SMD2 reads pseudouridines to regulate mRNA splicing and promote tumorigenesis [RIP-Seq]

Pseudouridines (?) in mRNA are linked to alternative splicing, but their regulatory mechanisms remain unclear due to the lack of identified reader proteins. Here, we identify SMD2, a core spliceosomal component, as a direct ? reader. Using in vitro and ex vivo assays, we show that SMD2 preferentially binds ? over unmodified uridines. SMD2 collaborates with PUS family enzymes to regulate alternative splicing by binding ? near exon-intron boundaries, modulating the splicing of numerous pre-mRNAs. Notably, the gene encoding SMD2, SNRPD2, is overexpressed across multiple cancers and is essential for tumor cell proliferation through the maturation of key transcripts. These findings uncover a direct mechanistic link between ? and spliceosomal function, establishing SMD2 as a critical regulator of ?-mediated splicing and a potential cancer therapeutic target. Overall design: Using RNA-Pull-Down technology, it was identified that the pseudouracil modification might have an interaction relationship with the SMD2 protein (gene name SNRPD2). We knocked out the pseudouracil modification enzyme PUS7 and the SNRPD2 gene in HEK293T cells through CRISPR-Cas9 technology (n=3). Meanwhile, the SMD2-RIP experiment was combined to explore its internal interaction relationship. Furthermore, we simultaneously conducted a CRISPR-Cas9 knockout similar to that in the SNRPD2 cancer high-expression CHOL cell line to investigate the specific functions of the interaction relationship between SNRPD2 and pseudouracil modification in cancer.