Figure 1. Transgenic expression of minor snRNAs in human cell lines.

(A) Schematics of viral constructs harboring cassettes for the expression of human U11, U12, and U4atac snRNAs driven by human U2 promoter with 3′ box at the 3′ end. (B) Semiquantitative RT-PCR analysis of U11, U12, and U4atac snRNA expression 72 hours after transduction of the U11/U12/U4atac lentiviral construct in HeLa, HEK293T, and SMA type I patient fibroblasts (GM03813). U1 and U2 major snRNAs as well as 5S rRNA were used as controls. Lanes that were run on the same gel but were noncontiguous are separated by a vertical line. (C) RT-PCR analysis of the total levels of minor snRNA overexpression in cell extracts from HEK293T cells transiently transfected with the U11/U12/U4atac lentiviral construct for 48 hours relative to mock-transfected cells used as controls. The scatter plot shows the fold change in the relative amounts of U11, U12, and U4atac overexpression over endogenous minor snRNA levels in mock-transfected cells, which were arbitrarily set to 1 (dotted line). Individual data points, mean, and SEM from 3 independent experiments are shown. (D) Equal amounts of cell extract from either U11/U12/U4atac or mock-transfected HEK293T cells as in C were immunoprecipitated with anti-SmB antibodies, followed by RNA purification and RT-PCR analysis. The scatter plot shows the fold change in the amounts of immunoprecipitated minor snRNAs from U11/U12/U4atac-transfected cells relative to the levels from mock-transfected cells, which were arbitrarily set to 1 (dotted line). Individual data points, mean, and SEM from 3 independent experiments are shown.