Fig 4.

Ras overactivation indirectly inhibits Nrd1. (A) The bcy1Δ mutation increases read-through transcription in nrd1-102 cells. Single and double mutant stains were grown to the log phase at the permissive temperature of 30°C in YPD. Total RNA was analyzed by qRT-PCR using primer sequences downstream of SNR13. (B) Phosphorylation of Nrd1HA is not affected by the perturbation of the Ras pathway. Total protein extracts from ira1Δ and pde2Δ strains, a strain overexpressing PDE2 (2μm PDE2), and wild-type control strains were collected and analyzed by Western blotting using antibodies to Nrd1 and to Pgk1, which served as a loading control. For comparison, an aliquot of the NRD1HA extract was treated with phosphatase (PPase) and run in lane 6.