RESUMO
During meiosis, germ cell and stage-specific components impose additional layers of regulation on the core cell cycle machinery to set up an extended G2 period termed meiotic prophase. In Drosophila males, meiotic prophase lasts 3.5â days, during which spermatocytes upregulate over 1800 genes and grow 25-fold. Previous work has shown that the cell cycle regulator Cyclin B (CycB) is subject to translational repression in immature spermatocytes, mediated by the RNA-binding protein Rbp4 and its partner Fest. Here, we show that the spermatocyte-specific protein Lut is required for translational repression of cycB in an 8-h window just before spermatocytes are fully mature. In males mutant for rbp4 or lut, spermatocytes enter and exit meiotic division 6-8â h earlier than in wild type. In addition, spermatocyte-specific isoforms of Syncrip (Syp) are required for expression of CycB protein in mature spermatocytes and normal entry into the meiotic divisions. Lut and Syp interact with Fest independent of RNA. Thus, a set of spermatocyte-specific regulators choreograph the timing of expression of CycB protein during male meiotic prophase.
Assuntos
Proteínas de Drosophila , Meiose , Animais , Masculino , Meiose/genética , Espermatogênese/fisiologia , Prófase , Mitose , Espermatócitos/metabolismo , Drosophila/genética , Ciclina B/genética , Ciclina B/metabolismo , Proteínas de Drosophila/metabolismoRESUMO
UV cross-linking-based methods are the most common tool to explore in vivo RNA-protein interactions. UV cross-linking enables the freezing of direct interactions in the cell, which can then be mapped by high-throughput sequencing through a family of methods termed CLIP-seq. CLIP-seq measures the distribution of cross-link events by purifying a protein of interest and sequencing the covalently bound RNA fragments. However, there are disagreements and ambiguities as to which proteins are RNA-binding proteins and what interactions are significant as all proteins contact all RNAs at some frequency. Here we describe a protocol for both determining RNA-protein interactions through a combination of RNA library preparation and the measurement of absolute cross-link rates, which helps determine what proteins are RNA-binding proteins and what interactions are significant. This protocol, comprising an updated form of the easyCLIP protocol, describes guidelines for RNA library preparation, oligo and protein standard construction, and the measurement of cross-link rates. These methods are easily visualizable through their fluorescent labels and can be adapted to study RNA-binding properties of both functional, high affinity RNA-binding proteins, and the accidental RNA interactions of non-RNA-binding proteins. © 2023 Wiley Periodicals LLC. Basic Protocol 1: RNA library construction Basic Protocol 2: Determining UV cross-link rates Support Protocol 1: Cross-linking and lysing cells Support Protocol 2: Adapter preparation Support Protocol 3: Preparation of cross-linked RBP standard.
Assuntos
Sequenciamento de Cromatina por Imunoprecipitação , Proteínas de Ligação a RNA , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , RNA/genética , RNA/química , RNA/metabolismo , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodosRESUMO
Glucose is a universal bioenergy source; however, its role in controlling protein interactions is unappreciated, as are its actions during differentiation-associated intracellular glucose elevation. Azido-glucose click chemistry identified glucose binding to a variety of RNA binding proteins (RBPs), including the DDX21 RNA helicase, which was found to be essential for epidermal differentiation. Glucose bound the ATP-binding domain of DDX21, altering protein conformation, inhibiting helicase activity, and dissociating DDX21 dimers. Glucose elevation during differentiation was associated with DDX21 re-localization from the nucleolus to the nucleoplasm where DDX21 assembled into larger protein complexes containing RNA splicing factors. DDX21 localized to specific SCUGSDGC motif in mRNA introns in a glucose-dependent manner and promoted the splicing of key pro-differentiation genes, including GRHL3, KLF4, OVOL1, and RBPJ. These findings uncover a biochemical mechanism of action for glucose in modulating the dimerization and function of an RNA helicase essential for tissue differentiation.
Assuntos
RNA Helicases DEAD-box , Glucose , Queratinócitos , Nucléolo Celular/metabolismo , Núcleo Celular/metabolismo , RNA Helicases DEAD-box/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Glucose/metabolismo , Queratinócitos/citologia , Queratinócitos/metabolismo , HumanosRESUMO
DNA-protein interactions mediate physiologic gene regulation and may be altered by DNA variants linked to polygenic disease. To enhance the speed and signal-to-noise ratio (SNR) in the identification and quantification of proteins associated with specific DNA sequences in living cells, we developed proximal biotinylation by episomal recruitment (PROBER). PROBER uses high-copy episomes to amplify SNR, and proximity proteomics (BioID) to identify the transcription factors and additional gene regulators associated with short DNA sequences of interest. PROBER quantified both constitutive and inducible association of transcription factors and corresponding chromatin regulators to target DNA sequences and binding quantitative trait loci due to single-nucleotide variants. PROBER identified alterations in regulator associations due to cancer hotspot mutations in the hTERT promoter, indicating that these mutations increase promoter association with specific gene activators. PROBER provides an approach to rapidly identify proteins associated with specific DNA sequences and their variants in living cells.
Assuntos
Cromatina , DNA , Biotinilação , Cromatina/genética , DNA/genética , DNA/metabolismo , Plasmídeos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Kinases play important roles in cell signaling, and adenosine monophosphate (AMP) is known to modulate cellular energy homeostasis through AMP-activated protein kinase (AMPK). Here, we explored novel AMP-binding kinases by employing a desthiobiotin-conjugated AMP acyl-phosphate probe to enrich efficiently AMP-binding proteins. Together with a parallel-reaction monitoring-based targeted proteomic approach, we uncovered 195 candidate AMP-binding kinases. We also enriched desthiobiotin-labeled peptides from adenine nucleotide-binding sites of kinases and analyzed them using LC-MS/MS in the multiple-reaction monitoring mode, which resulted in the identification of 44 peptides derived from 43 kinases displaying comparable or better binding affinities toward AMP relative to adenosine triphosphate (ATP). Moreover, our proteomic data revealed a potential involvement of AMP in the MAPK pathway through binding directly to the relevant kinases, especially MEK2 and MEK3. Together, we revealed the AMP-binding capacities of a large number of kinases, and our work built a strong foundation for understanding how AMP functions as a second messenger to modulate cell signaling.
Assuntos
Proteoma , Proteômica , Proteínas Quinases Ativadas por AMP/metabolismo , Monofosfato de Adenosina , Trifosfato de Adenosina/metabolismo , Cromatografia Líquida , Peptídeos , Proteoma/genética , Espectrometria de Massas em TandemRESUMO
Quantitative criteria to identify proteins as RNA-binding proteins (RBPs) are presently lacking, as are criteria to define RBP target RNAs. Here, we develop an ultraviolet (UV) cross-linking immunoprecipitation (CLIP)-sequencing method, easyCLIP. easyCLIP provides absolute cross-link rates, as well as increased simplicity, efficiency, and capacity to visualize RNA libraries during sequencing library preparation. Measurement of >200 independent cross-link experiments across >35 proteins identifies an RNA cross-link rate threshold that distinguishes RBPs from non-RBPs and defines target RNAs as those with a complex frequency unlikely for a random protein. We apply easyCLIP to the 33 most recurrent cancer mutations across 28 RBPs, finding increased RNA binding per RBP molecule for KHDRBS2 R168C, A1CF E34K and PCBP1 L100P/Q cancer mutations. Quantitating RBP-RNA interactions can thus nominate proteins as RBPs and define the impact of specific disease-associated RBP mutations on RNA association.
Assuntos
Proteínas de Ligação a RNA/química , RNA/química , Animais , Sítios de Ligação , Humanos , Imunoprecipitação , RNA/metabolismo , RNA/efeitos da radiação , Proteínas de Ligação a RNA/metabolismo , Proteínas de Ligação a RNA/efeitos da radiação , Raios UltravioletaRESUMO
RNA movements and localization pervade biology, from embryonic development to disease. To identify RNAs at specific locations, we developed a strategy in which a uridine-adding enzyme is anchored to subcellular sites, where it directly marks RNAs with 3' terminal uridines. This localized RNA recording approach yields a record of RNA locations, and is validated through identification of RNAs localized selectively to the endoplasmic reticulum (ER) or mitochondria. We identify a broad dual localization pattern conserved from yeast to human cells, in which the same battery of mRNAs encounter both ER and mitochondria in both species, and include an mRNA encoding a key stress sensor. Subunits of many multiprotein complexes localize to both the ER and mitochondria, suggesting coordinated assembly. Noncoding RNAs in the course of RNA surveillance and processing encounter both organelles. By providing a record of RNA locations over time, the approach complements those that capture snapshots of instantaneous positions.
Assuntos
RNA Fúngico , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Retículo Endoplasmático/química , Retículo Endoplasmático/metabolismo , Humanos , Mitocôndrias/química , Mitocôndrias/metabolismo , RNA Fúngico/química , RNA Fúngico/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Ribossomos/química , Ribossomos/metabolismo , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , UridinaRESUMO
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.
Assuntos
RNA Mensageiro/química , Proteínas de Ligação a RNA/química , Proteínas de Schizosaccharomyces pombe/química , Schizosaccharomyces/genética , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Biblioteca Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Modelos Moleculares , Motivos de Nucleotídeos , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Estrutura Terciária de Proteína , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Ribonucleotidyl transferases (rNTases) add untemplated ribonucleotides to diverse RNAs. We have developed TRAID-seq, a screening strategy in Saccharomyces cerevisiae to identify sequences added to a reporter RNA at single-nucleotide resolution by overexpressed candidate enzymes from different organisms. The rNTase activities of 22 previously unexplored enzymes were determined. In addition to poly(A)- and poly(U)-adding enzymes, we identified a cytidine-adding enzyme that is likely to be part of a two-enzyme system that adds CCA to tRNAs in a eukaryote; a nucleotidyl transferase that adds nucleotides to RNA without apparent nucleotide preference; and a poly(UG) polymerase, Caenorhabditis elegans MUT-2, that adds alternating uridine and guanosine nucleotides to form poly(UG) tails. MUT-2 is known to be required for certain forms of RNA silencing, and mutants of the enzyme that result in defective silencing did not add poly(UG) tails in our assay. We propose that MUT-2 poly(UG) polymerase activity is required to promote genome integrity and RNA silencing.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Nucleotidiltransferases/genética , Interferência de RNA , RNA Nucleotidiltransferases/genética , Saccharomyces cerevisiae/genética , Animais , Caenorhabditis elegans/enzimologia , Mutação , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
In the version of this paper originally published, three references were accidentally omitted: Schwartz, J. C. et al. Cell Rep. 5, 918-925 (2013); Tundup, S. et al. FEBS Lett. 580, 1285-1293 (2006); and Itri, F. et al. Biochem. Biophys. Res. Commun. 492, 67-73 (2017). The PDF and HTML versions of the paper now include these as references 58, 59, and 60, respectively, and subsequent references have been renumbered accordingly.
RESUMO
Noncoding RNA sequences, including long noncoding RNAs, small nucleolar RNAs, and untranslated mRNA regions, accomplish many of their diverse functions through direct interactions with RNA-binding proteins (RBPs). Recent efforts have identified hundreds of new RBPs that lack known RNA-binding domains, thus underscoring the complexity and diversity of RNA-protein complexes. Recent progress has expanded the number of methods for studying RNA-protein interactions in two general categories: approaches that characterize proteins bound to an RNA of interest (RNA-centric), and those that examine RNAs bound to a protein of interest (protein-centric). Each method has unique strengths and limitations, which makes it important to select optimal approaches for the biological question being addressed. Here we review methods for the study of RNA-protein interactions, with a focus on their suitability for specific applications.
Assuntos
Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Formaldeído/química , Ligação Proteica , Proteômica , RNA/química , Proteínas de Ligação a RNA/química , Análise de Sequência de RNA , Raios UltravioletaRESUMO
Metazoan PUF (Pumilio and FBF) RNA-binding proteins regulate various biological processes, but a common theme across phylogeny is stem cell regulation. In Caenorhabditis elegans, FBF (fem-3 Binding Factor) maintains germline stem cells regardless of which gamete is made, but FBF also functions in the process of spermatogenesis. We have begun to "disentangle" these biological roles by asking which FBF targets are gamete-independent, as expected for stem cells, and which are gamete-specific. Specifically, we compared FBF iCLIP binding profiles in adults making sperm to those making oocytes. Normally, XX adults make oocytes. To generate XX adults making sperm, we used a fem-3(gf) mutant requiring growth at 25°; for comparison, wild-type oogenic hermaphrodites were also raised at 25°. Our FBF iCLIP data revealed FBF binding sites in 1522 RNAs from oogenic adults and 1704 RNAs from spermatogenic adults. More than half of these FBF targets were independent of germline gender. We next clustered RNAs by FBF-RNA complex frequencies and found four distinct blocks. Block I RNAs were enriched in spermatogenic germlines, and included validated target fog-3, while Block II and III RNAs were common to both genders, and Block IV RNAs were enriched in oogenic germlines. Block II (510 RNAs) included almost all validated FBF targets and was enriched for cell cycle regulators. Block III (21 RNAs) was enriched for RNA-binding proteins, including previously validated FBF targets gld-1 and htp-1 We suggest that Block I RNAs belong to the FBF network for spermatogenesis, and that Blocks II and III are associated with stem cell functions.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Oogênese/genética , Proteínas de Ligação a RNA/genética , Espermatogênese/genética , Animais , Sítios de Ligação , Caenorhabditis elegans/genética , Caenorhabditis elegans/crescimento & desenvolvimento , Feminino , Masculino , Oócitos/crescimento & desenvolvimento , Oócitos/metabolismo , Filogenia , Ligação Proteica/genética , Processos de Determinação Sexual/genética , Espermatozoides/crescimento & desenvolvimento , Espermatozoides/metabolismo , Células-Tronco/metabolismoRESUMO
FOG-3 is a master regulator of sperm fate in Caenorhabditis elegans and homologous to Tob/BTG proteins, which in mammals are monomeric adaptors that recruit enzymes to RNA binding proteins. Here, we determine the FOG-3 crystal structure and in vitro demonstrate that FOG-3 forms dimers that can multimerize. The FOG-3 multimeric structure has a basic surface potential, suggestive of binding nucleic acid. Consistent with that prediction, FOG-3 binds directly to nearly 1,000 RNAs in nematode spermatogenic germ cells. Most binding is to the 3' UTR, and most targets (94%) are oogenic mRNAs, even though assayed in spermatogenic cells. When tethered to a reporter mRNA, FOG-3 represses its expression. Together these findings elucidate the molecular mechanism of sperm fate specification and reveal the evolution of a protein from monomeric to multimeric form with acquisition of a distinct mode of mRNA repression.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Espermatozoides/metabolismo , Regiões 3' não Traduzidas , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Dimerização , Masculino , Ligação Proteica , Multimerização Proteica , RNA/química , RNA/metabolismo , Processamento Pós-Transcricional do RNA , Espermatogênese , Espermatozoides/citologiaRESUMO
PUF (Pumilio/FBF) proteins are RNA-binding proteins and conserved stem cell regulators. The Caenorhabditis elegans PUF proteins FBF-1 and FBF-2 (collectively FBF) regulate mRNAs in germ cells. Without FBF, adult germlines lose all stem cells. A major gap in our understanding of PUF proteins, including FBF, is a global view of their binding sites in their native context (i.e., their "binding landscape"). To understand the interactions underlying FBF function, we used iCLIP (individual-nucleotide resolution UV crosslinking and immunoprecipitation) to determine binding landscapes of C. elegans FBF-1 and FBF-2 in the germline tissue of intact animals. Multiple iCLIP peak-calling methods were compared to maximize identification of both established FBF binding sites and positive control target mRNAs in our iCLIP data. We discovered that FBF-1 and FBF-2 bind to RNAs through canonical as well as alternate motifs. We also analyzed crosslinking-induced mutations to map binding sites precisely and to identify key nucleotides that may be critical for FBF-RNA interactions. FBF-1 and FBF-2 can bind sites in the 5'UTR, coding region, or 3'UTR, but have a strong bias for the 3' end of transcripts. FBF-1 and FBF-2 have strongly overlapping target profiles, including mRNAs and noncoding RNAs. From a statistically robust list of 1404 common FBF targets, 847 were previously unknown, 154 were related to cell cycle regulation, three were lincRNAs, and 335 were shared with the human PUF protein PUM2.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Células Germinativas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Animais , Proteínas de Caenorhabditis elegans/genética , Ligação Proteica , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genéticaRESUMO
Pumilio/fem-3 mRNA binding factor (PUF) proteins bind RNA with sequence specificity and modularity, and have become exemplary scaffolds in the reengineering of new RNA specificities. Here, we report the in vivo RNA binding sites of wild-type (WT) and reengineered forms of the PUF protein Saccharomyces cerevisiae Puf2p across the transcriptome. Puf2p defines an ancient protein family present throughout fungi, with divergent and distinctive PUF RNA binding domains, RNA-recognition motifs (RRMs), and prion regions. We identify sites in RNA bound to Puf2p in vivo by using two forms of UV cross-linking followed by immunopurification. The protein specifically binds more than 1,000 mRNAs, which contain multiple iterations of UAAU-binding elements. Regions outside the PUF domain, including the RRM, enhance discrimination among targets. Compensatory mutants reveal that one Puf2p molecule binds one UAAU sequence, and align the protein with the RNA site. Based on this architecture, we redesign Puf2p to bind UAAG and identify the targets of this reengineered PUF in vivo. The mutant protein finds its target site in 1,800 RNAs and yields a novel RNA network with a dramatic redistribution of binding elements. The mutant protein exhibits even greater RNA specificity than wild type. The redesigned protein decreases the abundance of RNAs in its redesigned network. These results suggest that reengineering using the PUF scaffold redirects and can even enhance specificity in vivo.
Assuntos
Motivos de Nucleotídeos/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Sequência de Bases , Sítios de Ligação/genética , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Modelos Genéticos , Mutação , Filogenia , Ligação Proteica , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/classificação , Proteínas de Ligação a RNA/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/classificação , Proteínas de Saccharomyces cerevisiae/metabolismo , Seleção GenéticaRESUMO
C. elegans germline stem cells exist within a stem cell pool that is maintained by a single-celled mesenchymal niche and Notch signaling. Downstream of Notch signaling, a regulatory network governs stem cells and differentiation. Central to that network is the FBF RNA-binding protein, a member of the widely conserved PUF family that functions by either of two broadly conserved mechanisms to repress its target mRNAs. Without FBF, germline stem cells do not proliferate and they do not maintain their naïve, undifferentiated state. Therefore, FBF is a pivotal regulator of germline self-renewal. Validated FBF targets include several key differentiation regulators as well as a major cell cycle regulator. A genomic analysis identifies many other developmental and cell cycle regulators as likely FBF targets and suggests that FBF is a broad-spectrum regulator of the genome with >1,000 targets. A comparison of the FBF target list with similar lists for human PUF proteins, PUM1 and PUM2, reveals â¼200 shared targets. The FBF hub works within a network controlling self-renewal vs. differentiation. This network consists of classical developmental cell fate regulators and classical cell cycle regulators. Recent results have begun to integrate developmental and cell cycle regulation within the network. The molecular dynamics of the network remain a challenge for the future, but models are proposed. We suggest that molecular controls of C. elegans germline stem cells provide an important model for controls of stem cells more broadly.
Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/metabolismo , Proteínas de Ligação a RNA/genética , Células-Tronco/metabolismo , Animais , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Ciclo Celular/genética , Diferenciação Celular , Proliferação de Células , Redes Reguladoras de Genes , Células Germinativas/citologia , Humanos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Transdução de Sinais , Nicho de Células-Tronco/genética , Células-Tronco/citologiaRESUMO
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.
Assuntos
Mapeamento de Interação de Proteínas/métodos , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/química , RNA/química , Regiões 3' não Traduzidas/genética , Animais , Sequência de Bases , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Mutação , Ligação Proteica , RNA/genética , RNA/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência do Ácido Nucleico , Técnicas do Sistema de Duplo-HíbridoRESUMO
Protein-protein interactions between domains within fatty acid and polyketide synthases are critical to catalysis, but their contributions remain incompletely characterized. A practical, quantitative system for establishing functional interactions between modifying enzymes and the acyl carrier protein that tethers the nascent polymer would offer a valuable tool for understanding and engineering these enzyme systems. Mechanism-based crosslinking of modular domains offers a potential diagnostic to highlight selective interactions between modular pairs. Here kinetic activity analysis and isothermal titration calorimetry are shown to correlate the efficiency of a ketosynthase-carrier protein crosslinking method to the binding affinity and transacylase activity that occurs in ketosynthase chain elongation.