RESUMEN
The structure of RNA molecules is absolutely critical to their functions in a biological system. RNA structure is dynamic and changes in response to cellular needs. Within the last few decades, there has been an increased interest in studying the structure of RNA molecules and how they change to support the needs of the cell in different conditions. Selective 2'-hydroxyl acylation-based mutational profiling using high-throughput sequencing is a powerful method to predict the secondary structure of RNA molecules both in vivo and in immunopurified samples. Selective 2'-hydroxyl acylation-based mutational profiling using high-throughput sequencing works by adding bulky groups onto accessible "flexible" bases in an RNA molecule that are not involved in any base-pairing or RNA-protein interactions. When the RNA is reverse transcribed into cDNA, the bulky groups are incorporated as base mutations, which can be compared to an unmodified control to identify the locations of flexible bases. The comparison of sequence data between modified and unmodified samples allows the computer software program (developed to generate reactivity profiles) to generate RNA secondary structure models. These models can be compared in a variety of conditions to determine how specific stimuli influence RNA secondary structures.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento , Mutación , Conformación de Ácido Nucleico , Pliegue del ARN , ARN , ARN/genética , ARN/química , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Programas Informáticos , AcilaciónRESUMEN
Telomerase is an enzyme involved in the maintenance of telomeres. Telomere shortening due to the end-replication problem is a threat to the genome integrity of all eukaryotes. Telomerase inside cells depends on a myriad of protein-protein and RNA-protein interactions to properly assemble and regulate the function of the telomerase holoenzyme. These interactions are well studied in model eukaryotes, like humans, yeast, and the ciliated protozoan known as Tetrahymena thermophila. Emerging evidence also suggests that deep-branching eukaryotes, such as the parasitic protist Trypanosoma brucei require conserved and novel RNA-binding proteins for the assembly and function of their telomerase. In this review, we will discuss telomerase regulatory pathways in the context of telomerase-interacting proteins, with special attention paid to RNA-binding proteins. We will discuss these interactors on an evolutionary scale, from parasitic protists to humans, to provide a broader perspective on the extensive role that protein-protein and RNA-protein interactions play in regulating telomerase activity in eukaryotes.
RESUMEN
Telomerase is a ribonucleoprotein enzyme responsible for maintaining the telomeric end of the chromosome. The telomerase enzyme requires two main components to function: the telomerase reverse transcriptase (TERT) and the telomerase RNA (TR), which provides the template for telomeric DNA synthesis. TR is a long non-coding RNA, which forms the basis of a large structural scaffold upon which many accessory proteins can bind and form the complete telomerase holoenzyme. These accessory protein interactions are required for telomerase activity and regulation inside cells. The interacting partners of TERT have been well studied in yeast, human, and Tetrahymena models, but not in parasitic protozoa, including clinically relevant human parasites. Here, using the protozoan parasite, Trypanosoma brucei (T. brucei) as a model, we have identified the interactome of T. brucei TERT (TbTERT) using a mass spectrometry-based approach. We identified previously known and unknown interacting factors of TbTERT, highlighting unique features of T. brucei telomerase biology. These unique interactions with TbTERT, suggest mechanistic differences in telomere maintenance between T. brucei and other eukaryotes.
RESUMEN
"Epitranscriptomics" is the new RNA code that represents an ensemble of posttranscriptional RNA chemical modifications, which can precisely coordinate gene expression and biological processes. There are several RNA base modifications, such as N6-methyladenosine (m6A), 5-methylcytosine (m5C), and pseudouridine (Ψ), etc. that play pivotal roles in fine-tuning gene expression in almost all eukaryotes and emerging evidences suggest that parasitic protists are no exception. In this review, we primarily focus on m6A, which is the most abundant epitranscriptomic mark and regulates numerous cellular processes, ranging from nuclear export, mRNA splicing, polyadenylation, stability, and translation. We highlight the universal features of spatiotemporal m6A RNA modifications in eukaryotic phylogeny, their homologs, and unique processes in 3 unicellular parasites-Plasmodium sp., Toxoplasma sp., and Trypanosoma sp. and some technological advances in this rapidly developing research area that can significantly improve our understandings of gene expression regulation in parasites.
Asunto(s)
Parásitos , ARN , Animales , ARN/metabolismo , Parásitos/genética , Parásitos/metabolismo , Regulación de la Expresión Génica , Procesamiento Postranscripcional del ARN , Eucariontes/genética , PoliadenilaciónRESUMEN
Telomerase has an established role in telomere maintenance in eukaryotes. However, recent studies have begun to implicate telomerase in cellular roles beyond telomere maintenance. Specifically, evidence is emerging of cross-talks between telomerase mediated telomere homeostasis and DNA repair pathways. Telomere shortening due to the end replication problem is a constant threat to genome integrity in eukaryotic cells. This poses a particular problem in unicellular parasitic protists because their major virulence genes are located at the subtelomeric loci. Although telomerase is the major regulator of telomere lengthening in eukaryotes, it is less studied in the ancient eukaryotes, including clinically important human pathogens. Recent research is highlighting interplay between telomerase and the DNA damage response in human parasites. The importance of this interplay in pathogen virulence is only beginning to be illuminated, including the potential to highlight novel developmental regulation of telomerase in parasites who transition between multiple developmental stages throughout their life cycle. In this review, we will discuss the telomerase ribonucleoprotein enzyme and DNA repair pathways with emerging views in human parasites to give a broader perspective of the possible connection of telomere, telomerase, and DNA repair pathways across eukaryotic lineages and highlight their potential role in pathogen virulence. This article is categorized under: RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.
Asunto(s)
Parásitos , Telomerasa , Animales , Eucariontes/genética , Eucariontes/metabolismo , Humanos , Parásitos/genética , Parásitos/metabolismo , ARN/metabolismo , Ribonucleoproteínas/genética , Telomerasa/química , Telómero/genética , Telómero/metabolismoRESUMEN
Nucleic acid nanoparticles (NANPs) are an exciting and innovative technology in the context of both basic and biomedical research. Made of DNA, RNA, or their chemical analogs, NANPs are programmed for carrying out specific functions within human cells. NANPs are at the forefront of preventing, detecting, and treating disease. Their nucleic acid composition lends them biocompatibility that provides their cargo with enhanced opportunity for coordinated delivery. Of course, the NANP system of targeting specific cells and tissues is not without its disadvantages. Accumulation of NANPs outside of the target tissue and the potential for off-target effects of NANP-mediated cargo delivery present challenges to research and medical professionals and these challenges must be effectively addressed to provide safe treatment to patients. Importantly, development of NANPs with regulated biological activities and immunorecognition becomes a promising route for developing versatile nucleic acid therapeutics. In a basic research context, NANPs can assist investigators in fine-tuning the structure-function relationship of final formulations and in this review, we explore the practical applications of NANPs in laboratory and clinical settings and discuss how we can use established nucleic acid research techniques to design effective NANPs.
Asunto(s)
Nanopartículas , Ácidos Nucleicos , Humanos , ARN/uso terapéutico , Ácidos Nucleicos/química , ADN/química , Nanopartículas/químicaRESUMEN
Telomerase is a unique ribonucleoprotein (RNP) reverse transcriptase that utilizes its cognate RNA molecule as a template for telomere DNA repeat synthesis. Telomerase contains the reverse transcriptase protein, TERT and the template RNA, TR, as its core components. The 5'-half of TR forms a highly conserved catalytic core comprising of the template region and adjacent domains necessary for telomere synthesis. However, how telomerase RNA folding takes place in vivo has not been fully understood due to low abundance of the native RNP. Here, using unicellular pathogen Trypanosoma brucei as a model, we reveal important regional folding information of the native telomerase RNA core domains, i.e. TR template, template boundary element, template proximal helix and Helix IV (eCR4-CR5) domain. For this purpose, we uniquely combined in-cell probing with targeted high-throughput RNA sequencing and mutational mapping under three conditions: in vivo (in WT and TERT-/- cells), in an immunopurified catalytically active telomerase RNP complex and ex vivo (deproteinized). We discover that TR forms at least two different conformers with distinct folding topologies in the insect and mammalian developmental stages of T. brucei. Also, TERT does not significantly affect the RNA folding in vivo, suggesting that the telomerase RNA in T. brucei exists in a conformationally preorganized stable structure. Our observed differences in RNA (TR) folding at two distinct developmental stages of T. brucei suggest that important conformational changes are a key component of T. brucei development.
Asunto(s)
Dominio Catalítico , Proteínas Protozoarias/genética , ARN Protozoario/genética , ARN/genética , Telomerasa/genética , Trypanosoma brucei brucei/genética , Secuencia de Bases , Biocatálisis , Pruebas de Enzimas/métodos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Mutación , Conformación de Ácido Nucleico , Unión Proteica , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , ARN/química , ARN/metabolismo , Pliegue del ARN , ARN Protozoario/química , ARN Protozoario/metabolismo , Telomerasa/química , Telomerasa/metabolismo , Termodinámica , Trypanosoma brucei brucei/metabolismoRESUMEN
Plasmodium falciparum is a deadly human pathogen responsible for the devastating disease called malaria. In this study, we measured the differential accumulation of RNA secondary structures in coding and non-coding transcripts from the asexual developmental cycle in P. falciparum in human red blood cells. Our comprehensive analysis that combined high-throughput nuclease mapping of RNA structures by duplex RNA-seq, SHAPE-directed RNA structure validation, immunoaffinity purification and characterization of antisense RNAs collectively measured differentially base-paired RNA regions throughout the parasite's asexual RBC cycle. Our mapping data not only aligned to a diverse pool of RNAs with known structures but also enabled us to identify new structural RNA regions in the malaria genome. On average, approximately 71% of the genes with secondary structures are found to be protein coding mRNAs. The mapping pattern of these base-paired RNAs corresponded to all regions of mRNAs, including the 5' UTR, CDS and 3' UTR as well as the start and stop codons. Histone family genes which are known to form secondary structures in their mRNAs and transcripts from genes which are important for transcriptional and post-transcriptional control, such as the unique plant-like transcription factor family, ApiAP2, DNA-/RNA-binding protein, Alba3 and proteins important for RBC invasion and malaria cytoadherence also showed strong accumulation of duplex RNA reads in various asexual stages in P. falciparum. Intriguingly, our study determined stage-specific, dynamic relationships between mRNA structural contents and translation efficiency in P. falciparum asexual blood stages, suggesting an essential role of RNA structural changes in malaria gene expression programs. Abbreviations: CDS: Coding Sequence; DNA: Deoxyribonucleic Acid; dsRNA: double-stranded RNA; IDC: Intra-erythrocytic Developmental Cycle (IDC); m6A: N6-methyladenosine; mRNA: Messenger RNA; ncRNA: Non-coding RNA; RBC: Red Blood cells; RBP: RNA-Binding Protein; REC: Relative Expression Counts; RNA-seq: RNA-sequencing; RNA: Ribonucleic Acid; RNP: Ribonucleoprotein; RPKM: Reads Per Kilobase of transcript Per Million; rRNA: Ribosomal RNA 16. RUFs: RNAs of Unknown Function; SHAPE: Selective 2'-hydroxyl acylation analysed by primer extension; snoRNA: Small Nucleolar RNA; snRNA: Small Nuclear RNA; SRP-RNA: Signal Recognition Particle RNA; ssRNA: (Single-stranded RNA); TE: Translation Efficiency; tRNA: transfer RNA; UTR: Untranslated Region.
Asunto(s)
Eritrocitos/metabolismo , Regulación de la Expresión Génica , Estadios del Ciclo de Vida , Malaria Falciparum/parasitología , Conformación de Ácido Nucleico , Plasmodium falciparum/genética , ARN Protozoario/química , Humanos , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/patogenicidad , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , TranscriptomaRESUMEN
Pre-mRNA processing and mRNA stability play direct roles in controlling protein abundance in a cell. Before the mRNA can be translated into a protein, the introns in the pre-mRNA transcripts need to be removed by splicing, such that exons can be ligated together and can code for a protein. In this process, the function of the RNA lariat debranching enzyme or Dbr1 provides a rate-limiting step in the intron turnover process and possibly regulating the production of translation competent mRNAs. Surprising new roles of Dbr1 are emerging in cellular metabolism which extends beyond intron turnover processes, ranging from splicing regulation to translational control. In this review, we highlight the importance of the Dbr1 enzyme, its structure and how anomalies in its function could relate to various human diseases.
Asunto(s)
ARN Nucleotidiltransferasas/genética , ARN Nucleotidiltransferasas/metabolismo , ARN Mensajero/metabolismo , Esclerosis Amiotrófica Lateral/enzimología , Esclerosis Amiotrófica Lateral/genética , Animales , Encefalitis Viral/enzimología , Encefalitis Viral/genética , VIH/enzimología , VIH/genética , Humanos , Intrones , Neoplasias/enzimología , Neoplasias/genética , ARN Nucleotidiltransferasas/químicaRESUMEN
1-Methyl-7-nitroisatoic anhydride (1M7) and 2-methylnicotinic acid imidazolide (NAI) are two of the most commonly applied RNA-SHAPE electrophiles; 1M7 due to its high reactivity and NAI for its solubility and cell permeability. While the addition of a nitro group yields desirable activation of the reagent, it also leads to poorer water solubility. This limited solubility has motivated the development of water-soluble reagents. We present alternative, isatoic anhydride-based reagents possessing variable reactivities that are simultaneously water-soluble. Solubility is gained by using a quaternary ammonium, while modulation of the reactivity is obtained by functionalization of the aryl ring. The syntheses of the reagents are discussed, and the electrophiles are demonstrated to be suitable for use for an in vitro RNA SHAPE experiment when directly compared to 1M7.
Asunto(s)
Oxazinas/química , ARN/química , Agua/química , Acilación , Secuencia de Bases , Radical Hidroxilo/química , ARN/genética , SolubilidadRESUMEN
We are delighted to share with you our seventh Journal Club and highlight some of the most interesting papers published recently [...].
RESUMEN
N-(3-Iodopropyl)isatoic anhydride (IPIA) has been demonstrated to serve as an efficient substrate for the development of an extended bioconjugation platform. Derivatives of IPIA are water-soluble and adaptable and share a common chromophore, rendering them easily quantifiable. We demonstrate the preparation of the readily diversified bioconjugation platform technology and application of the reagents in RNA-SHAPE analysis.
Asunto(s)
Oxazinas/química , Proteínas/química , ARN/química , Estructura Molecular , Solubilidad , Agua/químicaRESUMEN
Replicative capacity of a cell is strongly correlated with telomere length regulation. Aberrant lengthening or reduction in the length of telomeres can lead to health anomalies, such as cancer or premature aging. Telomerase is a master regulator for maintaining replicative potential in most eukaryotic cells. It does so by controlling telomere length at chromosome ends. Akin to cancer cells, most single-cell eukaryotic pathogens are highly proliferative and require persistent telomerase activity to maintain constant length of telomere and propagation within their host. Although telomerase is key to unlimited cellular proliferation in both cases, not much was known about the role of telomerase in human parasites (malaria, Trypanosoma, etc.) until recently. Since telomerase regulation is mediated via its own structural components, interactions with catalytic reverse transcriptase and several factors that can recruit and assemble telomerase to telomeres in a cell cycle-dependent manner, we compare and discuss here recent findings in telomerase biology in cancer, aging and parasitic diseases to give a broader perspective of telomerase function in human diseases.
Asunto(s)
Eucariontes/enzimología , Parásitos/enzimología , Telomerasa/química , Telomerasa/metabolismo , Animales , Senescencia Celular/genética , Reparación del ADN/genética , Susceptibilidad a Enfermedades , Eucariontes/genética , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Conformación de Ácido Nucleico , Parásitos/genética , Unión Proteica , ARN/química , ARN/genética , Relación Estructura-Actividad , Telomerasa/genética , Telómero/genética , Telómero/metabolismoRESUMEN
Telomerase is a ribonucleoprotein enzyme typically required for sustained cell proliferation. Although both telomerase activity and the telomerase catalytic protein component, TbTERT, have been identified in the eukaryotic pathogen Trypanosoma brucei, the RNA molecule that dictates telomere synthesis remains unknown. Here, we identify the RNA component of Trypanosoma brucei telomerase, TbTR, and provide phylogenetic and in vivo evidence for TbTR's native folding and activity. We show that TbTR is processed through trans-splicing, and is a capped transcript that interacts and copurifies with TbTERT in vivo. Deletion of TbTR caused progressive shortening of telomeres at a rate of 3-5 bp/population doubling (PD), which can be rescued by ectopic expression of a wild-type allele of TbTR in an apparent dose-dependent manner. Remarkably, introduction of mutations in the TbTR template domain resulted in corresponding mutant telomere sequences, demonstrating that telomere synthesis in T. brucei is dependent on TbTR. We also propose a secondary structure model for TbTR based on phylogenetic analysis and chemical probing experiments, thus defining TbTR domains that may have important functional implications in telomere synthesis. Identification and characterization of TbTR not only provide important insights into T. brucei telomere functions, which have been shown to play important roles in T. brucei pathogenesis, but also offer T. brucei as an attractive model system for studying telomerase biology in pathogenic protozoa and for comparative analysis of telomerase function with higher eukaryotes.
Asunto(s)
Proteínas Protozoarias/genética , ARN Protozoario , ARN/genética , Telomerasa/genética , Telómero/genética , Trypanosoma brucei brucei/genética , Secuencia de Bases , Prueba de Complementación Genética , Datos de Secuencia Molecular , Mutación , Filogenia , Pliegue de Proteína , Estructura Secundaria de Proteína , Proteínas Protozoarias/química , Proteínas Protozoarias/clasificación , Proteínas Protozoarias/metabolismo , ARN/química , ARN/clasificación , ARN/metabolismo , Empalme del ARN , Telomerasa/química , Telomerasa/clasificación , Telomerasa/metabolismo , Telómero/metabolismo , Trypanosoma brucei brucei/enzimologíaRESUMEN
The 2,2,7-trimethylguanosine caps of eukaryal snRNAs and snoRNA are formed by the enzyme Tgs1, which catalyzes sequential guanine-N2 methylations of m(7)G caps. Atypically, in the divergent unicellular eukaryote Trichomonas vaginalis, spliceosomal snRNAs lack a guanosine cap and the recombinant T. vaginalis trimethylguanosine synthase (TvTgs) produces only m(2,7)G in vitro. Here, we show by direct metabolic labeling that endogenous T. vaginalis RNAs contain m(7)G, m(2,7)G, and m(2,2,7)G caps. Immunodepletion of TvTgs from cell extracts and TvTgs add-back experiments demonstrate that TvTgs produces m(2,7)G and m(2,2,7)G caps. Expression of TvTgs in yeast tgs1Δ cells leads to the formation of m(2,7)G and m(2,2,7)G caps and complementation of the lethality of a tgs1Δ mud2Δ strain. Whereas TvTgs is present in the nucleus and cytosol of T. vaginalis cells, TMG-containing RNAs are localized primarily in the nucleolus. Molecular cloning of anti-TMG affinity-purified T. vaginalis RNAs identified 16 box H/ACA snoRNAs, which are implicated in guiding RNA pseudouridylation. The ensemble of new T. vaginalis H/ACA snoRNAs allowed us to predict and partially validate an extensive map of pseudouridines in T. vaginalis rRNA.
Asunto(s)
Metiltransferasas/metabolismo , ARN Nucleolar Pequeño/metabolismo , Trichomonas vaginalis/enzimología , Secuencia de Bases , Transporte Biológico , Guanosina/análogos & derivados , Guanosina/metabolismo , Metilación , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Caperuzas de ARN/metabolismo , ARN Nucleolar Pequeño/química , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Alineación de Secuencia , Especificidad por Sustrato , Trichomonas vaginalis/genéticaAsunto(s)
Proteínas de Unión al ADN/genética , ARN de Hongos/genética , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/enzimología , Schizosaccharomyces/genética , Telomerasa/metabolismo , Regulación Enzimológica de la Expresión Génica , Regulación Fúngica de la Expresión Génica , ARNRESUMEN
As the genomes of more eukaryotic pathogens are sequenced, understanding how molecular differences between parasite and host might be exploited to provide new therapies has become a major focus. Central to cell function are RNA-containing complexes involved in gene expression, such as the ribosome, the spliceosome, snoRNAs, RNase P, and telomerase, among others. In this article we identify by comparative genomics and validate by RNA analysis numerous previously unknown structural RNAs encoded by the Plasmodium falciparum genome, including the telomerase RNA, U3, 31 snoRNAs, as well as previously predicted spliceosomal snRNAs, SRP RNA, MRP RNA, and RNAse P RNA. Furthermore, we identify six new RNA coding genes of unknown function. To investigate the relationships of the RNA coding genes to other genomic features in related parasites, we developed a genome browser for P. falciparum (http://areslab.ucsc.edu/cgi-bin/hgGateway). Additional experiments provide evidence supporting the prediction that snoRNAs guide methylation of a specific position on U4 snRNA, as well as predicting an snRNA promoter element particular to Plasmodium sp. These findings should allow detailed structural comparisons between the RNA components of the gene expression machinery of the parasite and its vertebrate hosts.
Asunto(s)
Genoma de Protozoos , Genómica , Plasmodium falciparum/genética , ARN Protozoario/química , Animales , Emparejamiento Base , Secuencia de Bases , Humanos , Malaria/parasitología , Metilación , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Plasmodium falciparum/metabolismo , ARN/química , ARN/metabolismo , ARN Ribosómico/química , ARN Ribosómico/metabolismo , ARN Nuclear Pequeño/química , ARN Nucleolar Pequeño/química , ARN Nucleolar Pequeño/metabolismo , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Empalmosomas , Telomerasa/química , Telomerasa/metabolismoRESUMEN
RhoGEFs are central controllers of small G-proteins in cells and are regulated by several mechanisms. There are at least 22 human RhoGEFs that contain SH3 domains, raising the possibility that, like several other enzymes, SH3 domains control the enzymatic activity of guanine nucleotide exchange factor (GEF) domains through intra- and/or intermolecular interactions. The structure of the N-terminal SH3 domain of Kalirin was solved using NMR spectroscopy, and it folds much like other SH3 domains. However, NMR chemical shift mapping experiments showed that this Kalirin SH3 domain is unique, containing novel cooperative binding site(s) for intramolecular PXXP ligands. Intramolecular Kalirin SH3 domain/ligand interactions, as well as binding of the Kalirin SH3 domain to the adaptor protein Crk, inhibit the GEF activity of Kalirin. This study establishes a novel molecular mechanism whereby intramolecular and intermolecular Kalirin SH3 domain/ligand interactions modulate GEF activity, a regulatory mechanism that is likely used by other RhoGEF family members.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido/química , Proteínas Serina-Treonina Quinasas/química , Dominios Homologos src , Secuencias de Aminoácidos , Animales , Sitios de Unión , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Ligandos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Relación Estructura-ActividadRESUMEN
Kalirin is a multidomain guanine nucleotide exchange factor (GEF) that activates Rho proteins, inducing cytoskeletal rearrangement in neurons. Although much is known about the effects of Kalirin on Rho GTPases and neuronal morphology, little is known about the association of Kalirin with the receptor/signaling systems that affect neuronal morphology. Our experiments demonstrate that Kalirin binds to and colocalizes with the TrkA neurotrophin receptor in neurons. In PC12 cells, inhibition of Kalirin expression using antisense RNA decreased nerve growth factor (NGF)-induced TrkA autophosphorylation and process extension. Kalirin overexpression potentiated neurotrophin-stimulated TrkA autophosphorylation and neurite outgrowth in PC12 cells at a low concentration of NGF. Furthermore, elevated Kalirin expression resulted in catalytic activation of TrkA, as demonstrated by in vitro kinase assays and increased NGF-stimulated cellular activation of Rac, Mek, and CREB. Domain mapping demonstrated that the N-terminal Kalirin pleckstrin homology domain mediates the interaction with TrkA. The effects of Kalirin on TrkA provide a molecular basis for the requirement of Kalirin in process extension from PC12 cells and for previously observed effects on axonal extension and dendritic maintenance. The interaction of TrkA with the pleckstrin homology domain of Kalirin may be one example of a general mechanism whereby receptor/Rho GEF pairings play an important role in receptor tyrosine kinase activation and signal transduction.
Asunto(s)
Factores de Intercambio de Guanina Nucleótido/metabolismo , Factor de Crecimiento Nervioso/metabolismo , Receptor trkA/metabolismo , Transducción de Señal/fisiología , Animales , Diferenciación Celular/fisiología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Activación Enzimática , Factores de Intercambio de Guanina Nucleótido/genética , Humanos , MAP Quinasa Quinasa 1/metabolismo , Ratones , Factor de Crecimiento Nervioso/genética , Neuronas/metabolismo , Células PC12 , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas , Receptor trkA/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Unión al GTP rac/metabolismoRESUMEN
We describe the epidemiology of malaria in San Dulakudar, a village in Sundargarh District in the state of Orissa in eastern India. Malaria transmission is perennial with Plasmodium falciparum, accounting for greater than 80% of malaria cases. Transmission intensity varies with season with high transmission after the monsoon rains in autumn and winter, low transmission in summer, and intermediate transmission in spring. The anthropophagic mosquito Anopheles fluviatilis was identified as the main vector for malaria transmission. Based on observations of spleen rates and supported by data on malaria parasite prevalence and malaria incidence, San Dulakudar can be classified as a hyperendemic area for P. falciparum malaria. Parasite prevalence and malaria incidence rates decrease with age, suggesting that residents of San Dulakudar develop immunity to malaria. The study demonstrates the presence of regions in the Indian subcontinent such as Sundargarh District where P. falciparum is the primary cause of malaria and where malaria transmission rates are comparable to those found in many parts of Africa.