Experimental Validation of the U1 snRNA in Ustilago maydis

U1 small nuclear RNA (snRNA) is a highly conserved and abundant RNA found in eukaryotic cells and has a crucial role in recognizing the 5’ splice site during pre-mRNA intron splicing. The U1 snRNA in the phylum Basidiomycota has remained unexplored despite being well-studied in other groups of fungal species. This study aimed to provide experimental evidence to validate the authenticity of a putative U1 snRNA identified in Ustilago maydis, a fungal species in phylum Basidiomycota. Various molecular biology techniques including RT-PCR, 5’ and 3’ Rapid Amplification of cDNA Ends (RACE), and Immunoprecipitation were utilized towards this goal. To explore if the U1 snRNA candidate associates with the known U1 snRNA-associated proteins such as U1-70K and U1C, I cloned these protein cDNA from U. maydis and constructed affinity-tagged recombinant U1 protein genes for in vivo expression in U. maydis cells. Immunoprecipitation of the recombinant U1 protein and RT-PCR were performed to detect physical interactions between the U1 snRNA candidate and the U1 proteins. Despite the lack of conclusive evidence for direct interactions between U1 snRNA candidate and the recombinant U1 proteins, the results of 5’ and 3’ RACE analyses and Sanger sequencing of the RACE PCR products revealed the size and sequence of this RNA transcript. Moreover, this study generates valuable systems and reagents that allow future investigation on U. maydis U1 snRNP biogenesis and intron splicing mechanisms in Basidiomycota.