Rif-Correct, a software tool for bacterial mRNA half-life estimation

Aretakis, James R.; Schrader, Jared M.

doi:10.1101/2022.02.25.481994

Cited by 2 publications

(4 citation statements)

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“…Consistent with a role in stimulating mRNA decay, RNase EΔIDR mutants have been shown to have slow mRNA decay in different bacterial species, including E. coli and C. crescentus (10, 32). To test whether the S. meliloti BR-body deficient mutant (RNase EΔIDR) also exhibits a slow-down in mRNA decay, we performed Rif-seq experiments to measure global mRNA half-lives (10, 33) (Fig 3A,B). In this assay, wild-type (termed RNase E henceforth) and RNase EΔIDR cells were incubated with rifampicin to block transcription, and RNA abundance was measured prior to and 1, 3, and 9 minutes after addition of rifampicin.…”

Section: Resultsmentioning

confidence: 99%

“…In this assay, wild-type (termed RNase E henceforth) and RNase EΔIDR cells were incubated with rifampicin to block transcription, and RNA abundance was measured prior to and 1, 3, and 9 minutes after addition of rifampicin. The half-lives of RNAs can then be calculated in a genome-wide manner (10, 33). Importantly, each time-course was collected in duplicate, and tRNA is not removed from the RNA samples, as this stable RNA acts as a loading control.…”

Section: Resultsmentioning

confidence: 99%

“…The bulk mRNA half-lives show that mRNA is significantly stabilized in the RNase EΔIDR mutant (from half-life of 11.7 minutes in RNase E to 25.4 minutes in the RNase EΔIDR mutant) (Fig 3B). By using the Rifcorrect software package (33), we calculated the half-lives of individual mRNAs expressed in mid-log cells (Fig 3C). Rifcorrect yielded a total of 665 mRNA half-lives that were measured in all replicates of both the RNase E and RNase EΔIDR Rif-seq datasets (Fig 3C).…”

Section: Sinorhizobium Meliloti Rnase E Forms Br-bodies That Promote ...mentioning

confidence: 99%

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Sinorhizobium melilotiBR-bodies promote fitness during host colonization

Mallikaarachchi,

Huang,

Madras

et al. 2024

Preprint

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Biomolecular condensates, such as the nucleoli or P-bodies, are non-membrane-bound assemblies of proteins and nucleic acids that facilitate specific cellular processes. Like eukaryotic P-bodies, the recently discovered bacterial ribonucleoprotein bodies (BR-bodies) organize the mRNA decay machinery, yet the similarities in molecular and cellular functions across species have been poorly explored. Here, we examine the functions of BR-bodies in the nitrogen-fixing endosymbiont Sinorhizobium meliloti, which colonizes the roots of compatible legume plants. Assembly of BR-bodies into visible foci in S. meliloti cells requires the C-terminal intrinsically disordered region (IDR) of RNase E, and foci fusion is readily observed in vivo, suggesting they are liquid-like condensates that form via mRNA sequestration. Using Rif-seq to measure mRNA lifetimes, we found a global slowdown in mRNA decay in a mutant deficient in BR-bodies, indicating that compartmentalization of the degradation machinery promotes efficient mRNA turnover. While BR-bodies are constitutively present during exponential growth, the abundance of BR-bodies increases upon cell stress, whereby they promote stress resistance. Finally, using Medicago truncatula as host, we show that BR-bodies enhance competitiveness during colonization and appear to be required for effective symbiosis, as mutants without BR-bodies failed to stimulate plant growth. These results suggest that BR-bodies provide a fitness advantage for bacteria during infection, perhaps by enabling better resistance against the host immune response.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Sinorhizobium Meliloti Rnase E Forms Br-bodies That Promote ...mentioning

confidence: 99%

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Sinorhizobium melilotiBR-bodies promote fitness during host colonization

Mallikaarachchi,

Huang,

Madras

et al. 2024

Preprint

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“…RNA-seq samples were stripped of adapter sequences using a custom Python script, reads were aligned to rRNA using bowtie, and the unaligned reads were then aligned to the genome using bowtie. RNA levels were calculated for each sample using Reads per kilobase per million (RPKM), and half-lives were calculated from the time series using Rif-Correct ( 62 ).…”

Section: Methodsmentioning

confidence: 99%

The DEAD-box RNA helicase RhlB is required for efficient RNA processing at low temperature in Caulobacter

de Araújo,

Picinato,

Lorenzetti

et al. 2023

Microbiol Spectr

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The RNA helicases from the DEAD-box family participate in several biochemical processes, with special relevance in the assembly of ribosome ribonucleoparticles and in RNA turnover as part of the multi-enzymatic RNA degradosome complex. Caulobacter crescentus has three DEAD-box RNA helicases, including RhlB, the main RNA helicase associated with the RNA degradosome. In this work, we evaluated the impact of RhlB on C. crescentus gene expression both at normal and low temperatures and its role in global RNA decay. The rhlB null mutant showed a freezing-sensitive phenotype after pre-incubation at low temperature, indicating a failure in assembling a proper cryotolerance response. A global transcriptomic profile of the wild-type and rhlB mutants was carried out at 30°C and 10°C and showed 73 and 477 differentially expressed genes, with the most affected gene categories being translation and posttranslational modifications. The rhlB mutant is more sensitive to streptonigrin, suggesting that the downregulation of iron-regulated genes may be due to a higher intracellular iron concentration. A high throughput screening for RhlB-binding RNAs identified 220 transcripts at 30°C, and global RNA decay analyses identified several mRNAs and sRNAs that displayed altered profiles of RNA decay rates. Analyses of the transcripts’ 5′-ends from the C. crescentus rhlB mutant and from Pseudomonas aeruginosa PAO1 rhlE1 and rhlE2 mutants confirm that in the absence of the DEAD-box RNA helicase associated with RNase E, there is an accumulation of RNA degradation intermediates. IMPORTANCE One of the most important control points in gene regulation is RNA stability, which determines the half-life of a transcript from its transcription until its degradation. Bacteria have evolved a sophisticated multi-enzymatic complex, the RNA degradosome, which is dedicated mostly to RNA turnover. The combined activity of RNase E and the other RNA degradosome enzymes provides an efficient pipeline for the complete degradation of RNAs. The DEAD-box RNA helicases are very often found in RNA degradosomes from phylogenetically distant bacteria, confirming their importance in unwinding structured RNA for subsequent degradation. This work showed that the absence of the RNA helicase RhlB in the free-living Alphaproteobacterium Caulobacter crescentus causes important changes in gene expression and cell physiology. These are probably due, at least in part, to inefficient RNA processing by the RNA degradosome, particularly at low-temperature conditions.

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Rif-Correct, a software tool for bacterial mRNA half-life estimation

Cited by 2 publications

References 4 publications

Sinorhizobium melilotiBR-bodies promote fitness during host colonization

Sinorhizobium melilotiBR-bodies promote fitness during host colonization

The DEAD-box RNA helicase RhlB is required for efficient RNA processing at low temperature in Caulobacter

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