Protocol to study the effects of mutations near splicing sites on pre-mRNA splicing

Mutations at RNA splicing sites or regulatory elements can alter splicing efficiency or patterns, affecting RNA functionality and tissue-specific expression. Here, we present a protocol to study the impact of mutations near splicing sites on precursor mRNA (pre-mRNA) splicing. We describe steps for constructing plasmids by cloning the target gene into the pEGFP-N1 vector, performing site-directed mutagenesis, and transiently transfecting HEK293 cells. We then detail procedures for conducting mini-gene splicing assays to analyze splicing patterns influenced by mutations. For complete details on the use and execution of this protocol, please refer to Peng et al.¹.