Regulatory roles of Escherichia coli 5' UTR and ORF-internal RNAs detected by 3' end mapping

Adams, Philip P.; Baniulyte, Gabriele; Esnault, Caroline; Chegireddy, Kavya; Singh, Navjot; Monge, Molly; Dale, Ryan; Storz, Gisela; Wade, Joseph T.

doi:10.7554/elife.62438

Cited by 91 publications

(147 citation statements)

References 102 publications

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“…4 b). This finding goes in line with the increasing recognition of the prevalence of small transcripts from the aforementioned regions [ 36 , 45 , 46 ].…”

Section: Discussionsupporting

confidence: 75%

Methyltransferase-directed orthogonal tagging and sequencing of miRNAs and bacterial small RNAs

et al. 2021

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Background Targeted installation of designer chemical moieties on biopolymers provides an orthogonal means for their visualisation, manipulation and sequence analysis. Although high-throughput RNA sequencing is a widely used method for transcriptome analysis, certain steps, such as 3′ adapter ligation in strand-specific RNA sequencing, remain challenging due to structure- and sequence-related biases introduced by RNA ligases, leading to misrepresentation of particular RNA species. Here, we remedy this limitation by adapting two RNA 2′-O-methyltransferases from the Hen1 family for orthogonal chemo-enzymatic click tethering of a 3′ sequencing adapter that supports cDNA production by reverse transcription of the tagged RNA. Results We showed that the ssRNA-specific DmHen1 and dsRNA-specific AtHEN1 can be used to efficiently append an oligonucleotide adapter to the 3′ end of target RNA for sequencing library preparation. Using this new chemo-enzymatic approach, we identified miRNAs and prokaryotic small non-coding sRNAs in probiotic Lactobacillus casei BL23. We found that compared to a reference conventional RNA library preparation, methyltransferase-Directed Orthogonal Tagging and RNA sequencing, mDOT-seq, avoids misdetection of unspecific highly-structured RNA species, thus providing better accuracy in identifying the groups of transcripts analysed. Our results suggest that mDOT-seq has the potential to advance analysis of eukaryotic and prokaryotic ssRNAs. Conclusions Our findings provide a valuable resource for studies of the RNA-centred regulatory networks in Lactobacilli and pave the way to developing novel transcriptome and epitranscriptome profiling approaches in vitro and inside living cells. As RNA methyltransferases share the structure of the AdoMet-binding domain and several specific cofactor binding features, the basic principles of our approach could be easily translated to other AdoMet-dependent enzymes for the development of modification-specific RNA-seq techniques.

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“…4 b). This finding goes in line with the increasing recognition of the prevalence of small transcripts from the aforementioned regions [ 36 , 45 , 46 ].…”

Section: Discussionsupporting

confidence: 75%

Methyltransferase-directed orthogonal tagging and sequencing of miRNAs and bacterial small RNAs

et al. 2021

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“…Agarose northern blots were performed using total wild-type B . burgdorferi RNA as described previously [ 28 , 72 ]. 10 μg of RNA were fractionated on a 2% NuSieve 3:1 agarose (Lonza), 1X MOPS, 2% formaldehyde gel and transferred to a Zeta-Probe GT membrane (Bio-Rad) via capillary action overnight.…”

Section: Methodsmentioning

confidence: 99%

BB0562 is a nutritional virulence determinant with lipase activity important for Borrelia burgdorferi infection and survival in fatty acid deficient environments

et al. 2021

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The Lyme disease spirochete Borrelia burgdorferi relies on uptake of essential nutrients from its host environments for survival and infection. Therefore, nutrient acquisition mechanisms constitute key virulence properties of the pathogen, yet these mechanisms remain largely unknown. In vivo expression technology applied to B. burgdorferi (BbIVET) during mammalian infection identified gene bb0562, which encodes a hypothetical protein comprised of a conserved domain of unknown function, DUF3996. DUF3996 is also found across adjacent encoded hypothetical proteins BB0563 and BB0564, suggesting the possibility that the three proteins could be functionally related. Deletion of bb0562, bb0563 and bb0564 individually and together demonstrated that bb0562 alone was important for optimal disseminated infection in immunocompetent and immunocompromised mice by needle inoculation and tick bite transmission. Moreover, bb0562 promoted spirochete survival during the blood dissemination phase of infection. Gene bb0562 was also found to be important for spirochete growth in low serum media and the growth defect of Δbb0562 B. burgdorferi was rescued with the addition of various long chain fatty acids, particularly oleic acid. In mammals, fatty acids are primarily stored in fat droplets in the form of triglycerides. Strikingly, addition of glyceryl trioleate, the triglyceride form of oleic acid, to the low serum media did not rescue the growth defect of the mutant, suggesting bb0562 may be important for the release of fatty acids from triglycerides. Therefore, we searched for and identified two canonical GXSXG lipase motifs within BB0562, despite the lack of homology to known bacterial lipases. Purified BB0562 demonstrated lipolytic activity dependent on the catalytic serine residues within the two motifs. In sum, we have established that bb0562 is a novel nutritional virulence determinant, encoding a lipase that contributes to fatty acid scavenge for spirochete survival in environments deficient in free fatty acids including the mammalian host.

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“…RhlS not only controls the translation of its downstream gene ( rhll ), but also regulates the translation of fpvA mRNAs transcribed from a distant locus and which encode a siderophore pyoverdine receptor, although the mechanism of action here is still unclear. Finally, recent work on E. coli focusing on the identification of 3' ends from 5' UTRs revealed the presence of trans -acting sRNA sponges ( Adams et al, 2021 ).…”

Section: Unconventional Regulatory Rna Biogenesis and Functionsmentioning

confidence: 99%

“…RybB is also sponged by RbsZ, the sRNA associated with Hfq and ProQ ( Melamed et al, 2020 ). Recent studies in the model organism E. coli demonstrated the presence of sRNA sponges ChiZ and IspZ deriving from 5' UTRs ( Adams et al, 2021 ), suggesting that sponge RNAs can be produced from any part of the genome. They can thus be produced independently since they contain their own promoters, they can be processed from an existing transcript, they may belong to intergenic spacer regions or intergenic tRNA spacers, or they can be generated from 3' UTRs ( Denham, 2020 ).…”

Section: Unconventional Regulatory Rna Biogenesis and Functionsmentioning

confidence: 99%

Diversity and Versatility in Small RNA-Mediated Regulation in Bacterial Pathogens

Felden

Augagneur

2021

Front. Microbiol.

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Bacterial gene expression is under the control of a large set of molecules acting at multiple levels. In addition to the transcription factors (TFs) already known to be involved in global regulation of gene expression, small regulatory RNAs (sRNAs) are emerging as major players in gene regulatory networks, where they allow environmental adaptation and fitness. Developments in high-throughput screening have enabled their detection in the entire bacterial kingdom. These sRNAs influence a plethora of biological processes, including but not limited to outer membrane synthesis, metabolism, TF regulation, transcription termination, virulence, and antibiotic resistance and persistence. Almost always noncoding, they regulate target genes at the post-transcriptional level, usually through base-pair interactions with mRNAs, alone or with the help of dedicated chaperones. There is growing evidence that sRNA-mediated mechanisms of actions are far more diverse than initially thought, and that they go beyond the so-called cis- and trans-encoded classifications. These molecules can be derived and processed from 5' untranslated regions (UTRs), coding or non-coding sequences, and even from 3' UTRs. They usually act within the bacterial cytoplasm, but recent studies showed sRNAs in extracellular vesicles, where they influence host cell interactions. In this review, we highlight the various functions of sRNAs in bacterial pathogens, and focus on the increasing examples of widely diverse regulatory mechanisms that might compel us to reconsider what constitute the sRNA.

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Regulatory roles of Escherichia coli 5' UTR and ORF-internal RNAs detected by 3' end mapping

Cited by 91 publications

References 102 publications

Methyltransferase-directed orthogonal tagging and sequencing of miRNAs and bacterial small RNAs

Methyltransferase-directed orthogonal tagging and sequencing of miRNAs and bacterial small RNAs

BB0562 is a nutritional virulence determinant with lipase activity important for Borrelia burgdorferi infection and survival in fatty acid deficient environments

Diversity and Versatility in Small RNA-Mediated Regulation in Bacterial Pathogens

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