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Affinity chromatography-based identification of sRNA protein partners in Rhizobia

ABSTRACT: This project aims to identify novel protein partners of conserved trans-sRNAs in the α-proteobacterium Sinorhizobium meliloti. Trans-sRNAs (AbcR2, NfeR1 and EcpR1) were tagged at their 5’ ends with the MS2 aptamer. To identify the proteins associated with the trans-sRNAs we: i) expressed and purified the MS2 coat protein fused to maltose-binding protein (MS2-MBP); ii) purified MS2 tagged sRNAs conjugated with MS2-MBP via amylase column; and iii) subjected retained proteins to mass spectrometry analysis. To discard unspecific interactions we also analysed several control samples: i) samples expressing untagged trans-sRNAs; ii) samples expressing an MS2 tagged Rho-independent transcriptional terminator.

INSTRUMENT(S): amaZon Speed ETD

ORGANISM(S): Rhizobium Meliloti (ensifer Meliloti) (sinorhizobium Meliloti)

SUBMITTER: Ana Matia Gonzalez

LAB HEAD: José Ignacio Jiménez Zurdo

PROVIDER: PXD006410 | Pride | 2020-10-19

REPOSITORIES: Pride

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Dataset's files

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			Action	DRS
	ABCR2.dat	Other
	ABCR2.yep	Other
	ABCR2_MSII.dat	Other
	ABCR2_MSII.yep	Other
	ECPR1.dat	Other

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Publications

Synthetase of the methyl donor S-adenosylmethionine from nitrogen-fixing α-rhizobia can bind functionally diverse RNA species.

Robledo Marta M García-Tomsig Natalia I NI Matia-González Ana M AM García-Rodríguez Fernando M FM Jiménez-Zurdo José I JI

RNA biology 20201010 8

Function of bacterial small non-coding RNAs (sRNAs) and overall RNA metabolism is largely shaped by a vast diversity of RNA-protein interactions. However, in non-model bacteria with defined non-coding transcriptomes the sRNA interactome remains almost unexplored. We used affinity chromatography to capture proteins associated <i>in vivo</i> with MS2-tagged <i>trans</i>-sRNAs that regulate nutrient uptake (AbcR2 and NfeR1) and cell cycle (EcpR1) mRNAs by antisense-based translational inhibition in ...[more]

PMID: 33043803

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Project description:During ribosomal and transfer RNA maturation, external transcribed spacer (ETS) and internal transcribed spacer (ITS) sequences are excised and, as non-functional by-products, are rapidly degraded. The 3’ETS of the glyW-cysT-leuZ polycistronic tRNA precursor was highly and specifically enriched by co-purification with at least two different small regulatory RNAs (sRNAs), RyhB and RybB. Both sRNAs were shown to base pair with the same region in the 3’ETS of leuZ (3’ETSleuZ). Disrupting the pairing by mutating 3’ETSleuZ significantly increased the activity of sRNAs, even under non-inducing conditions. Our results indicate that 3’ETSleuZ prevents sRNA-dependent remodeling of tricarboxylic acid (TCA) cycle fluxes and increases antibiotic sensitivity when sRNAs are transcriptionally repressed. This suggests that 3’ETSleuZ functions as a sponge to absorb transcriptional noise from repressed sRNAs. Finally, the fact that RybB and MicF sRNAs are co-purified with ITSmetZ-metW and ITSmetW-metV strongly suggests a much broader phenomenon. Identification of sRNAs co-purified with MS2-ITSmetZW and MS2-ITSmetWV. ITSmetZW and ITSmetWV (without MS2) were used as control

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MS2-affinity purification coupled with RNA sequencing (MAPS) reveals RyhB sRNA targetome

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