Figure 4.

Circular permutations in the ARC of Mini-T cause senescence and repositioning the Sm site is tolerated but causes RNA abundance and processing defects. (A) Haploid yeast strains harbouring the indicated telomerase RNA allele, expressed from a 2μ plasmid, were passaged on plates up to 275 generations. (B) Mini-T circular permutant RNAs are detectable by northern blotting when expressed from 2μ plasmids. Control CEN samples were included to compare overexpression. Quantitation of the amounts of total and full-length RNA (black triangle) for two independent biological replicates can be found in Supplementary Figure S4. (C) Telomeres in ARC mutant strains begin to shorten at early time points. Southern blot on XhoI-digested genomic DNA probed for telomeric sequence and an internal chromosome IV fragment. Samples are shown at 300 generations, except for underlined samples where cells senesced (note generation listed above for each). For comparison, samples from CEN plasmids were also included.