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 5'-phosphorylation. 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 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 called RppH. Overall design: The mutT4 gene of M. tuberculosis strain mc26030 (H37Rv ?RD1 ?panCD) was partially replaced by a zeocin resistance gene by recombineering. Care was taken to disrupt the function of mutT4 without disrupting adjacent genes. The deletion strain was complemented by integration of a plasmid containing mutT4 with its native promoter and 5' UTR into the Giles phage integration site. Triplicate cultures of the parental, deletion, and complemented strains strains were grown to log phase in 7H9 media supplemented with OADC (0.05 g/L oleic acid, 5 g/L bovine serum albumin fraction V, 2 g/L glucose, 0.85 g/L NaCl, and 4 mg/L catalase), 0.2% glycerol, 0.05% Tween 80, and 24 µg/mL pantothenate. RNA was harvested and rRNA was depleted as described in (Culviner, P.H., Guegler, C.K. and Laub, M.T. (2020) A simple, cost-effective, and robust method for rRNA depletion in RNA-sequencing studies. MBio, 11, 10.1128/mbio. 00010-00020.). Samples were then treated with E. coli RppH or mock-treated, and 5'-end directed RNAseq libraries were constructed as described (Martini, M.C., Sun, H. and Shell, S.S. (2021) RNA Sequencing for Transcript 5'-End Mapping in Mycobacteria. Methods Mol Biol, 2314, 513-531).