Gene expression can be controlled at the levels of transcription and translation. Translational regulation offers a more rapid response to environmental conditions than regulation at the transcriptional level1
. PUF family proteins, named for founding members Drosophila melanogaster
Pumilio and Caenorhabditis elegans fem-3
mRNA binding factor (FBF), are translational regulators that are important for germline stem cell maintenance and embryonic development and have been identified in yeast, plants and animals (reviewed in refs. 2,3
). PUF proteins are characterized by an RNA binding domain, called a Pumilio homology domain, typically formed from eight α-helical repeats4–8
The crystal structures of the RNA binding domains of the D. melanogaster
and human Pumilio proteins revealed that the eight repeats, each with three α-helices, are nestled together to form a crescent shape9–11
. The structures of human Pumilio homolog 1 protein (PUM1) in complex with RNA ligand showed a simple pattern of sequence-specific RNA recognition that could be used to design RNA sequence specificity10,12–14
. Each repeat of PUM1 recognizes a single RNA base using three side chains from conserved positions. Thus, the eight repeats of PUM1 specifically recognize an 8-nt sequence, UGUA-U or C-AUA.
PUF proteins share the recognition sequence of UGUR (where R represents a purine) at the 5′ end, which is well conserved, followed by 3′ sequences that are specific to each PUF protein2
. Saccharomyces cerevisiae
encode six PUF proteins, Jsn1p (also known as Puf1p), Puf2p, Puf3p, Puf4p, Mpt5p (also known as Puf5p) and Puf6p. Previous work identified the mRNAs associated with TAP-tagged PUF proteins Puf1p to Puf5p (Mpt5p) and from these data consensus sequences for Puf3p, Puf4p and Mpt5p were derived15
. The consensus sequences suggest that each of these proteins binds to RNA target sequences beginning with a UGUA sequence at the 5′ end and a UA sequence at the 3′ end. However, the Puf3p, Puf4p and Mpt5p consensus sequences have two, three or four bases between the conserved 5′ and 3′ motifs, respectively. Nevertheless, each of the proteins has eight repeats, but they recognize 8-nt (Puf3p), 9-nt (Puf4p) or 10-nt (Mpt5p) sequences. This suggests that the RNA binding domains of Puf4p and Mpt5p must accommodate binding of one or two additional nucleotides, respectively.
A similar situation exists in C. elegans
, where Puf-8 and FBF proteins have differing specificities. Opperman, et al
showed that Puf-8 recognizes 8-nt RNA sequences whereas FBF recognizes 9-nt RNA sequences, although both proteins have eight repeats2
. To determine whether the extra base in the FBF recognition sequence was accommodated within the RNA binding surface or at the 3′ end, they engineered mutant FBF proteins that would lead to predictable changes in RNA binding specificity depending on where the extra RNA base was accommodated. These results allowed them to conclude that the additional base was accommodated within the RNA binding surface between the fourth and fifth repeats. A chimeric protein of Puf-8 in which repeat 5 and its flanking sequences were replaced by those from FBF bound preferentially to FBF's 9-nt recognition sequence, suggesting that modifications allowing binding of a longer recognition sequence can be contained in a relatively short stretch of the protein sequence.
Puf4p seems to bind to many mRNAs encoding nuclear or nucleolar proteins15
. It was recently reported that Puf4p is important for post-transcriptional regulation of HO endonuclease expression16
. HO endonuclease controls mating-type switching in yeast by introducing double-stranded DNA breaks that initiate recombination17
. Both Mpt5p and Puf4p bind to sequences in the 3′ untranslated region (3′ UTR) of HO
mRNA, collaborating to repress its expression16,18
To determine how Puf4p accommodates an additional nucleotide in its binding surface, we have solved the crystal structure of Puf4p in complex with a target RNA sequence from HO mRNA. This structure reveals subtle changes in the architecture of the PUF RNA binding domain that allow binding to a 9-nt RNA sequence. Furthermore, the structure led us to identify mutations that changed the preference of Puf4p from a 9-nt target to an 8-nt target. The structure of the mutant shows that the protein binds to the 8-nt target RNA, as was seen in the PUM1 structures, with the same regular one-repeat–one RNA base pattern.