** P ≤ .001; **** P ≤ .0001.

Figure 5.

RppH_Mtb forms dynamic condensate-like bodies that appear to localize with RNase E and RNase J. Genes encoding the indicated fluorescent protein fusions were integrated in single copy into phage integration sites in the genome of otherwise WT M. smegmatis strain mc²155. Live cells were imaged during mid-log phase. All genes are from M. smegmatis except in panel (B). (A) RppH_Msmeg localizes with RNase E and RNase J condensates. Protein localization was analyzed using ImageJ by tracing a line along the center of each bacterium. Fluorescence intensity profiles for the red and green channels were plotted, and peaks within 0.2 µm were counted as colocalized. Percentage localization for each bacterium was calculated as RppH_Msmeg colocalizing peaks divided by total RppH_Msmeg peaks. The significance of the colocalization was assessed by flipping the x-axis values for one of the two colors, calculating percentage localization, and comparing those values to the real data by Mann–Whitney test. * P < .05, **** P < .0001 (B) RppH_Mtb forms condensate-like bodies when expressed heterologously in M. smegmatis. (C) Treatment with rifampicin disassembles RppH_Msmeg condensates. Mycolicibacterium smegmatis expressing RppH_Msmeg-Dendra2 was treated with 100 µg/ml rifampicin (RIF) or DMSO as a control for 30 min and imaged. The coefficient of variance (CV) of fluorescence signal within each cell was used as a metric of condensate formation. More punctate signal results in a higher CV. Each dot indicates the CV of fluorescence intensity along a straight line from one cell pole to the other. Scale bar 2 µm for all images. In the plots in panels (A) and (C), data are from three biological replicates, 50 bacteria each. Medians from each replicate culture are shown as squares and data from individual bacteria as circles. Mann–Whitney test was performed for panel (C). **** P ≤ .0001.

Figure 6.

The N-terminal intrinsically disordered region of RppH_Msmeg is sufficient for focus formation. (A) Schematic representation of Dendra2 constructs (not to scale). Amino acids are indicated in the RppH_Msmeg full-length Dendra2 construct. (B) CV of fluorescence signal within each cell was used as a metric of condensate formation. More punctate signal results in a higher CV. Each dot indicates the CV of fluorescence intensity along a straight line from one cell pole to the other. Data are from three biological replicates, 50 bacteria each. Medians from each replicate are shown as squares and data from individual bacteria as circles. (C) Representative microscopy images. Scale bar 2 µm. Kruskal–Wallis test was performed for panel (B). **** P ≤ .0001.

Figure 7.

Deletion of rppH_Mtb leads to increased vancomycin sensitivity and mycomembrane permeability. (A) Antibiotic susceptibility testing of Mtb strains against vancomycin. A representative result performed with technical duplicates of three biological replicates is shown. (B) Permeability to a fluorescent derivative of vancomycin (FL-VAN). Mtb was incubated with FL-VAN, and permeability to this molecule was measured by flow cytometry and normalized to the WT strain. Higher values indicate higher permeability. Mean and standard deviation of three biological replicates. (C) Permeability to an azide–rifamycin conjugate measured by PAC-MAN competition assay. Mtb peptidoglycan layer was tagged with DBCO groups. Next, cells were incubated with an azide–rifamycin conjugate that covalently reacts with the DBCO groups via SPAAC. Finally, unreacted DBCO epitopes were linked to a highly permeable azide fluorophore and total cellular fluorescence was measured by flow cytometry. Fluorescence was normalized to the control untreated with azide–rifamycin. Higher competition values indicate lower permeability. Mean and standard deviation of three biological replicates. A one-way ANOVA was performed with Tukey’s test for panel (B). * P ≤ .05, ** P ≤ .01.

Figure 8.

Deletion of rppH_Mtb leads to increased resistance to H₂O₂ in Mtb. (A) Resistance to H₂O₂ measured by disk diffusion assay. (B) Acute treatment of Mtb with H₂O₂ in liquid media. Mtb was exposed to 10 mM H₂O₂ for 24 h and plated to enumerate CFU/ml. Data from three biological replicates are expressed as survival normalized to CFU/ml at time 0. A one-way ANOVA was performed with Tukey’s test for panels (A) and (B). * P ≤ .05, ** P ≤ .01, *** P ≤ .001, **** P ≤ .0001.