Figure 6. Xrn2p degrades cas3Δi mRNA in a Pab2p-dependent fashion.

A. Simple depletion of XRN2 does not lead to cas3Δi mRNA stabilisation. Cells from different mutant strains grown overnight in galactose were washed and transferred to glucose for 10 h. RNA was extracted and 5 µg were loaded on a denaturing electrophoresis agarose gel, transferred on a nylon membrane and probed with CAS3 and XRN2 specific probes. B. Depletion of XRN2 leads to increased expression of RRP44 and vice versa. Cells from different mutant strains grown overnight in galactose were washed and transferred to glucose for 10 h. RNA was extracted and 5 µg were loaded on a denaturing electrophoresis agarose gel, transferred on a nylon membrane and probed with RRP44 and XRN2 specific probes. C. and D. The degradation of cas3Δi mRNA by Rrp44p is largely independent of Pab2p whereas that by Xrn2p rather depends on Pab2p. RNA was extracted from cells growing in YPG (5·10⁷ cells/mL). 5 µg were loaded on a denaturing electrophoresis agarose gel, transferred on a nylon membrane and probed with CAS3, RRP44 and XRN2 specific probes C. RNA was treated with Dnase I and 1 µg were used to synthesise cDNA. Each quantitative PCR run was assayed in triplicate. For qPCR analyses primers were chosen that amplify 131 bp of the 5′ part of the cas3Δi transcript ensuring the capture of all isoforms of cas3Δi mRNA stabilised upon PAB2 deletion. The reported values are the means ± SD of three independent experiments.