RNA regulatory networks diversified through curvature of the PUF protein scaffold.
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ABSTRACT:
SUBMITTER: Wilinski D
PROVIDER: S-EPMC4570272 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description: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.
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Project description:Protein-RNA networks are ubiquitous and central in biological control. We present an approach termed RNA Tagging that enables the user to identify protein-RNA interactions in vivo by analyzing purified cellular RNA, without protein purification or cross-linking. An RNA-binding protein of interest is fused to an enzyme that adds uridines to the end of RNA. RNA targets bound by the chimeric protein in vivo are covalently marked with uridines and subsequently identified from extracted RNA via high-throughput sequencing. We used this approach to identify hundreds of RNAs bound by a Saccharomyces cerevisiae PUF protein, Puf3p. The results showed that although RNA-binding proteins productively bind specific RNAs to control their function, they also 'sample' RNAs without exerting a regulatory effect. We used the method to uncover hundreds of new and likely regulated targets for a protein without canonical RNA-binding domains, Bfr1p. RNA Tagging is well suited to detect and analyze protein-RNA networks in vivo.
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Project description:Protein-protein interaction (PPI) networks, providing a comprehensive landscape of protein interaction patterns, enable us to explore biological processes and cellular components at multiple resolutions. For a biological process, a number of proteins need to work together to perform a job. Proteins densely interact with each other, forming large molecular machines or cellular building blocks. Identification of such densely interconnected clusters or protein complexes from PPI networks enables us to obtain a better understanding of the hierarchy and organization of biological processes and cellular components. However, most existing graph clustering algorithms on PPI networks often cannot effectively detect densely connected subgraphs and overlapped subgraphs. In this article, we formulate the problem of complex detection as diversified dense subgraph mining and introduce a novel approximation algorithm to efficiently enumerate putative protein complexes from biological networks. The key insight of our algorithm is that instead of enumerating all dense subgraphs, we only need to find a small diverse subset of subgraphs that cover as many proteins as possible. The problem is modeled as finding a diverse set of maximal dense subgraphs where we develop highly effective pruning techniques to guarantee efficiency. To scale up to large networks, we devise a divide-and-conquer approach to speed up the algorithm in a distributed manner. By comparing with existing clustering and dense subgraph-based algorithms on several yeast and human PPI networks, we demonstrate that our method can detect more putative protein complexes and achieves better prediction accuracy.
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Project description:PUF proteins are a conserved group of sequence specific RNA-binding proteins that bind to RNA in a modular fashion. The RNA-binding domain of PUF proteins typically consists of eight clustered Puf repeats. Plant genomes code for large families of PUF proteins that show significant variability in their predicted Puf repeat number, organization, and amino acid sequence. Here we sought to determine whether the observed variability in the RNA-binding domains of four plant PUFs results in a preference for nonclassical PUF RNA target sequences. We report the identification of a novel RNA binding sequence for a nucleolar Arabidopsis PUF protein that contains an atypical RNA-binding domain. The Arabidopsis PUM23 (APUM23) binding sequence was 10 nucleotides in length, contained a centrally located UUGA core element, and had a preferred cytosine at nucleotide position 8. These RNA sequence characteristics differ from those of other PUF proteins, because all natural PUFs studied to date bind to RNAs that contain a conserved UGU sequence at their 5' end and lack specificity for cytosine. Gel mobility shift assays validated the identity of the APUM23 binding sequence and supported the location of 3 of the 10 predicted Puf repeats in APUM23, including the cytosine-binding repeat. The preferred 10-nucleotide sequence bound by APUM23 is present within the 18S rRNA sequence, supporting the known role of APUM23 in 18S rRNA maturation. This work also reveals that APUM23, an ortholog of yeast Nop9, could provide an advanced structural backbone for Puf repeat engineering and target-specific regulation of cellular RNAs.
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Project description:mRNA control networks depend on recognition of specific RNA sequences. Pumilio-fem-3 mRNA binding factor (PUF) RNA-binding proteins achieve that specificity through variations on a conserved scaffold. Saccharomyces cerevisiae Puf3p achieves specificity through an additional binding pocket for a cytosine base upstream of the core RNA recognition site. Here we demonstrate that this chemically simple adaptation is prevalent and contributes to the diversity of RNA specificities among PUF proteins. Bioinformatics analysis shows that mRNAs associated with Caenorhabditis elegans fem-3 mRNA binding factor (FBF)-2 in vivo contain an upstream cytosine required for biological regulation. Crystal structures of FBF-2 and C. elegans PUF-6 reveal binding pockets structurally similar to that of Puf3p, whereas sequence alignments predict a pocket in PUF-11. For Puf3p, FBF-2, PUF-6, and PUF-11, the upstream pockets and a cytosine are required for maximal binding to RNA, but the quantitative impact on binding affinity varies. Furthermore, the position of the upstream cytosine relative to the core PUF recognition site can differ, which in the case of FBF-2 originally masked the identification of this consensus sequence feature. Importantly, other PUF proteins lack the pocket and so do not discriminate upstream bases. A structure-based alignment reveals that these proteins lack key residues that would contact the cytosine, and in some instances, they also present amino acid side chains that interfere with binding. Loss of the pocket requires only substitution of one serine, as appears to have occurred during the evolution of certain fungal species.
| S-EPMC3307254 | biostudies-literature
Project description:PUF proteins specifically bind mRNAs to regulate their stability and translation. Here we focus on the RNA-binding specificity of a C. elegans PUF protein, PUF-11. Our findings reveal that PUF-11 binds RNA in multiple modes, in which the protein can accommodate variable spacings between two distinct recognition elements. We propose a structural model in which flexibility in the central region of the protein enables the protein to adopt at least two distinct structures, one of which results in base flipping.
| S-EPMC2685523 | biostudies-literature
Project description:Differences in biomolecular sequence and function underlie dramatic ranges of appearance and behavior among species. We studied the basic region-leucine zipper (bZIP) transcription factors and quantified bZIP dimerization networks for five metazoan and two single-cell species, measuring interactions in vitro for 2891 protein pairs. Metazoans have a higher proportion of heteromeric bZIP interactions and more network complexity than the single-cell species. The metazoan bZIP interactomes have broadly similar structures, but there has been extensive rewiring of connections compared to the last common ancestor, and each species network is highly distinct. Many metazoan bZIP orthologs and paralogs have strikingly different interaction specificities, and some differences arise from minor sequence changes. Our data show that a shifting landscape of biochemical functions related to signaling and gene expression contributes to species diversity.
| S-EPMC4115154 | biostudies-literature
Project description:PUF proteins, named for Drosophila Pumilio (PUM) and Caenorhabditis elegans fem-3-binding factor (FBF), recognize specific sequences in the mRNAs they bind and control. RNA binding by classical PUF proteins is mediated by a characteristic PUM homology domain (PUM-HD). The Puf1 and Puf2 proteins possess a distinct architecture and comprise a highly conserved subfamily among fungal species. Puf1/Puf2 proteins contain two types of RNA-binding domain: a divergent PUM-HD and an RNA recognition motif (RRM). They recognize RNAs containing UAAU motifs, often in clusters. Here, we report a crystal structure of the PUM-HD of a fungal Puf1 in complex with a dual UAAU motif RNA. Each of the two UAAU tetranucleotides are bound by a Puf1 PUM-HD forming a 2:1 protein-to-RNA complex. We also determined crystal structures of the Puf1 RRM domain that identified a dimerization interface. The PUM-HD and RRM domains act in concert to determine RNA-binding specificity: the PUM-HD dictates binding to UAAU, and dimerization of the RRM domain favors binding to dual UAAU motifs rather than a single UAAU. Cooperative action of the RRM and PUM-HD identifies a new mechanism by which multiple RNA-binding modules in a single protein collaborate to create a unique RNA-binding specificity.
| S-EPMC7145691 | biostudies-literature
Project description: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 3' UTR (3'-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.
| S-EPMC3062182 | biostudies-literature
Project description:Caenorhabditis elegans fem-3 binding factor (FBF) is a founding member of the PUMILIO/FBF (PUF) family of mRNA regulatory proteins. It regulates multiple mRNAs critical for stem cell maintenance and germline development. Here, we report crystal structures of FBF in complex with 6 different 9-nt RNA sequences, including elements from 4 natural mRNAs. These structures reveal that FBF binds to conserved bases at positions 1-3 and 7-8. The key specificity determinant of FBF vs. other PUF proteins lies in positions 4-6. In FBF/RNA complexes, these bases stack directly with one another and turn away from the RNA-binding surface. A short region of FBF is sufficient to impart its unique specificity and lies directly opposite the flipped bases. We suggest that this region imposes a flattened curvature on the protein; hence, the requirement for the additional nucleotide. The principles of FBF/RNA recognition suggest a general mechanism by which PUF proteins recognize distinct families of RNAs yet exploit very nearly identical atomic contacts in doing so.
| S-EPMC2787170 | biostudies-literature