RESUMO
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.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Polinucleotídeo Adenililtransferase/metabolismo , Biossíntese de Proteínas , Proteínas de Ligação a RNA/metabolismo , Animais , Western Blotting , Proteínas de Caenorhabditis elegans/genética , Feminino , Expressão Gênica , Humanos , Luciferases/genética , Luciferases/metabolismo , Mutação , Oócitos/metabolismo , Poli A/genética , Poli A/metabolismo , Polinucleotídeo Adenililtransferase/genética , Ligação Proteica , RNA/genética , RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Xenopus laevisRESUMO
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.
Assuntos
Proteínas de Caenorhabditis elegans/química , Caenorhabditis elegans/metabolismo , Proteínas de Ligação a RNA/química , RNA/química , Animais , Sequência de Bases , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Cristalografia por Raios X , Conformação de Ácido Nucleico , Estrutura Secundária de Proteína , Proteínas de Ligação a RNA/genéticaRESUMO
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.
Assuntos
Proteínas de Caenorhabditis elegans/química , Caenorhabditis elegans/metabolismo , Modelos Moleculares , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/química , Animais , Proteínas de Caenorhabditis elegans/metabolismo , Cristalografia , Ensaio de Desvio de Mobilidade Eletroforética , Proteínas de Ligação a RNA/metabolismo , Técnicas do Sistema de Duplo-HíbridoRESUMO
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.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Proteínas de Caenorhabditis elegans/genética , Modelos Biológicos , Modelos Moleculares , Conformação Proteica , Proteínas de Ligação a RNA/genéticaRESUMO
RNA-protein interactions play an essential role in the maturation and regulation of RNAs within eukaryotic organisms. The three-hybrid system provides a simple, yet powerful means to study RNA-protein interactions within the eukaryote Saccharomyces cerevisiae. This chapter describes the basis of the system and applications in both examining specific RNA-protein interactions and screening libraries for novel interactions. We provide a detailed discussion on affinity versus reporter output, variations on library screening (e.g., randomization studies), some adaptations of the system, and updated reagents and protocols.
Assuntos
Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Ligação Proteica/genética , RNA/genética , Proteínas de Ligação a RNA/genéticaRESUMO
Regulation of messenger RNA is crucial in many contexts, including development, memory and cell growth. The 3' untranslated region is a rich repository of regulatory elements that bind proteins and microRNAs. Here we focus on PUF proteins, an important family of mRNA regulatory proteins crucial in stem-cell proliferation, pattern formation and synaptic plasticity. We show that two Caenorhabditis elegans PUF proteins, FBF and PUF-8, differ in RNA-binding specificity. FBF requires the presence of a single 'extra' nucleotide in the middle of an eight-nucleotide site, whereas PUF-8 requires its absence. A discrete protein segment is responsible for the difference. We propose that a structural distortion in the central region of FBF imposes the requirement for the additional nucleotide and that this mode of PUF specificity may be common. We suggest that new specificities can be designed and selected using the PUF scaffold.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Regulação da Expressão Gênica/genética , Modelos Moleculares , Nucleotídeos/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Sequência de Bases , Sítios de Ligação/genética , Proteínas de Caenorhabditis elegans/genética , Ensaio de Desvio de Mobilidade Eletroforética , Dados de Sequência Molecular , Nucleotídeos/química , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Relação Estrutura-Atividade , Especificidade por Substrato , Técnicas do Sistema de Duplo-Híbrido , LevedurasRESUMO
Sequence-specific RNA-protein interactions underlie regulation of many mRNAs. Here we analyze the RNA sequence specificity of Caenorhabditis elegans FBF-1, a founding member of the PUF protein family. Like other PUF proteins, FBF-1 binds to the 3' UTR of target mRNAs and decreases expression of those target genes. Here, we show that FBF-1 and its close relative, FBF-2, bind with similar affinity to multiple RNA sites. We use mutagenesis and in vivo selection experiments to identify nucleotides that are essential for FBF-1 binding. The binding elements comprise a "core" central region and flanking sequences. The core region is similar but distinct from the binding sites of other PUF proteins. We combine the identification of binding elements with informatics to predict new FBF-1 binding sites in a C. elegans 3' UTR database. These data identify a set of new candidate mRNA targets of FBF-1 and FBF-2.