Wildtype and methyl-deficient Puf4 RIP-seq in Cryptococcus neoformans

Puf4 is a pumilio/fbf domain containing RNA binding protein. Our previous work revelated that Puf4 modulates ER stress and anrifungal resistance in C. neoformans. Our recent work showed that Puf4 is targeted by arginine methyltransferases. We investigated the RNA binding network of wildtype Puf4 as well as two mutants that have methyl-deficient (arginine to lysine) mutations. Cells were grown overnight to OD600 of 1.5-1.8 in 2 L YPD at 30˚C. Wildtype Puf4-FLAG and RtoK mutants where Arg462 or Arg783/Arg785 are mutated were analyzed. Puf4 was immunoprecipitated using FLAG beads and RNA was extracted using low ph phenol chloroform. Untagged cells were included as a negative control to assess non-specific binding.