RNA Sequencing for Transcript 5′-End Mapping in Mycobacteria

Martini, María Carla; Sun, Huaming; Shell, Scarlet S.

doi:10.1007/978-1-0716-1460-0_22

“…For RNAseq libraries, 5 mL of log phase cultures of Mtb H37Rv WT, Δ rnj , and Δ rnj :: rnj OE strains grown in 7H9 were placed in liquid nitrogen and stored at -80°C. RNA purification and construction of both RNA expression and 5’ end-directed (non-5’ pyrophosphohydrolase-converted) libraries were performed as previously reported [ 38 , 39 ]. For both types of libraries, Illumina HiSeq 2000 paired-end sequencing producing 50 nt reads was used.…”

Section: Methodsmentioning

confidence: 99%

Loss of RNase J leads to multi-drug tolerance and accumulation of highly structured mRNA fragments in Mycobacterium tuberculosis

Martini

¹

,

Hicks

²

,

Xiao

³

et al. 2022

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Despite the existence of well-characterized, canonical mutations that confer high-level drug resistance to Mycobacterium tuberculosis (Mtb), there is evidence that drug resistance mechanisms are more complex than simple acquisition of such mutations. Recent studies have shown that Mtb can acquire non-canonical resistance-associated mutations that confer survival advantages in the presence of certain drugs, likely acting as stepping-stones for acquisition of high-level resistance. Rv2752c/rnj, encoding RNase J, is disproportionately mutated in drug-resistant clinical Mtb isolates. Here we show that deletion of rnj confers increased tolerance to lethal concentrations of several drugs. RNAseq revealed that RNase J affects expression of a subset of genes enriched for PE/PPE genes and stable RNAs and is key for proper 23S rRNA maturation. Gene expression differences implicated two sRNAs and ppe50-ppe51 as important contributors to the drug tolerance phenotype. In addition, we found that in the absence of RNase J, many short RNA fragments accumulate because they are degraded at slower rates. We show that the accumulated transcript fragments are targets of RNase J and are characterized by strong secondary structure and high G+C content, indicating that RNase J has a rate-limiting role in degradation of highly structured RNAs. Taken together, our results demonstrate that RNase J indirectly affects drug tolerance, as well as reveal the endogenous roles of RNase J in mycobacterial RNA metabolism.

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“…We thus compiled all sequences preceded by a start tag (TSS or PSS) that also aligned to one of our genes of interest as follows (68): First, we normalized the TSS hit list against the list of PSS-tagged 5’-ends, retaining only the ends that had were 10-fold or more abundantly TSS-tagged vs. PSS-tagged. 5’-ends tagged with predominantly PSS sequences were eliminated from our hit list as processed starts.…”

Section: Methodsmentioning

confidence: 99%

“…5’-ends tagged with predominantly PSS sequences were eliminated from our hit list as processed starts. Of the remaining hits, we further selected only 5’-ends with the highest coverage in a 10 nt window to eliminate any redundant start sites resulting from imprecise transcription initiation (68). This was done prior to inferring transcription start site vs. processed sites from the 5’-end of an aligned sequences ( Supplemental Tables S5-S7 ).…”

Section: Methodsmentioning

confidence: 99%

Gene recoding by synonymous mutations creates promiscuous intragenic transcription initiation in mycobacteria

Hegelmeyer

¹

,

Previti

²

,

Andrade

³

et al. 2023

Preprint

0

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Each genome encodes some codons more frequently than their synonyms (codon usage bias), but codons are also arranged more frequently into specific pairs (codon pair bias). Recoding viral genomes and yeast or bacterial genes with non-optimal codon pairs has been shown to decrease gene expression. Gene expression is thus importantly regulated not only by the use of particular codons but by their proper juxtaposition. We therefore hypothesized that non-optimal codon pairing could likewise attenuate Mtb genes. We explored the role of codon pair bias by recoding Mtb genes (rpoB, mmpL3, ndh) and assessing their expression in the closely related and tractable model organism M. smegmatis. To our surprise, recoding caused the expression of multiple smaller protein isoforms from all three genes. We confirmed that these smaller proteins were not due to protein degradation, but instead issued from new transcription initiation sites positioned within the open reading frame. New transcripts gave rise to intragenic translation initiation sites, which in turn led to the expression of smaller proteins. We next identified the nucleotide changes associated with these new sites of transcription and translation. Our results demonstrated that apparently benign, synonymous changes can drastically alter gene expression in mycobacteria. More generally, our work expands our understanding of the codon-level parameters that control translation and transcription initiation.

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“…For RNAseq libraries, 5 mL of log phase cultures of Mtb H37Rv WT, Δ rnj , and Δ rnj::rnj OE strains grown in 7H9 were placed in liquid nitrogen and stored at −80 °C. RNA purification and construction of both RNA expression and 5’ end-directed (non-5’ pyrophosphohydrolase-converted) libraries, were performed as previously reported (Martini et al ., 2019, Martini et al ., 2021). For both types of libraries, Illumina HiSeq 2000 paired-end sequencing producing 50 nt reads was used.…”

Section: Methodsmentioning

confidence: 99%

Loss of RNase J leads to multi-drug tolerance and accumulation of highly structured mRNA fragments in Mycobacterium tuberculosis

Martini

¹

,

Hicks

²

,

Xiao

³

et al. 2022

Preprint

Self Cite

View full text Add to dashboard Cite

Despite the existence of well-characterized, canonical mutations that confer high-level drug resistance to Mycobacterium tuberculosis (Mtb), there is evidence that drug resistance mechanisms are more complex than simple acquisition of such mutations. Recent studies have shown that Mtb can acquire non-canonical resistance-associated mutations that confer survival advantages in the presence of certain drugs, likely acting as stepping-stones for acquisition of high-level resistance. Rv2752c/rnj, encoding RNase J, is disproportionately mutated in drug-resistant clinical Mtb isolates. Here we show that deletion of rnj confers increased tolerance to lethal concentrations of several drugs. RNAseq revealed that RNase J affects expression of a subset of genes enriched for PE/PPE genes and stable RNAs and is key for proper 23S rRNA maturation. Gene expression differences implicated two sRNAs and ppe50-ppe51 as important contributors to the drug tolerance phenotype. In addition, we found that in the absence of RNase J, many short RNA fragments accumulate because they are degraded at slower rates. We show that the accumulated transcript fragments are targets of RNase J and are characterized by strong secondary structure and high G+C content, indicating that RNase J has a rate-limiting role in degradation of highly structured RNAs. Taken together, our results demonstrate that RNase J indirectly affects drug tolerance, as well as reveal the endogenous roles of RNase J in mycobacterial RNA metabolism.

show abstract

RNA Sequencing for Transcript 5′-End Mapping in Mycobacteria

Cited by 5 publications

References 8 publications

Loss of RNase J leads to multi-drug tolerance and accumulation of highly structured mRNA fragments in Mycobacterium tuberculosis

Loss of RNase J leads to multi-drug tolerance and accumulation of highly structured mRNA fragments in Mycobacterium tuberculosis

Gene recoding by synonymous mutations creates promiscuous intragenic transcription initiation in mycobacteria

Loss of RNase J leads to multi-drug tolerance and accumulation of highly structured mRNA fragments in Mycobacterium tuberculosis

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