Method for identifying type and site of RNA binding with protein in plant

Abstract

The invention relates to a method for identifying the type and site of a RNA binding with protein in a plant. The method mainly comprises the following steps: immobilizing the in-vivo binding state of protein and nucleic acid through ultraviolet crosslinking; subjecting immunoprecipitated RNAs to digestion with nuclease into small fragments; removing non-specifically bound RNAs by using SDS-PAGE and transferring methods; and carrying out reverse-transcription PCR amplification and large-scale sequencing by using adaptors connected with two ends of the RNA. The method has the advantages that 1) ultraviolet crosslinking is carried out in vivo, so real in-vivo binding conditions can be reflected; 2) since a covalent bond formed by ultraviolet crosslinking is irreversible, the length of nucleic acid binding with protein can be reduced through digestion by nuclease, and subsequent multi-step purification can greatly improve a ratio of signal to noise; 3) most non-specifically bound RNAs in a precipitation sample are removed through SDS-PAGE separation and subsequent transferring process removes free RNAs; and 4) the two ends of the RNA are connected with the adaptors, which makes subsequent PCR amplification and large-scale sequencing to be possible.

Description

technical field [0001] The invention relates to the research on protein-nucleic acid interaction methods in the field of biotechnology, in particular to a method for identifying RNA species and sites bound to a specific RNA-binding protein within the entire genome. Background technique [0002] Among the human annotated genes, 23% of the proteins are nucleic acid binding proteins, suggesting that these proteins play an important role in the regulation of gene expression. In order to study the regulatory network of these proteins, scientists have tried to establish multiple experimental systems to study the method of protein-nucleic acid interaction. SELEX (Selected evolution of ligands through exponential enrichment) is a commonly used method for verifying the interaction between proteins and RNA in vitro. The key to the success of this experiment depends on the selection of specific RNA domains with strong affinity from random RNA sequence libraries ( motif). But sometime...

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Problems solved by technology

At present, the HITS-CLIP method has been very well applied in animals, but there is no corresponding report in the plant system, which greatly limits the research on the function of RNA-binding proteins in

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