A 5' cytosine binding pocket in Puf3p specifies regulation of mitochondrial mRNAs

Abstract

A single regulatory protein can control the fate of many mRNAs with related functions. The Puf3 protein of Saccharomyces cerevisiae is exemplary, as it binds and regulates more than 100 mRNAs that encode proteins with mitochondrial function. Here we elucidate the structural basis of that specificity. To do so, we explore the crystal structures of Puf3p complexes with 2 cognate RNAs. The key determinant of Puf3p specificity is an unusual interaction between a distinctive pocket of the protein with an RNA base outside the "core" PUF-binding site. That interaction dramatically affects binding affinity in vitro and is required for regulation in vivo. The Puf3p structures, combined with those of Puf4p in the same organism, illuminate the structural basis of natural PUF-RNA networks. Yeast Puf3p binds its own RNAs because they possess a -2C and is excluded from those of Puf4p which contain an additional nucleotide in the core-binding site.

Fig. 1.

Crystal structures of the Puf3p RNA-binding domain in complex with COX17 mRNA sequences. (A) Ribbon diagram of the RNA-binding domain of S. cerevisiae Puf3p in complex with a COX17 site B RNA fragment (CCUGUAAAUA). Protein repeats are colored alternately blue and gold. The RNA is colored according to atom type (carbon, white; nitrogen, blue; oxygen, red; sulfur, yellow; phosphorus, orange). Protein side chains that contact the RNA are shown. Two loop regions that were disordered in the crystal structures are indicated by dotted lines. This figure and Fig. 2 were prepared with PyMol (31). (B) Superposition of CA traces of the Puf3p:COX17 site B RNA complex (blue) and PUM1:RNA complex (gray; PDB ID: 1M8Y) aligned over the entire protein structures. The COX17 site A RNA from the structure of a complex with Puf3p is shown in gold.

Fig. 2.

Recognition of RNA by the Puf3p RNA-binding domain. The protein and RNA are colored as in Fig. 1A. (A) Puf3p recognition of bases A4–A6 in COX17 site B RNA. (B) Recognition of a cytosine 2 positions 5′ of the conserved UGUA sequence.

Fig. 3.

A cytosine at the −2 position is critical for Puf3p-mediated mRNA decay in vivo. Representative Northern blots illustrating the decay from the steady state of COX17 mRNAs bearing either the wild-type or mutant 3′-UTR in wild-type (A) and Δpuf3 (B) strains. The mutant 3′-UTR carries C(−2)A mutations in both of its Puf3p-binding sites. The control RNA is ScR1.

Fig. 4.

Key determinants of Puf3p and Puf4p binding and exclusion. Bar graph illustrating relative binding of Puf3p and Puf4p to RNAs with either C or U at position −2 and with or without the +6′ nucleotide. RNA sequences are given in Table S1.

Fig. 5.

Structural basis of the RNA selectivity of Puf3p and Puf4p. A core PUF-RNA complex, with 8 helices (tan circles, R1–R8) aligned with 8 bases (purple hexagons, +1 to + 8), depicted in the center, represents the “base” PUF-RNA structure, as seen in human PUM1 (21). The 2 key determinants for Puf3p and Puf4p are expanded above and to the right, respectively. See the text for details.