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A plant tethering system for the functional study of protein-RNA interactions in vivo

ABSTRACT: The sorting of RNA transcripts dictates their ultimate post-transcriptional fates, such as translation, decay or degradation by RNA interference (RNAi). This sorting of RNAs into distinct fates is mediated by their interaction with RNA-binding proteins. While hundreds of RNA binding proteins have been identified, which act to sort RNAs into different pathways is largely unknown. Particularly in plants, this is due to the lack of reliable protein-RNA artificial tethering tools necessary to determine the mechanism of protein action on an RNA in vivo. Here we used minimal Arabidopsis BRN1 RNA binding domains to generate a protein-RNA tethering system which functions on an endogenous Arabidopsis RNA called SOC1 that is tracked by the quantitative flowering time phenotype. Unlike other protein-RNA tethering systems that have been attempted in plants, our system circumvents the inadvertent triggering of RNAi. We successfully in vivo tethered a protein epitope to the RNA and identified new proteins that were previously unknown to interact during BRN1-RNA binding. This protein-RNA tethering system enables the future determination of any protein’s function upon recruitment to an RNA and can be used to discover new interactions with RNA-binding proteins.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Leaf

SUBMITTER: Yu-Hung Hung

LAB HEAD: R. Keith Slotkin

PROVIDER: PXD031072 | Pride | 2022-05-09

REPOSITORIES: Pride

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			Action	DRS
	20210206_Slotkin05_YH_IP_A_R1.raw	Raw
	20210206_Slotkin05_YH_IP_A_R1__F020944_.mzid.gz	Mzid
	20210206_Slotkin05_YH_IP_A_R1__F020944_.mzid_20210206_Slotkin05_YH_IP_A_R1__F020944_.MGF	Mzid
	20210206_Slotkin05_YH_IP_A_R2.raw	Raw
	20210206_Slotkin05_YH_IP_A_R2__F020945_.mzid.gz	Mzid

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A plant tethering system for the functional study of protein-RNA interactions in vivo

Project description:The sorting of RNA transcripts dictates their ultimate post-transcriptional fates, such as translation, decay or degradation by RNA interference (RNAi). This sorting of RNAs into distinct fates is mediated by their interaction with RNA-binding proteins. While hundreds of RNA binding proteins have been identified, which act to sort RNAs into different pathways is largely unknown. Particularly in plants, this is due to the lack of reliable protein-RNA artificial tethering tools necessary to determine the mechanism of protein action on an RNA in vivo. Here we generated a protein-RNA tethering system which functions on an endogenous Arabidopsis RNA that is easily tracked by the quantitative flowering time phenotype. Unlike other protein-RNA tethering systems that have been attempted in plants, our system circumvents the inadvertent triggering of RNAi. We successfully in vivo tethered a protein epitope, deadenylase protein and translation factor to the RNA, which function to tag, decay and boost protein production, respectively. We demonstrated that our tethering system 1) is sufficient to engineer the downstream fate of an RNA, 2) enables the determination of any protein’s function upon recruitment to an RNA, and 3) can be used to discover new interactions with RNA-binding proteins.

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