Method for regulating RNA splicing by inducing splice site base mutation or polypyrimidine region base substitution

Abstract

A method of regulating RNA splicing by inducing splice site base mutation or polypyrimidine region base substitution is disclosed, the method comprises expressing targeted cytidine deaminase in cellsto induce AG to AA mutation of 3' splicing site of intron of interested of gene of interested in the cells, or GT to AT mutation of 5' splicing site of the intron of interested of the gene of interested in the cells, or respective mutation of multiple C in a polypyrimidine region of the intron of interested of the gene of interested to T. The method can specifically block the exon recognition process, regulates the alternative splicing process of endogenous mRNA, induces exon skipping, activates alternative splicing sites, induces mutual exclusive exon conversion, induces intron retention andenhances exon retention.

Description

technical field [0001] The invention relates to a method for regulating RNA splicing by inducing base mutations at splicing sites or base substitutions in polypyrimidine regions. Background technique [0002] Correct expression of eukaryotic genes requires intron splicing in pre-mRNA and exon splicing to form mature mRNA. More than 98% of introns are excised by a highly dynamic protein complex called the spliceosome. The spliceosome is composed of more than 150 small nuclear ribonucleoproteins (snRNPs), such as U1, U2, U4, U5 and U6. During splicing, U1snRNP recognizes the GU sequence at the 5' splice site of the intron, splicing factor 1 (SF1) binds to the bifurcation point of the intron, and the 35KD subunit of the U2 accessory protein (U2AF) binds to the intron's The AG sequence at the 3' splicing site, the 65KD subunit is combined with the polypyrimidine region sequence to complete the exon recognition process; then the U5 and U6 proteins catalyze the intron recognitio...

AI Technical Summary

Problems solved by technology

However, there is still a lack of convenient and effective methods to regulate the alternative splicing process, and the functions of most alternative splicing protein isoforms are not clear

[0004] Antisense oligonucleotides can bind to RNA cis-acting elements (such as exon splicing enhancers) to block exon splicing, but splicing regulation using antisense oligonucleotides requires careful design And strict screening, continuous drug administration is required in the course of disease treatment, and its synthesis process is also very expensive, consuming time and money

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