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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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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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