Stacking interactions in PUF–RNA complexes

The half-a-tetratricopeptide repeat (HAT) motif is a helical repeat motif found in proteins that influence various aspects of RNA metabolism, including rRNA biogenesis, RNA splicing, and polyadenylation. This functional association with RNA suggested that HAT repeat tracts might bind RNA. However, RNA binding activity has not been reported for any HAT repeat tract, and recent literature has emphasized a protein binding role. In this study, we show that a chloroplast-localized HAT protein, HCF107, is a sequence-specific RNA binding protein. HCF107 consists of 11 tandem HAT repeats and short flanking regions that are also predicted to form helical hairpins. The minimal HCF107 binding site spans ∼11 nt, consistent with the possibility that HAT repeats bind RNA through a modular one repeat-1 nt mechanism. Binding of HCF107 to its native RNA ligand in the psbH 5′ UTR remodels local RNA structure and protects the adjacent RNA from exonucleases in vitro. These activities can account for the RNA stabilizing, RNA processing, and translational activation functions attributed to HCF107 based on genetic data. We suggest that analogous activities contribute to the functions of HAT domains found in ribonucleoprotein complexes in the nuclear-cytosolic compartment.helical repeat protein | plastid | pentatricopeptide repeat T he half-a-tetratricopeptide (HAT) motif is a helical repeat motif that has been functionally linked to RNA because of its presence exclusively in complexes that influence RNA metabolism (1). Examples of HAT domain proteins include Utp6, which is involved in nuclear pre-rRNA processing, Prp6, which is involved in nuclear pre-mRNA splicing, and CstF-77, which is involved in pre-mRNA cleavage and polyadenylation. The HAT motif is related to the tetratricopeptide repeat (TPR), a degenerate 34-aa motif that forms a pair of antiparallel α-helices (reviewed in ref.2). TPR motifs are typically found in tandem arrays, which stack to form a broad surface that binds protein ligands. Crystal structures of CstF-77 confirmed that HAT repeat tracts adopt a TPR-like structure (3, 4). The possibility that HAT repeat tracts might bind RNA has been suggested (1, 5-7), but the notion that HAT repeat tracts serve as a scaffold for binding other proteins dominates recent literature (3,(8)(9)(10)). This view is reflected by the annotation of the HAT motif at InterPro, which states only that "the repeats may be involved in protein-protein interactions" (http://www.ebi. ac.uk/interpro/IEntry?ac=IPR003107).Although TPR, HEAT, and several other helical repeat motifs form protein binding surfaces, similar structures have been shown to bind nucleic acids. Puf and pentatricopeptide repeat (PPR) motifs have been shown to bind RNA (reviewed in refs. 11 and 12), and mTERF, ALK, and TAL repeat motifs have been shown to bind DNA (reviewed in refs. 13 and 14). Thus, it seemed quite plausible that the presence of HAT domains exclusively in ribonucleoprotein complexes reflects the fact that HAT repeat tracts interact directly with RNA. In this ...

Section: Discussionmentioning

confidence: 99%

RNA binding and RNA remodeling activities of the half-a-tetratricopeptide (HAT) protein HCF107 underlie its effects on gene expression

Hammani

Cook

Barkan

2012

“…pACT2 FBF-2 was as described (47). Puf4p (amino acids 536-888), Puf3p (amino acids 511-879), and Puf5p (amino acids 28-860) were cloned into pGADT7.…”

Section: Methodsmentioning

confidence: 99%

“…WT and mutant FBF-2 purifications, EMSA, and filter binding experiments were performed essentially as described (47,49). Minor changes are described in SI Materials and Methods.…”

Section: Methodsmentioning

confidence: 99%

Patterns and plasticity in RNA-protein interactions enable recruitment of multiple proteins through a single site

Valley¹,

Porter

Qiu

et al. 2012

Self Cite

mRNA control hinges on the specificity and affinity of proteins for their RNA binding sites. Regulatory proteins must bind their own sites and reject even closely related noncognate sites. In the PUF [Pumilio and fem-3 binding factor (FBF)] family of RNA binding proteins, individual proteins discriminate differences in the length and sequence of binding sites, allowing each PUF to bind a distinct battery of mRNAs. Here, we show that despite these differences, the pattern of RNA interactions is conserved among PUF proteins: the two ends of the PUF protein make critical contacts with the two ends of the RNA sites. Despite this conserved "two-handed" pattern of recognition, the RNA sequence is flexible. Among the binding sites of yeast Puf4p, RNA sequence dictates the pattern in which RNA bases are flipped away from the binding surface of the protein. Small differences in RNA sequence allow new modes of control, recruiting Puf5p in addition to Puf4p to a single site. This embedded information adds a new layer of biological meaning to the connections between RNA targets and PUF proteins.mRNA turnover | RNA regulation | translation | 3′UTR elements

“…Studies of multiple natural and mutant PUF proteins have led to a biochemical and structural understanding of how RNA selectivity can be achieved (12,16,18,21,22). Furthermore, introducing rationally designed or selected mutations can alter specificities (12,16,18,21,23). In some instances, mutations shift the balance in affinities for two RNAs, rather than cause a complete switch in specificity.…”

Section: A Hs Pum1mentioning

confidence: 99%

“…Mutations can be rationally designed to alter RNA specificity, based on the known structures of natural PUF-RNA complexes (12,16,18,(21)(22)(23)(24). New specificities can be obtained by transferring a segment from one protein to another, by targeted mutagenesis, or by genetic selections from protein libraries (18,23).…”

mentioning

confidence: 99%

Targeted translational regulation using the PUF protein family scaffold

Cooke¹,

Prigge

Opperman³

et al. 2011

Self Cite

Regulatory complexes formed on mRNAs control translation, stability, and localization. These complexes possess two activities: one that binds RNA and another-the effector-that elicits a biological function. The Pumilio and FBF (PUF) protein family of RNA binding proteins provides a versatile scaffold to design and select proteins with new specificities. Here, the PUF scaffold is used to target translational activation and repression of specific mRNAs, and to induce specific poly(A) addition and removal. To do so, we linked PUF scaffold proteins to a translational activator, GLD2, or a translational repressor, CAF1. The chimeric proteins activate or repress the targeted mRNAs in Xenopus oocytes, and elicit poly(A) addition or removal. The magnitude of translational control relates directly to the affinity of the RNA-protein complex over a 100-fold range of K d . The chimeric proteins act on both reporter and endogenous mRNAs: an mRNA that normally is deadenylated during oocyte maturation instead receives poly(A) in the presence of an appropriate chimera. The PUF-effector strategy enables the design of proteins that affect translation and stability of specific mRNAs in vivo.CAF1/RNA stability | GLD2/polyadenylation