Biology subjects

Rapiejko, A. R.

Publications and source records attributed to Rapiejko, A. R..

4 recordsLinked to original sources

bioRxiv2026

In vitro Cleavage Requirements and Specificities of Mycobacterial RNase E

Regulation of RNA pools allows for adaptation to changing environments and stress, which is especially important in pathogenic bacteria such as Mycobacterium tuberculosis. RNA degradation is a significant contributor to RNA abundance, and Ribonuclease (RNase) E has a rate-limiting role in degradation of a majority of mycobacterial transcripts. However, many open questions remain about the RNA substrate requirements and specificities for efficient cleavage by mycobacterial RNase E. Here, using both Mycolicibacterium smegmatis and M. tuberculosis RNase E, we demonstrate that this enzyme is only active on substrates with a minimum length of approximately 27 nt. Furthermore, we show that mycobacterial RNase E prefers substrates with 5 monophosphates rather than 5 triphosphates, and that the positions of cleavage events within substrates are dictated by both sequence and distance from the RNA ends. Our results also suggest that RNase E may be affected by product inhibition. Finally, we show that M. smegmatis RNase E behaves similarly to M. tuberculosis RNase E, validating the use of this model organism for RNA degradation studies.

molecular biology↗

bioRxiv2025

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

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 have defined the interactions among major mycobacterial mRNA degradation enzymes and identified the functions 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 impact on steady-state 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, including stimulation of RhlE1 activity. 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.

molecular biology↗

bioRxiv2025

Mycobacterium tuberculosis MutT4 is an RNA pyrophosphohydrolase that forms biomolecular condensates and sensitizes mRNAs to degradation

Bacterial adaptation to stress involves changes in transcription and mRNA degradation rates. 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 deleted each non-essential Nudix gene from Mtb to determine their impacts on mRNA phosphorylation states. Deletion of mutT4 (Rv3908) increased the relative abundance of 5-triphosphates on myriad mRNAs across the transcriptome. Purified MutT4 converted mRNA 5-triphosphates into monophosphates, and stimulated degradation by RNase E and RNase J. MutT4 has intrinsically disordered regions (IDRs), a common domain for biomolecular condensate formation. Microscopy showed that MutT4 forms condensates that dissociate upon addition of rifampicin, and that the N-terminal IDR is sufficient for condensate formation. These MutT4 condensates localize with RNase E and RNase J. Deletion of mutT4 in Mtb leads to a higher outer membrane permeability and resistance to oxidative stress. We conclude that MutT4 is the RppH homolog of Mtb, assembling in condensates that may act as degradation hubs. Our data indicate that MutT4 is unlikely to participate in DNA repair or nucleotide pool cleansing, and as such would more accurately be named RppH.

microbiology↗

bioRxiv2023

Mycobacterial RNase E cleaves with a distinct sequence preference and controls the degradation rates of most Mycolicibacterium smegmatis mRNAs

The mechanisms and regulation of RNA degradation in mycobacteria have been subject to increased interest following the identification of interplay between RNA metabolism and drug resistance. Mycobacteria encode multiple ribonucleases that are predicted to participate in mRNA degradation and/or processing of stable RNAs. RNase E is an endoribonuclease hypothesized to play a major role in mRNA degradation due to its essentiality in mycobacteria and its role in mRNA degradation in gram- negative bacteria. Here, we defined the impact of RNase E on mRNA degradation rates transcriptome- wide in the non-pathogenic model Mycolicibacterium smegmatis. RNase E played a rate-limiting role in the degradation of at least 89% of protein-coding genes, with leadered transcripts generally being more affected by RNase E repression than leaderless transcripts. There was an apparent global slowing of transcription in response to knockdown of RNase E, suggesting that M. smegmatis regulates transcription in responses to changes in mRNA degradation. This compensation was incomplete, as the abundance of most transcripts increased upon RNase E knockdown. We assessed the sequence preferences for cleavage by RNase E transcriptome-wide in both M. smegmatis and M. tuberculosis, and found a consistent bias for cleavage in C-rich regions. Purified RNase E had a clear preference for cleavage immediately upstream of cytidines, distinct from the sequence preferences of RNase E in gram-negatives. We furthermore report a high-resolution map of mRNA cleavage sites in M. tuberculosis, which occur primarily within the RNase E-preferred sequence context, confirming RNase E as a broad contributor to M. tuberculosis transcriptome structure.

molecular biology↗