Characterization of the splicing functions of the two pseudouridines Ψ5 and Ψ6 in U1 small nuclear RNA

Abstract

Pseudouridylation is the most abundant type of Ribonucleic acid (RNA) modification where uracil is converted into pseudouridine (Ψ). The 5’ end of U1 small nuclear RNA (snRNA) contains two evolutionary conserved pseudouridines, Ψ5 and Ψ6. It is still a challenge to understand the splicing roles of Ψ5 and Ψ6 in helping U1 to recognise thousands of 5’ splice site (5’ss) variants. Hence, our project is to characterize the effects of removing the Ψ5 and Ψ6 in U1 snRNA on splicing changes via scaRNAs ACA47 and U109 Knockout (KO) which act as guide RNAs for pseudouridylation to occur. Differentially expressed genes (DEGs) and time-course of rescue plasmids experiments were conducted to investigate if Ψ5 and Ψ6 affect the gene expression after ACA47 and U109 KO. Minigene experiments were also performed to observe the splicing effects of Ψ5 and Ψ6. Results showed that indeed Ψ5 and Ψ6 do affect the gene expression and longer transfection time would increase the percentage of rescue DEGs. The splicing pattern of Ψ5 and Ψ6 can also cause an indirect effect of gene regulation. In summary, our findings are useful for better understanding of the 5’ splice site (5’ss)/U1 recognition.