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 these enzymes, RNase E, contains intrinsically disordered regions (IDRs) and initiates mRNA degradation. 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. Overall design: M. smegmatis strains were constructed in which the native copy of rne (encoding RNase E) was deleted and a copy of rne with its native promoter and 5' UTR was inserted into the L5 phage integration site. All rne versions had N-terminal 6xhis-3xFLAG tags. The N-terminal deletion lacked codons 2-329, the C-terminal deletion lacked codons 824-1037, and the N- and C-terminal double deletion lacked both.