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An Aptamer-based mRNA Affinity Purification Procedure (RaPID) for the Identification of Associated RNAs (RaPID-seq)
Rohini R Nair1, Gal Haimovich1, Jeffrey E Gerst1
1Department of Molecular Genetics, Weizmann Institute of Science, Rehovot 7610001, Israel.
Abstract:
RNA-RNA and RNA-protein interactions are involved in the regulation of gene expression. Here, we describe an updated and extended version of our RNA purification and protein identification (RaPID) protocol for the pulldown of aptamer-tagged mRNAs by affinity purification. The method takes advantage of the high affinity interaction between the MS2 RNA aptamer and the MS2 coat protein (MCP), as well as that between streptavidin-binding peptide (SBP) and streptavidin. Thus, it employs MCP-SBP fusions to affinity purify MS2-tagged target RNAs of interest over immobilized streptavidin. Purified aptamer-tagged mRNAs, along with any associated RNAs and proteins, are then sent for RNA sequencing (RaPID-seq) or mass spectrometry (RaPID-MS), which allows for the identification of bound cohort RNAs and proteins, respectively.
Insights
This study presents an updated RNA purification and protein identification (RaPID) protocol. The enhanced RaPID method efficiently pulls down aptamer-tagged mRNAs to identify interacting RNAs and proteins.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- RNA-RNA and RNA-protein interactions are crucial for gene expression regulation.
- Identifying these interactions is key to understanding cellular mechanisms.
- Existing methods may have limitations in efficiency or scope.
Purpose of the Study:
- To present an updated and extended RNA purification and protein identification (RaPID) protocol.
- To enable efficient pulldown of aptamer-tagged mRNAs for subsequent analysis.
- To facilitate the identification of interacting RNA and protein partners.
Main Methods:
- Utilizes the high-affinity MS2 RNA aptamer and MS2 coat protein (MCP) interaction.
- Employs a fusion protein of MCP and streptavidin-binding peptide (SBP).
- Immobilized streptavidin is used for affinity purification of MS2-tagged target RNAs.
- Purified complexes are analyzed via RNA sequencing (RaPID-seq) or mass spectrometry (RaPID-MS).
Main Results:
- The updated RaPID protocol successfully purifies aptamer-tagged mRNAs.
- Associated RNAs and proteins are co-purified with the target mRNA.
- RaPID-seq and RaPID-MS allow for the identification of bound RNA and protein cohorts.
- The method provides a robust approach for studying RNA-centric interactions.
Conclusions:
- The enhanced RaPID protocol offers an efficient method for studying RNA-RNA and RNA-protein interactions.
- This technique aids in the comprehensive identification of RNA-binding proteins and co-purifying RNAs.
- RaPID is a valuable tool for investigating gene expression regulation at the post-transcriptional level.