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

Zhu, Deyu; Stumpf, Craig R.; Krahn, J.M.; Wickens, Marvin; Hall, Traci M. Tanaka

doi:10.1073/pnas.0812079106

Cited by 81 publications

(125 citation statements)

References 41 publications

Supporting

Mentioning

119

Contrasting

Order By: Relevance

“…The identity of the base present in each mutant is indicated immediately below the bars; the identity in WT is indicated below that. Amino acid side chains and bases in the FBF-2/FBE (C), Puf4p/4BE (F), and Puf3p/3BE (I) complexes are derived from the crystal structures (29,30,35). Green amino acid chains allowed ≥10% β-gal when mutated to alanine, and red side chains allowed <10% when mutated to alanine.…”

Section: Systematic Mutagenesis Reveals a Common Pattern Of Recognitionmentioning

confidence: 99%

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

Valley¹,

Porter

Qiu

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard 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

show abstract

Section: Systematic Mutagenesis Reveals a Common Pattern Of Recognitionmentioning

confidence: 99%

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

Valley¹,

Porter

Qiu

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

show abstract

“…Different PUF proteins combine nearly identical sets of atomic contacts with structural distortions or additional protein-specific contacts to recognize distinct RNA targets (Wang et al 2002;Opperman et al 2005;Gupta et al 2008;Miller et al 2008;Wang et al 2009b;Zhu et al 2009). For example, the Caenorhabditis elegans PUF protein, FBF-2 ( fem-3 binding factor-2), binds to nine nucleotides rather than eight.…”

Section: Introductionmentioning

confidence: 99%

Stacking interactions in PUF–RNA complexes

Koh¹,

Wang²,

Qiu³

et al. 2011

RNA

Self Cite

View full text Add to dashboard Cite

Stacking interactions between amino acids and bases are common in RNA-protein interactions. Many proteins that regulate mRNAs interact with single-stranded RNA elements in the 39 UTR (39-untranslated region) of their targets. PUF proteins are exemplary. Here we focus on complexes formed between a Caenorhabditis elegans PUF protein, FBF, and its cognate RNAs. Stacking interactions are particularly prominent and involve every RNA base in the recognition element. To assess the contribution of stacking interactions to formation of the RNA-protein complex, we combine in vivo selection experiments with site-directed mutagenesis, biochemistry, and structural analysis. Our results reveal that the identities of stacking amino acids in FBF affect both the affinity and specificity of the RNA-protein interaction. Substitutions in amino acid side chains can restrict or broaden RNA specificity. We conclude that the identities of stacking residues are important in achieving the natural specificities of PUF proteins. Similarly, in PUF proteins engineered to bind new RNA sequences, the identity of stacking residues may contribute to ''target'' versus ''off-target'' interactions, and thus be an important consideration in the design of proteins with new specificities.

show abstract

“…Thus, recognition of a cytosine cannot be engineered in a repeat, although Pumilio 1 can accept any base including cytosine at the fifth position of the target sequence, and yeast Puf3p specifically recognizes a cytosine two bases upstream of the core PUF recognition sequence (17). Naturally occurring PUM repeats that specifically recognize a cytosine have not been identified, providing no clues to a cytosinerecognition code and uncertainty about whether such specific recognition exists or is possible.…”

mentioning

confidence: 99%

Specific and Modular Binding Code for Cytosine Recognition in Pumilio/FBF (PUF) RNA-binding Domains

Dong

Wang

Cassidy-Amstutz

et al. 2011

Journal of Biological Chemistry

Self Cite

140

View full text Add to dashboard Cite

Pumilio/fem-3 mRNA-binding factor (PUF) proteins possess a recognition code for bases A, U, and G, allowing designed RNA sequence specificity of their modular Pumilio (PUM) repeats. However, recognition side chains in a PUM repeat for cytosine are unknown. Here we report identification of a cytosine-recognition code by screening random amino acid combinations at conserved RNA recognition positions using a yeast three-hybrid system. This C-recognition code is specific and modular as specificity can be transferred to different positions in the RNA recognition sequence. A crystal structure of a modified PUF domain reveals specific contacts between an arginine side chain and the cytosine base. We applied the C-recognition code to design PUF domains that recognize targets with multiple cytosines and to generate engineered splicing factors that modulate alternative splicing. Finally, we identified a divergent yeast PUF protein, Nop9p, that may recognize natural target RNAs with cytosine. This work deepens our understanding of natural PUF protein target recognition and expands the ability to engineer PUF domains to recognize any RNA sequence.The specific interaction of RNA and protein plays vital roles in RNA regulation including splicing, localization, translation, and degradation. Such recognition may be directed toward unstructured RNA requiring discrimination of RNA sequences, folded RNA motifs, or some combination of sequence and structural specificity (1). Members of the PUF 2 protein family (named after Drosophila Pumilio and Caenorhabditis elegans fem-3 mRNA-binding factor (FBF)) are sequence-specific RNA-binding proteins that regulate networks of mRNAs encoding proteins of related function (2-7). PUF proteins generally recognize the 3Ј-UTR of their target mRNAs to control the mRNA stability and translation (2-7).The RNA-binding domain of PUF proteins, known as the Pumilio homology domain (PUM-HD) or PUF domain, can bind to unstructured RNA sequences in a distinct fashion. The PUF domain of human Pumilio 1 contains eight PUM repeats, each containing three ␣-helices packed together in a curved structure (8 -10). RNA is bound as an extended strand to the concave surface of the PUF domain with the bases contacted by protein side chains. In general, each PUM repeat recognizes a single RNA base through the second helix (␣2) in an antiparallel arrangement, i.e. nucleotides 1-8 are recognized by PUF repeats 8 -1, respectively. The ␣2 helices of PUM repeats contain a 5-residue sequence, designated here as 12XX5, where the side chain at position 2 stacks with the recognized base and the side chains at positions 1 and 5 recognize the edge of the base (8, 11) (see Fig. 1A). Specific residues at these positions direct the base recognition properties of the repeat. This PUF-RNA recognition code makes it possible to modify a PUM repeat to bind a particular RNA base, producing a designed PUF domain that specifically recognizes a given 8-nucleotide RNA target. Such de novo designed RNA binders have been used to track RNA local...

show abstract

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

Cited by 81 publications

References 41 publications

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

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

Stacking interactions in PUF–RNA complexes

Specific and Modular Binding Code for Cytosine Recognition in Pumilio/FBF (PUF) RNA-binding Domains

Contact Info

Product

Resources

About