Defining the role of RNase E in the mycobacterial degradosome-like network

Abstract

mRNA degradation is a fundamentally important process that is regulated in response to stress in the globally important pathogen Mycobacterium tuberculosis. Several mycobacterial ribonucleases (RNases) are hypothesized to function together to coordinate mRNA degradation, but the interactions among them are mostly undefined. One of the rate-limiting enzymes, RNase E, contains intrinsically disordered regions (IDRs). Here, we aimed to define the interactions between major mycobacterial mRNA degradation enzymes and identify the function(s) of the two IDRs of RNase E in the nonpathogenic model Mycolicibacterium smegmatis. We found that the two IDRs differentially impact mRNA degradation rates in vivo but are largely functionally redundant in their impacts on steady-steady transcript abundance. In vitro, the IDRs are uninvolved in catalysis but play major roles in RNA binding and interactions with other mRNA degradation enzymes, namely PNPase, RNase J, and RhlE1. In vivo, these enzymes localize with RNase E, but its IDRs play only a minor role, suggesting substantial redundancy in subcellular localization mechanisms. Collectively, we propose a degradosome-like network model in mycobacteria, held together by dynamic, transient interactions among RNA degradation enzymes and RNA that can be disrupted during physiologically relevant stress to allow for adaptability.

Keywords: RNase E; degradosome; degradosome-like network; mRNA decay; mRNA degradation; mRNA processing.

Figure 1.. The structure of RNase E contains two intrinsically disordered regions.

A) AphaFold3 structure of RNase E from M. smegmatis. Blue indicates areas of high confidence and red/yellow indicate areas of low confidence. B) IUPred2 disorder prediction of RNase E. Multiple sequence alignments in ClustalOmega show location and relative size and position of RNase E IDRs in a few species of bacteria. C) Schematic of IDR deletion mutants of RNase E constructed and used in this study.

Figure 2.. Deletion of the two IDRs of RNase E in M. smegmatis has differential impacts on cell size, growth rate, mRNA degradation, and gene expression.

A) Cell length of RNase E IDR mutants. Cells were imaged on a spinning disk microscope, and cell length was measured on ImageJ with the FIJI plugin. The circle colors represent biological replicates, and the squares represent the median of that biological replicate. Black line represents the mean of the medians. Significance stars represent a one-way ANOVA with Kruskal-Wallis multiple comparisons test comparing each IDR mutant to full length. B) Growth curve of the IDR deletion strains. Points represent the average of three biological replicate strains. Linear regression was used for significance testing. C) RNA half-lives of selected genes in the IDR mutants. Longer half-lives indicate less efficient RNA degradation. Transcript half-lives for the indicated genes were measured by blocking transcription with 150 μg/mL rifampicin and measuring RNA abundance at several timepoints by quantitative PCR. Error bars denote 95% confidence interval. Half-lives were compared using linear regression analysis. D-E) RNAseq was used to compare the steady-state transcriptomes of strains expressing the indicated mutants or full-length RNase E. Genes were classified as differentially expressed if their log₂ foldchange was <-1 (downregulated) or >1 (upregulated) in the truncations compared to full-length, with adjusted p <0.05. In A-C, ns p > 0.05, *** p ≤ 0.001, **** p ≤ 0.0001

Figure 3.. The IDRs of RNase E are dispensable for catalytic activity but have partially redundant roles in RNA binding.

A) Kinetic analysis of IDR deletion RNase E mutants in vitro using a 29 nt FAM labeled substrate, quantified in B). Initial rates were calculated by simple linear regression of the disappearance of substrate, quantified on ImageJ with the FIJI plugin. Points represent the average of three replicates. Nonlinear regression with a K_cat model was used for determining kinetic constants, and each mutant was compared to full-length using the extra sum of squares F-test. C) EMSA binding assays with a 721 nt fluorocein-UTP labeled RNA and catalytically dead RNase E, fit with the one-site specific binding equation quantified in D). The fraction bound was quantified on ImageJ with the FIJI plugin, and each point represents the average of three replicates. Comparisons were made using the extra sum of squares F-test.

Figure 4.. RNase E IDRs play a minor role in condensate formation in vivo.

A) Representative cells from spinning disk microscopy of mCherry::RNase E with or without 100 μg/mL rifampicin. B) In ImageJ with the FIJI plugin, a line was drawn through each cell and gray value versus distance was plotted. Cell regions where the signal intensity was greater than the mean were counted as foci, and the number of foci was normalized by cell length. A Mann-Whitney test was used for significance testing. C) A schematic of mCherry tagged RNase E constructs imaged by spinning disk microscopy with representative cells shown in D). Cells were quantified in E) using the same method for foci per micron calculations as in B) and in F) using coefficient of variation of signal intensity for a line drawn through the length of each cell. A one-way ANOVA with the Kruskal-Wallis multiple comparisons test was performed. G) A schematic of constructs used to assess the ability of RNase E IDRs to induce foci formation by mScarlet. Full-length RNase E was used as a positive control. Images obtained by spinning disk microscopy with representative cells are shown in H) and quantified in I) and J) in the same way as panels B) and F), respectively. For panels B, D-E, and I-J, the circle colors represent biological replicates, and the squares represent the median of that biological replicate. Black line represents the mean of the medians. ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001

Figure 5.. Defining the protein-protein interactions of the degradosome-like network in vitro.

Representative images of pull downs of Flag-tagged RNase E and coimmunoprecipitation of HA-tagged A) PNPase, C) RNase J, and E) PNPase, detected by western blot. In all cases “IP” indicates immunoprecipitation, and samples incubated in buffer without beads were run in parallel for comparison. Pull downs were quantified in B), D), and F), respectively, using ImageJ with the FIJI plugin. Pull down by IDR deletions were expressed as percentages of the full-length pull down. Dots represent replicates, and bars represent averages. A one-way ANOVA was performed with Sidak’s multiple comparison test. Representative images of Coomassie SDS-PAGE gels of HA coimmunoprecipitations of G) HA-RNase J and untagged PNPase, H) HA-RhlE1and untagged PNPase, and I) HA-tagged RhlE1 and untagged RNase J. J) Summary of the verified 2-way protein-protein interactions and a model of the possible degradosome-like network in mycobacteria. ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001

Figure 6.. Localization of degradosome-like network proteins in vivo.

Representative cells from spinning disk microscopy of RNase E::mCherry with IDR mutants and A) PNPase::eGFP, C) RNase J::dendra2, and E) RhlE1::dendra2. Quantification of localization in B), D), and F), respectively, was done by drawing a line through a cell in ImageJ with the FIJI plugin, plotting green versus red channels, and computing the Pearson’s R value. Values at +1 indicate complete correlation, 0 indicates no correlation, and −1 indicates anticorrelation. The circle colors represent biological replicates, and the squares represent the median of that biological replicate. Black line represents the mean of the medians. A one-way ANOVA with the Kruskal-Wallis multiple comparisons test was performed. ns p > 0.05, **** p ≤ 0.0001

Figure 7.. Localization of degradosome-like network proteins in vivo during carbon starvation stress.

A) Single-tagged strains of mCherry::RNase E, PNPase::eGFP, RNase J::dendra2, and RhlE1::dendra2 were imaged by spinning disk microscopy during both carbon rich and carbon starvation conditions. Foci per micron was calculated as in Figure 4B. Representative cells from spinning disk microscopy of RNase E::mCherry with IDR mutants and B) PNPase::eGFP, D) RNase J::dendra2, and F) RhlE1::dendra2 are shown during carbon rich and carbon starvation conditions. Quantification of localization in C), E), and G), respectively, was done as in Figure 6 B, D, and F. The circle colors represent biological replicates, and the squares represent the median of that biological replicate. Black line represents the mean of the medians. A one-way ANOVA with the Kruskal-Wallis multiple comparisons test was performed. ns p > 0.05, **** p ≤ 0.0001

Figure 8.. Model of the degradosome-like network in M. smegmatis during log phase with RNase E IDR deletion mutants and carbon starvation.

The circles in the cells show the foci identified in vivo with teal representing RNase E, blue representing RNase J, red representing PNPase, and yellow representing RhlE1. Fuzzy circles represent diffuse foci. Black dashed lines represent transient interactions with RNase E identified in vitro.