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Engaging in research experiences as a high school or undergraduate student interested in science, technology, engineering, and mathematics (STEM) is pivotal for their academic and professional development. A structured teaching framework can help cultivate a student's curiosity and passion for learning and research. In this study, an eight-week training program was created to encompass fundamental molecular biology principles and hands-on laboratory activities. This curriculum focuses on using clustered regularly interspaced short palindromic repeats (CRISPR) gene editing in the Caenorhabditis elegans model organism. Through pre- and post-program assessments, enhancements in students' molecular biology proficiency and enthusiasm for scientific exploration were observed. Overall, this training module demonstrated its accessibility and ability to engage inexperienced students in molecular biology and gene editing methodologies.
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RNA modifications shape gene expression through a smorgasbord of chemical changes to canonical RNA bases. Although numbering in the hundreds, only a few RNA modifications are well characterized, in part due to the absence of methods to identify modification sites. Antibodies remain a common tool to identify modified RNA and infer modification sites through straightforward applications. However, specificity issues can result in off-target binding and confound conclusions. This work utilizes in silico λ-dynamics to efficiently estimate binding free energy differences of modification-targeting antibodies between a variety of naturally occurring RNA modifications. Crystal structures of inosine and N6-methyladenosine (m6A) targeting antibodies bound to their modified ribonucleosides were determined and served as structural starting points. λ-Dynamics was utilized to predict RNA modifications that permit or inhibit binding to these antibodies. In vitro RNA-antibody binding assays supported the accuracy of these in silico results. High agreement between experimental and computed binding propensities demonstrated that λ-dynamics can serve as a predictive screen for antibody specificity against libraries of RNA modifications. More importantly, this strategy is an innovative way to elucidate how hundreds of known RNA modifications interact with biological molecules without the limitations imposed by in vitro or in vivo methodologies.
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Robust methods are critical for testing the in vivo regulatory mechanism of RNA binding proteins. Here we report improvement of a protein-mRNA tethering assay to probe the function of an RNA binding protein in its natural context within the C. elegans adult germline. The assay relies on a dual reporter expressing two mRNAs from a single promoter and resolved by trans-splicing. The gfp reporter 3'UTR harbors functional binding elements for λN22 peptide, while the mCherry reporter 3'UTR carries mutated nonfunctional elements. This strategy enables internally controlled quantitation of reporter protein by immunofluorescence and mRNA by smFISH. To test the new system, we analyzed a C. elegans Nanos protein, NOS-3, which serves as a post-transcriptional regulator of germ cell fate. Unexpectedly, tethered NOS-3 enhanced reporter expression. We confirmed this enhancement activity with a second reporter engineered at an endogenous germline gene. NOS-3 enhancement of reporter expression was associated with its amino-terminal intrinsically disordered region, not its carboxy-terminal zinc fingers. RNA quantitation revealed that tethered NOS-3 enhances stability of the reporter mRNA. We suggest that this direct NOS-3 enhancement activity may explain a paradox: Classically Nanos proteins are expected to repress RNA, but nos-3 had been found to promote gld-1 expression, an effect that could be direct. Regardless, the new dual reporter dramatically improves in situ quantitation of reporter expression after RNA binding protein tethering to determine its molecular mechanism in a multicellular tissue.
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Proteínas de Caenorhabditis elegans/metabolismo , Hibridación Fluorescente in Situ/métodos , Estabilidad del ARN , ARN Mensajero/metabolismo , Animales , Caenorhabditis elegans , Regulación de la Expresión Génica , Células Germinativas , Imagen Individual de MoléculaRESUMEN
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.
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Proteínas de Caenorhabditis elegans/metabolismo , Espermatozoides/metabolismo , Regiones no Traducidas 3' , Animales , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Dimerización , Masculino , Unión Proteica , Multimerización de Proteína , ARN/química , ARN/metabolismo , Procesamiento Postranscripcional del ARN , Espermatogénesis , Espermatozoides/citologíaRESUMEN
Cellular RNA-protein (RNP) granules are ubiquitous and have fundamental roles in biology and RNA metabolism, but the molecular basis of their structure, assembly, and function is poorly understood. Using nematode "P-granules" as a paradigm, we focus on the PGL granule scaffold protein to gain molecular insights into RNP granule structure and assembly. We first identify a PGL dimerization domain (DD) and determine its crystal structure. PGL-1 DD has a novel 13 α-helix fold that creates a positively charged channel as a homodimer. We investigate its capacity to bind RNA and discover unexpectedly that PGL-1 DD is a guanosine-specific, single-stranded endonuclease. Discovery of the PGL homodimer, together with previous results, suggests a model in which the PGL DD dimer forms a fundamental building block for P-granule assembly. Discovery of the PGL RNase activity expands the role of RNP granule assembly proteins to include enzymatic activity in addition to their job as structural scaffolds.
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Gránulos Citoplasmáticos/metabolismo , Ribonucleasas/metabolismo , Secuencia de Aminoácidos , Animales , Caenorhabditis/metabolismo , Cristalografía por Rayos X , Gránulos Citoplasmáticos/química , Modelos Moleculares , Datos de Secuencia Molecular , Ribonucleasas/química , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
Germ cell specification as sperm or oocyte is an ancient cell fate decision, but its molecular regulation is poorly understood. In Caenorhabditis elegans, the FOG-1 and FOG-3 proteins behave genetically as terminal regulators of sperm fate specification. Both are homologous to well-established RNA regulators, suggesting that FOG-1 and FOG-3 specify the sperm fate post-transcriptionally. We predicted that FOG-1 and FOG-3, as terminal regulators of the sperm fate, might regulate a battery of gamete-specific differentiation genes. Here we test that prediction by exploring on a genomic scale the messenger RNAs (mRNAs) associated with FOG-1 and FOG-3. Immunoprecipitation of the proteins and their associated mRNAs from spermatogenic germlines identifies 81 FOG-1 and 722 FOG-3 putative targets. Importantly, almost all FOG-1 targets are also FOG-3 targets, and these common targets are strongly biased for oogenic mRNAs. The discovery of common target mRNAs suggested that FOG-1 and FOG-3 work together. Consistent with that idea, we find that FOG-1 and FOG-3 proteins co-immunoprecipitate from both intact nematodes and mammalian tissue culture cells and that they colocalize in germ cells. Taking our results together, we propose a model in which FOG-1 and FOG-3 work in a complex to repress oogenic transcripts and thereby promote the sperm fate.
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Proteínas de Caenorhabditis elegans/fisiología , Caenorhabditis elegans/genética , ARN Mensajero/metabolismo , Espermatogénesis/genética , Animales , Caenorhabditis elegans/citología , Proteínas de Caenorhabditis elegans/genética , Femenino , Masculino , Oogénesis/genética , Procesamiento Postranscripcional del ARN , TransgenesRESUMEN
RT-SHIV is a chimera of simian immunodeficiency virus (SIV) containing the reverse transcriptase (RT)-encoding region of human immunodeficiency virus type 1 (HIV-1) within the backbone of SIVmac239. It has been used in a non-human primate model for studies of non-nucleoside RT inhibitors (NNRTI) and highly active antiretroviral therapy (HAART). We and others have identified several mutations that arise in the "foreign" HIV-1 RT of RT-SHIV during in vivo replication. In this study we catalogued amino acid substitutions in the HIV-1 RT and in regions of the SIV backbone with which RT interacts that emerged 30 weeks post-infection from seven RT-SHIV-infected rhesus macaques. The virus set points varied from relatively high virus load, moderate virus load, to undetectable virus load. The G196R substitution in RT was detected from 6 of 7 animals at week 4 post-infection and remained in virus from 4 of 6 animals at week 30. Virus from four high virus load animals showed several common mutations within RT, including L74V or V75L, G196R, L214F, and K275R. The foreign RT from high virus load isolates exhibited as much variation as that of the highly variable envelope surface glycoprotein, and 10-fold higher than that of the native RT of SIVmac239. Isolates from moderate virus load animals showed much less variation in the foreign RT than the high virus load isolates. No variation was found in SIVmac239 genes known to interact with RT. Our results demonstrate substantial adaptation of the foreign HIV-1 RT in RT-SHIV-infected macaques, which most likely reflects selective pressure upon the foreign RT to attain optimal activity within the context of the chimeric RT-SHIV and the rhesus macaque host.
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Transcriptasa Inversa del VIH/genética , Mutación , Proteínas Recombinantes de Fusión/genética , Síndrome de Inmunodeficiencia Adquirida del Simio/genética , Virus de la Inmunodeficiencia de los Simios/genética , Sustitución de Aminoácidos , Animales , Terapia Antirretroviral Altamente Activa , Células Cultivadas , Femenino , Genoma Viral/genética , Transcriptasa Inversa del VIH/química , Transcriptasa Inversa del VIH/metabolismo , Interacciones Huésped-Patógeno/genética , Humanos , Macaca mulatta , Masculino , Modelos Moleculares , Estructura Terciaria de Proteína , ARN Viral/sangre , ARN Viral/genética , ADN Polimerasa Dirigida por ARN/genética , ADN Polimerasa Dirigida por ARN/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Inhibidores de la Transcriptasa Inversa , Síndrome de Inmunodeficiencia Adquirida del Simio/enzimología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/enzimología , Carga Viral/genética , Proteínas Virales/genética , Proteínas Virales/metabolismo , Replicación Viral/genéticaRESUMEN
Simian immunodeficiency virus (SIV) infection of rhesus macaques has become an important surrogate model for evaluating HIV vaccine strategies. The extreme resistance to neutralizing antibody (NAb) of many commonly used strains, such as SIVmac251/239 and SIVsmE543-3, limits their potential relevance for evaluating the role of NAb in vaccine protection. In contrast, SIVsmE660 is an uncloned virus that appears to be more sensitive to neutralizing antibody. To evaluate the role of NAb in this model, we generated full-length neutralization-sensitive molecular clones of SIVsmE660 and evaluated two of these by intravenous inoculation of rhesus macaques. All animals became infected and maintained persistent viremia that was accompanied by a decline in memory CD4(+) T cells in blood and bronchoalveolar lavage fluid. High titers of autologous NAb developed by 4 weeks postinoculation but were not associated with control of viremia, and neutralization escape variants were detected concurrently with the generation of NAb. Neutralization escape was associated with substitutions and insertion/deletion polymorphisms in the V1 and V4 domains of envelope. Analysis of representative variants revealed that escape variants also induced NAbs within a few weeks of their appearance in plasma, in a pattern that is reminiscent of the escape of human immunodeficiency virus type 1 (HIV-1) isolates in humans. Although early variants maintained a neutralization-sensitive phenotype, viruses obtained later in infection were significantly less sensitive to neutralization than the parental viruses. These results indicate that NAbs exert selective pressure that drives the evolution of the SIV envelope and that this model will be useful for evaluating the role of NAb in vaccine-mediated protection.
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Síndrome de Inmunodeficiencia Adquirida del Simio/inmunología , Síndrome de Inmunodeficiencia Adquirida del Simio/virología , Virus de la Inmunodeficiencia de los Simios/genética , Virus de la Inmunodeficiencia de los Simios/inmunología , Sustitución de Aminoácidos , Animales , Anticuerpos Neutralizantes/sangre , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/sangre , Anticuerpos Antivirales/inmunología , Recuento de Linfocito CD4 , Modelos Animales de Enfermedad , Evolución Molecular , Evasión Inmune , Macaca mulatta , Datos de Secuencia Molecular , Mutagénesis Insercional , Mutación , Mutación Puntual , Polimorfismo Genético , ARN Viral/genética , Análisis de Secuencia de ADN , Eliminación de Secuencia , Virus de la Inmunodeficiencia de los Simios/aislamiento & purificaciónRESUMEN
Antibodies that neutralize rotavirus infection target outer coat proteins VP4 and VP7 and inhibit viral entry. The structure of a VP7-Fab complex (S. T. Aoki, et al., Science 324:1444-1447, 2009) led us to reclassify epitopes into two binding regions at inter- and intrasubunit boundaries of the calcium-dependent trimer. It further led us to show that antibodies binding at the intersubunit boundary inhibit uncoating of the virion outer layer. We have now tested representative antibodies for each of the defined structural epitope regions and find that antibodies recognizing epitopes in either binding region neutralize by cross-linking VP7 trimers. Antibodies that bind at the intersubunit junction neutralize as monovalent Fabs, while those that bind at the intrasubunit region require divalency. The VP7 structure has also allowed us to design a disulfide cross-linked VP7 mutant which recoats double-layered particles (DLPs) as efficiently as does wild-type VP7 but which yields particles defective in cell entry as determined both by lack of infectivity and by loss of α-sarcin toxicity in the presence of recoated particles. We conclude that dissociation of the VP7 trimer is an essential step in viral penetration into cells.
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Antígenos Virales/metabolismo , Proteínas de la Cápside/metabolismo , Rotavirus/fisiología , Internalización del Virus , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Antígenos Virales/química , Antígenos Virales/inmunología , Proteínas de la Cápside/química , Proteínas de la Cápside/inmunología , Línea Celular , Disulfuros/metabolismo , Macaca mulattaRESUMEN
Rotaviruses, major causes of childhood gastroenteritis, are nonenveloped, icosahedral particles with double-strand RNA genomes. By the use of electron cryomicroscopy and single-particle reconstruction, we have visualized a rotavirus particle comprising the inner capsid coated with the trimeric outer-layer protein, VP7, at a resolution (4 A) comparable with that of X-ray crystallography. We have traced the VP7 polypeptide chain, including parts not seen in its X-ray crystal structure. The 3 well-ordered, 30-residue, N-terminal "arms" of each VP7 trimer grip the underlying trimer of VP6, an inner-capsid protein. Structural differences between free and particle-bound VP7 and between free and VP7-coated inner capsids may regulate mRNA transcription and release. The Ca(2+)-stabilized VP7 intratrimer contact region, which presents important neutralizing epitopes, is unaltered upon capsid binding.
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Microscopía por Crioelectrón/métodos , Rotavirus/ultraestructura , Virión/ultraestructura , Antígenos Virales/química , Antígenos Virales/metabolismo , Antígenos Virales/ultraestructura , Calcio/metabolismo , Proteínas de la Cápside/química , Proteínas de la Cápside/metabolismo , Proteínas de la Cápside/ultraestructura , Cristalografía por Rayos X , Modelos Biológicos , Modelos Moleculares , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Rotavirus/metabolismo , Proteínas del Núcleo Viral/química , Proteínas del Núcleo Viral/metabolismo , Proteínas del Núcleo Viral/ultraestructura , Virión/metabolismoRESUMEN
Rotavirus outer-layer protein VP7 is a principal target of protective antibodies. Removal of free calcium ions (Ca2+) dissociates VP7 trimers into monomers, releasing VP7 from the virion, and initiates penetration-inducing conformational changes in the other outer-layer protein, VP4. We report the crystal structure at 3.4 angstrom resolution of VP7 bound with the Fab fragment of a neutralizing monoclonal antibody. The Fab binds across the outer surface of the intersubunit contact, which contains two Ca2+ sites. Mutations that escape neutralization by other antibodies suggest that the same region bears the epitopes of most neutralizing antibodies. The monovalent Fab is sufficient to neutralize infectivity. We propose that neutralizing antibodies against VP7 act by stabilizing the trimer, thereby inhibiting the uncoating trigger for VP4 rearrangement. A disulfide-linked trimer is a potential subunit immunogen.