Fig. 5

TGS1 loss leads to an accumulation of 3′-extended U2 and U4atac snRNAs in human cells. (A) 3′ RACE and sequencing on total RNA showing that TGS1 mutant HeLa cells accumulate more U2-extended snRNA molecules than either parental (CTR) or TGS1 M1R cells. UMUC3 cells treated with TGS1 siRNAs (siTGS1) also accumulate more U2-extended snRNAs than cells exposed to non-targeting siRNAs (siSCR). Nascent and steady-state RNA fractions were purified from control HeLa cells (see the Materials and Methods). Extended U2 molecules carry extra 3′ sequences of templated and/or untemplated nucleotides. Average reads per sample > 78 000 ± 41 000. Data are mean values of two independent clones + SEM. P-values: two-sided Student's t-test. (B) Plots showing the percentage of the different sequence reads of U2 snRNAs in the same HeLa cell samples as in (A). Numbers on the x-axis: additional nucleotides beyond the 189 nt of the mature form. Position 0 includes mature U2 snRNA species of 188 nt or 188 nt plus a post-transcriptionally added A. y-axis: percentage of total reads. Blue, genomic-templated nucleotides; green, untemplated adenosine; orange, untemplated uridine. (C) Characterization of U2 snRNA molecules from nuclear and cytoplasmic fractions from TGS1 M1, TGS1 M2, TGS1 M1R and control (CTR) cells. (D) Tail length and composition of U2 snRNAs in the samples shown in (C). y-axis: percentage of total reads. (E) Percentages of U2 molecules with tails ending with post-transcriptionally added uridines in the HeLa cell RNA samples described in (A). Total reads per sample > 75 000. (F) Percentages of 3′-extended U4atac snRNAs in the RNA samples described in (A). Average reads per sample > 106 000 ± 21 000 (Nascent: >11 000). (G) Tail lengths and composition of the 3′ ends of U4atac snRNAs in TGS1 M1, TGS1 M2, TGS1 M1R and control (CTR) cells. Numbers on the x-axis: additional nucleotides beyond the 130 nt of mature U4atac (position 0). y-axis: percentage of total reads. (H) Percentages of U4atac snRNA molecules with extended tails ending with untemplated uridines. Total reads per sample > 75 000.