Analysis of vU1.1 + 10 associated sequence alterations on interactions with U1-specific proteins and splicing. (A) Secondary structure of U1 snRNA indicating sequence alterations in vU1.7 + 9 and vU1.1 + 10. (B) Western analysis of RAP complexes isolated using biotinylated SL1, SL3, and SL4 RNAs from canonical U1 and vU1.1 + 10. (C) Levels of U1-70K, UAP56, and SF3A1 in the vU1.1 + 10-SL1, -SL3, and -SL4 complexes were calculated relative to stem–loop complexes of the canonical U1 snRNA. n = 3; (*) P < 0.05, (**) P < 0.01, (***) P < 0.001. (D) RT-qPCR analysis of canonical snRNAs in nuclear and cytoplasmic fractions from HeLa cells cotransfected with Dup51p reporter and either pcDNA or U1-5a constructs for canonical and variant U1 snRNA. The level of snRNAs in the total or nuclear fraction RNA was calculated relative to the cytoplasmic fraction (±SD; n = 3). (E) Primer extension analysis to monitor splicing of the Dup51p reporter pre-mRNA in HeLa cells coexpressing either pcDNA control or plasmids for either the canonical U1-5a, vU1.1 + 10-5a, or U1-5a(C25G) snRNA. PSI at increasing U1:Dup51p ratios were tested. The full-length and exon 2 skipped Dup51p mRNA products are depicted to the left. (F) PSI values for exon 2 in the full-length Dup51p mRNA (±SD) are represented (n = 3). Statistical significance was determined using t-test.
