Identification of unknown RNA partners using MAPS

Lalaouna, David; Prévost, Karine; Eyraud, Alex; Massé, Éric

doi:10.1016/j.ymeth.2016.11.011

Cited by 37 publications

(46 citation statements)

References 19 publications

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“…In order to characterize RNAs that were targeted in vivo by CcnA and confirm whether CtrA and GcrA mRNAs were indeed targets of CcnA, we performed the technique called MAPS (MS2 Affinity Purification high-throughput Sequencing) as previously described (Lalaouna et al, 2017) (Figure 2A).…”

Section: Resultsmentioning

confidence: 99%

Pulling the strings of cell cycle: a non-coding RNA, CcnA, modulates the master regulators CtrA and GcrA in Caulobacter crescentus

Beroual

Prévost

Lalaouna

et al. 2019

Preprint

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20Bacterial cells are powerful models for understanding how cells divide and accomplish global 21 regulatory programs. In Caulobacter crescentus a cascade of essential master regulators regulate the 22 correct and sequential activation of DNA replication, cell division and development of different cell 23 types. Among them CtrA plays a crucial role coordinating all those functions. Despite decades of 24 investigation, no control by non-coding RNAs (ncRNAs) has been linked to Caulobacter cell cycle. 25Here, for the first time we describe the role of a novel essential factor named CcnA, a ncRNA located 26 at the origin of replication, activated by CtrA and responsible for the rapid and strong accumulation of 27 CtrA itself. In addition CcnA is also responsible for the inhibition of GcrA translation by direct 28 interaction with its UTR region. By a combination of probing experiments and mutagenesis, we 29 propose a new mechanism by liberation (CtrA) or sequestration (GcrA) of the Ribosome Binding Site 30 (RBS). CcnA role is conserved in other alphaproteobacterial species, such as Sinorhizobium meliloti, 31 representing indeed a conserved and fundamental process regulating cell cycle in Rhizobiales and32 Caulobacterales.73 responsible for the specific and highly regulated proteolysis of CtrA. 74In Caulobacter, the regulation of gene expression by ncRNAs has revealed few examples. Initially 75 only 27 ncRNAs were described in this organism (Landt et al., 2008). CrfA is an sRNA involved in 76 adaptation to carbon starvation (Landt et al., 2010). GsrN is involved in the response to σ T -dependent 77 multiple stresses (Tien et al., 2017). Finally ChvR has been recently characterized as a ncRNA that is 78 expressed in response to DNA damage, low pH, and growth in minimal medium (Fröhlich et al.,79 2018). However as new recent approaches using RNAseq and post-genomic techniques expanded the 80 plethora of ncRNA candidates to more than 100 (Zhou et al., 2015), predictions of their integration 81 into the cell cycle circuit (Beroual et al., 2018) has suggested that several new candidate ncRNAs 82 should be deeply studied. 83Here we investigated the role of a ncRNA, named CcnA, that belongs to the origin of replication of 84 Caulobacter chromosome. We studied its role by the construction of deletion mutants, silencing by 85 expression of its antisense and over expression. Results presented in this work identified the mRNAs 86 of CtrA and GcrA, two master regulators of cell cycle, as main targets of this ncRNA. Data were 87 88 closely related organism such as Sinorhizobium meliloti suggested its potential conservation across 89 bacteria. 91 Results 92CcnA expression is regulated by CtrA 94Based on previous results (Zhou et al., 2015) we observed that CCNA_R0094, here named Cell Cycle 95 non-coding RNA A (CcnA), expression peaks after few minutes from the accumulation of ctrA 96 transcript and protein, in the second half of the S-phase, when P2, the second ctrA promoter, is 97 activated (Fig 1A). 98We designed primers to dete...

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Section: Resultsmentioning

confidence: 99%

Pulling the strings of cell cycle: a non-coding RNA, CcnA, modulates the master regulators CtrA and GcrA in Caulobacter crescentus

Beroual

Prévost

Lalaouna

et al. 2019

Preprint

Self Cite

View full text Add to dashboard Cite

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“…Affinity purification assays were performed as previously described (Lalaouna et al ., ; Lalaouna et al ., ). Briefly, we used a Δ gcvB rne131 background to maximize the co‐purification of target mRNAs.…”

Section: Methodsmentioning

confidence: 97%

“…A recent report indicates that GcvB binds to potentially more than the 24 characterized target mRNAs (Melamed et al ., ). To address this, we performed MAPS for MS2‐affinity purification coupled with RNAseq (Lalaouna et al ., ; Lalaouna et al ., ), enabling to co‐purifying all RNAs interacting with in vivo expressed MS2‐tagged GcvB sRNA. Thus, we revealed and validated seven additional GcvB target mRNAs which are mainly involved in amino acids metabolism.…”

Section: Introductionmentioning

confidence: 99%

GcvB small RNA uses two distinct seed regions to regulate an extensive targetome

Lalaouna

Eyraud

Devinck

et al. 2018

Molecular Microbiology

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GcvB small RNA is described as post-transcriptional regulator of 1-2% of all mRNAs in Escherichia coli and Salmonella Typhimurium. At least 24 GcvB:mRNA interactions have been validated in vivo, establishing the largest characterized sRNA targetome. By performing MS2-affinity purification coupled with RNA sequencing (MAPS) technology, we identified seven additional mRNAs negatively regulated by GcvB in E. coli. Contrary to the vast majority of previously known targets, which pair to the wellconserved GcvB R1 region, we validated four mRNAs targeted by GcvB R3 region. This indicates that base-pairing through R3 seed sequence seems relatively common. We also noticed unusual GcvB pairing sites in the coding sequence of two target mRNAs. One of these target mRNAs has a pairing site displaying a unique ACA motif, suggesting that GcvB could hijack a translational enhancer element. The second target mRNA is likely regulated via an active RNase E-mediated mRNA degradation mechanism. Remarkably, we confirmed the importance of the sRNA sponge SroC in the fine-tuning control of GcvB activity in function of growth conditions such as growth phase and nutrient availability.

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“…To identify new targets of SraL, we used the recently developed in vivo technology MS2-affinity purification coupled with RNA sequencing (MAPS) (23)(24)(25)(26). For this, SraL was fused to an MS2 RNA aptamer, which binds the MS2 coat protein with high affinity, enabling copurification of SraL with its mRNA(s) target(s).…”

Section: Ms2 Affinity Purification Coupled With Rna Sequencing To Idementioning

confidence: 99%

SraL sRNA interaction regulates the terminator by preventing premature transcription termination of rho mRNA

Silva

Barahona

Eyraud

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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Identification of unknown RNA partners using MAPS

Cited by 37 publications

References 19 publications

Pulling the strings of cell cycle: a non-coding RNA, CcnA, modulates the master regulators CtrA and GcrA in Caulobacter crescentus

Pulling the strings of cell cycle: a non-coding RNA, CcnA, modulates the master regulators CtrA and GcrA in Caulobacter crescentus

GcvB small RNA uses two distinct seed regions to regulate an extensive targetome

SraL sRNA interaction regulates the terminator by preventing premature transcription termination of rho mRNA

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