Fission yeast RNA-binding proteins Puf2 and Puf4 are involved in repression of ferrireductase Frp1 expression in response to iron

This study identifies a post-transcriptional mechanism of iron uptake regulation by Puf2 and Puf4 of the Pumilio and FBF (Puf) family of RNA-binding proteins in Schizosaccharomyces pombe. Cells expressing Puf2 and Puf4 stimulate decay of the frp1(+) mRNA encoding a key enzyme of the reductive iron uptake pathway. Results consistently showed that frp1(+) mRNA is stabilized in puf2 Delta puf4 Delta mutant cells under iron-replete conditions. As a result, puf2 Delta puf4 Delta cells exhibit an increased sensitivity to iron accompanied by enhanced ferrireductase activity. A pool of GFP-frp1(+) 3 ' UTR RNAs was generated using a reporter gene containing the 3 ' untranslated region (UTR) of frp1(+) that was under the control of a regulatable promoter. Results showed that Puf2 and Puf4 accelerate the destabilization of mRNAs containing the frp1(+) 3 ' UTR which harbors two Pumilio response elements (PREs). Binding studies revealed that the PUM-homology RNA-binding domain of Puf2 and Puf4 expressed in Escherichia coli specifically interacts with PREs in the frp1(+) 3 ' UTR. Using RNA immunoprecipitation in combination with reverse transcription qPCR assays, results showed that Puf2 and Puf4 interact preferentially with frp1(+) mRNA under basal and iron-replete conditions, thereby contributing to inhibit Frp1 production and protecting cells against toxic levels of iron.