Two Yeast PUF Proteins Negatively Regulate a Single mRNA

Brad A Hook,Daniel J Seay,Marvin Wickens,Aaron C Goldstrohm

doi:10.1074/jbc.m611253200

Brad A Hook, Daniel J Seay + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m611253200

Copy DOI

Abstract

mRNA stability and translation are regulated by protein repressors that bind 3'-untranslated regions. PUF proteins provide a paradigm for these regulatory molecules: like other repressors, they inhibit translation, enhance mRNA decay, and promote poly(A) removal. Here we show that a single mRNA in Saccharomyces cerevisiae, encoding the HO endonuclease, is regulated by two distinct PUF proteins, Puf4p and Mpt5p. These proteins bind to adjacent sites and can co-occupy the mRNA. Both proteins are required for full repression and deadenylation in vivo; their removal dramatically stabilizes the mRNA. The two proteins act through overlapping but non-identical mechanisms: repression by Puf4p is dependent on deadenylation, whereas repression by Mpt5p can occur through additional mechanisms. Combinatorial action of the two regulatory proteins may allow responses to specific environmental cues and be common in 3'-untranslated region-mediated control.

Highlights

Regulation of mRNA translation and stability commonly is controlled by elements in the 3Ј-untranslated region (UTR)2 of the mRNA, to which specific regulatory proteins bind [1,2,3,4]
All PUF proteins examined to date either repress translation, induce decay, or both; their activity is correlated with poly(A) shortening [13]
The “HO mt” reporter (Fig. 4) has the UGU ried out with Pop2p-TAP complexes purified from yeast and trinucleotides in the Puf4 and Mpt5 binding sites changed to recombinant PUF protein purified from E. coli as previously

Summary

EXPERIMENTAL PROCEDURES

Modest difference in Mpt5p binding affinities between Fig. 1C and that reported in Fig. 2A are likely due to different. The wild-type BY4742 yeast strain and isogenic constants reported in Fig. 2A are the statistically determined strains with gene-specific deletions of CCR4 and POP2 were results of three independent binding experiments. These deletion strains were The following synthetic RNAs were used in the assays (ones created by PCR-mediated gene modification using the kanamy- not given in figures): Mpt5p BS, Ј-AGUUUAAAAAGUUGUA-. The “HO mt” reporter (Fig. 4) has the UGU ried out with Pop2p-TAP complexes purified from yeast and trinucleotides in the Puf and Mpt binding sites changed to recombinant PUF protein purified from E. coli as previously. The reactions contained 10 ng of Pop2p-TAP nM, consistent with our previous findings [7], whereas Puf4p bound the Puf BS with an apparent Kd of 10 nM. We conclude that Puf4p binds the newly defined Puf BS in the HO 3Ј-UTR

Both PUF Proteins Can Bind

RESULTS

Findings

DISCUSSION