Figure 4.

Inefficient splicing of SPIN pre-U6 RNA in vivo. Primer extension analysis of total cellular RNA from strains containing SNR6–SPIN+B alleles on low-copy-number or high-copy-number plasmids. Primer extension was done using ³²P-labeled oligonucleotide 6D (complementary to the 3′ end of U6 RNA) and the resulting cDNA products were run on a denaturing 6% polyacrylamide gel. The positions of pre-U6, U6 and Ψ-WT U6 cDNAs are indicated on the right. Lane 1, RNA from a strain containing only wild-type (WT) SNR6 on a centromere plasmid. Lane 2, RNA from a strain containing only pseudo-wild-type (Ψ-WT) SNR6 on a centromere plasmid. Lanes 3–6, RNA from strains containing Ψ-WT SNR6 on a centromere plasmid and SNR6–SPIN+B (lanes 3 and 5) or SNR6–SPIN+B+9 (lanes 4 and 6) on a TRP1-marked centromere (lanes 3 and 4) or 2 μm (lanes 5 and 6) plasmid.