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1.
Nature ; 619(7968): 193-200, 2023 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-37344590

RESUMO

Lymphocytes of vertebrate adaptive immune systems acquired the capability to assemble, from split genes in the germline, billions of functional antigen receptors1-3. These receptors show specificity; unlike the broadly tuned receptors of the innate system, antibodies (Ig) expressed by B cells, for instance, can accurately distinguish between the two enantiomers of organic acids4, whereas T cell receptors (TCRs) reliably recognize single amino acid replacements in their peptide antigens5. In developing lymphocytes, antigen receptor genes are assembled from a comparatively small set of germline-encoded genetic elements in a process referred to as V(D)J recombination6,7. Potential self-reactivity of some antigen receptors arising from the quasi-random somatic diversification is suppressed by several robust control mechanisms8-12. For decades, scientists have puzzled over the evolutionary origin of somatically diversifying antigen receptors13-16. It has remained unclear how, at the inception of this mechanism, immunologically beneficial expanded receptor diversity was traded against the emerging risk of destructive self-recognition. Here we explore the hypothesis that in early vertebrates, sequence microhomologies marking the ends of recombining elements became the crucial targets of selection determining the outcome of non-homologous end joining-based repair of DNA double-strand breaks generated during RAG-mediated recombination. We find that, across the main clades of jawed vertebrates, TCRα repertoire diversity is best explained by species-specific extents of such sequence microhomologies. Thus, selection of germline sequence composition of rearranging elements emerges as a major factor determining the degree of diversity of somatically generated antigen receptors.


Assuntos
Evolução Molecular , Rearranjo Gênico da Cadeia alfa dos Receptores de Antígenos dos Linfócitos T , Receptores de Antígenos de Linfócitos T alfa-beta , Recombinação V(D)J , Animais , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Recombinação V(D)J/genética , Vertebrados/classificação , Vertebrados/genética , Reparo do DNA por Junção de Extremidades , Quebras de DNA de Cadeia Dupla , Genes RAG-1 , Especificidade da Espécie , Homologia de Sequência , Rearranjo Gênico da Cadeia alfa dos Receptores de Antígenos dos Linfócitos T/genética , Linfócitos/metabolismo
2.
Nucleic Acids Res ; 51(8): 4078-4085, 2023 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-36928676

RESUMO

Many prokaryotic argonautes (pAgos) mediate DNA interference by using small DNA guides to cleave target DNA. A recent study shows that CbAgo, a pAgo from Clostridium butyricum, induces DNA interference between homologous sequences and generates double-stranded breaks (DSBs) in target DNAs. This mechanism enables the host to defend against invading DNAs such as plasmids and viruses. However, whether such a CbAgo-mediated DNA cleavage is mutagenic remains unexplored. Here we demonstrate that CbAgo, directed by plasmid-encoded guide sequences, can cleave genome target sites and induce chromosome recombination between downstream homologous sequences in Escherichia coli. The recombination rate correlates well with pAgo DNA cleavage activity and the mechanistic study suggests the recombination involves DSBs and RecBCD processing. In RecA-deficient E. coli strain, guide-directed CbAgo cleavage on chromosomes severely impairs cell growth, which can be utilized as counter-selection to assist Lambda-Red recombineering. These findings demonstrate the guide-directed cleavage of pAgo on the host genome is mutagenic and can lead to different outcomes according to the function of the host DNA repair machinery. We anticipate this novel DNA-guided interference to be useful in broader genetic manipulation. Our study also provides an in vivo assay to characterize or engineer pAgo DNA cleavage activity.


Assuntos
DNA , Escherichia coli , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Plasmídeos , Células Procarióticas/metabolismo , Homologia de Sequência , Genoma Bacteriano
3.
J Proteome Res ; 22(2): 420-431, 2023 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-36696582

RESUMO

Neuropeptides are a class of endogenous peptides that have key regulatory roles in biochemical, physiological, and behavioral processes. Mass spectrometry analyses of neuropeptides often rely on protein informatics tools for database searching and peptide identification. As neuropeptide databases are typically experimentally built and comprised of short sequences with high sequence similarity to each other, we developed a novel database searching tool, HyPep, which utilizes sequence homology searching for peptide identification. HyPep aligns de novo sequenced peptides, generated through PEAKS software, with neuropeptide database sequences and identifies neuropeptides based on the alignment score. HyPep performance was optimized using LC-MS/MS measurements of peptide extracts from various Callinectes sapidus neuronal tissue types and compared with a commercial database searching software, PEAKS DB. HyPep identified more neuropeptides from each tissue type than PEAKS DB at 1% false discovery rate, and the false match rate from both programs was 2%. In addition to identification, this report describes how HyPep can aid in the discovery of novel neuropeptides.


Assuntos
Neuropeptídeos , Espectrometria de Massas em Tandem , Sequência de Aminoácidos , Cromatografia Líquida , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Peptídeos/análise , Software , Homologia de Sequência , Bases de Dados de Proteínas
4.
Nat Commun ; 13(1): 6968, 2022 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-36379955

RESUMO

Multiple sequence alignments are widely used to infer evolutionary relationships, enabling inferences of structure, function, and phylogeny. Standard practice is to construct one alignment by some preferred method and use it in further analysis; however, undetected alignment bias can be problematic. I describe Muscle5, a novel algorithm which constructs an ensemble of high-accuracy alignment with diverse biases by perturbing a hidden Markov model and permuting its guide tree. Confidence in an inference is assessed as the fraction of the ensemble which supports it. Applied to phylogenetic tree estimation, I show that ensembles can confidently resolve topologies with low bootstrap according to standard methods, and conversely that some topologies with high bootstraps are incorrect. Applied to the phylogeny of RNA viruses, ensemble analysis shows that recently adopted taxonomic phyla are probably polyphyletic. Ensemble analysis can improve confidence assessment in any inference from an alignment.


Assuntos
Algoritmos , Evolução Biológica , Filogenia , Alinhamento de Sequência , Homologia de Sequência
6.
Sci Adv ; 8(18): eabm3948, 2022 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-35507661

RESUMO

Broadly HIV-1-neutralizing VRC01-class antibodies bind the CD4-binding site of Env and contain VH1-2*02-derived heavy chains paired with light chains expressing five-amino acid-long CDRL3s. Their unmutated germline forms do not recognize HIV-1 Env, and their lack of elicitation in human clinical trials could be due to the absence of activation of the corresponding naïve B cells by the vaccine immunogens. To address this point, we examined Env-specific B cell receptor sequences from participants in the HVTN 100 clinical trial. Of all the sequences analyzed, only one displayed homology to VRC01-class antibodies, but the corresponding antibody (FH1) recognized the C1C2 gp120 domain. For FH1 to switch epitope recognition to the CD4-binding site, alterations in the CDRH3 and CDRL3 were necessary. Only germ line-targeting Env immunogens efficiently activated VRC01 B cells, even in the presence of FH1 B cells. Our findings support the use of these immunogens to activate VRC01 B cells in humans.


Assuntos
HIV-1 , Vacinas , Anticorpos Neutralizantes , Anticorpos Amplamente Neutralizantes , Anticorpos Anti-HIV/química , Humanos , Homologia de Sequência
7.
Curr Protoc ; 2(5): e449, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35612494

RESUMO

Cross-species exome sequencing approaches provide unprecedented avenues for obtaining genetic diversity, evolutionary relationships, and functional information from a variety of organisms including non-model species. These approaches offer cost-effective opportunities to study multiple individuals or species in parallel, but also create bioinformatics challenges in the application of multiple but powerful bioinformatics tools for the identification of homologous gene families across individual or species boundaries. Popular tools of this kind include SPAdes for sequence assembly, AUGUSTUS for ab initio gene prediction, and BLAST for building homologous sequence families. These tools can also be sophisticated in terms of installation and usage. Here, we present detailed steps on how to run these tools for the recovery and clustering of exon sequences from cross-species raw exome-capture data into homologous sequence families. We also present a utility pipeline, CODSEQCP, that automates these steps to cluster exon sequences, facilitating population genomics and evolutionary studies. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Reads assembly using SPAdes Basic Protocol 2: Coding sequence extraction using AUGUSTUS Basic Protocol 3: Sequence clustering using BLAST Alternate Protocol: How to run CODSEQCP.


Assuntos
Biologia Computacional , Exoma , Análise por Conglomerados , Biologia Computacional/métodos , Exoma/genética , Humanos , Homologia de Sequência
8.
Plant J ; 110(6): 1592-1602, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35365907

RESUMO

The activation of plant immunity is mediated by resistance (R)-gene receptors, also known as nucleotide-binding leucine-rich repeat (NB-LRR) genes, which in turn trigger the authentic defense response. R-gene identification is a crucial goal for both classic and modern plant breeding strategies for disease resistance. The conventional method identifies NB-LRR genes using a protein motif/domain-based search (PDS) within an automatically predicted gene set of the respective genome assembly. PDS proved to be imprecise since repeat masking prior to automatic genome annotation unwittingly prevented comprehensive NB-LRR gene detection. Furthermore, R-genes have diversified in a species-specific manner, so that NB-LRR gene identification cannot be universally standardized. Here, we present the full-length Homology-based R-gene Prediction (HRP) method for the comprehensive identification and annotation of a genome's R-gene repertoire. Our method has substantially addressed the complex genomic organization of tomato (Solanum lycopersicum) NB-LRR gene loci, proving to be more performant than the well-established RenSeq approach. HRP efficiency was also tested on three differently assembled and annotated Beta sp. genomes. Indeed, HRP identified up to 45% more full-length NB-LRR genes compared to previous approaches. HRP also turned out to be a more refined strategy for R-gene allele mining, testified by the identification of hitherto undiscovered Fom-2 homologs in five Cucurbita sp. genomes. In summary, our high-performance method for full-length NB-LRR gene discovery will propel the identification of novel R-genes towards development of improved cultivars.


Assuntos
Genes de Plantas , Solanum lycopersicum , Resistência à Doença/genética , Genes de Plantas/genética , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Melhoramento Vegetal , Doenças das Plantas/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Homologia de Sequência
9.
Sci Rep ; 12(1): 6297, 2022 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-35428787

RESUMO

miRNAs form a class of noncoding RNAs, involved in post-transcriptional regulation of gene expression, broadly studied for their involvement in physiological and pathological context. Inhibition of mature miRNA transcripts, commonly used in miRNA loss-of-function experiments, may not be specific in case of miRNAs with high sequence homology, e.g. miRNAs from the same seed family. Phenotypic effects of miRNA repression might be biased by the repression of highly similar miRNAs. Another challenge is simultaneous inhibition of multiple miRNAs encoded within policistronic clusters, potentially co-regulating common biological processes. To elucidate roles of miRNA clusters and miRNAs with high sequence homology, it is of key importance to selectively repress only the miRNAs of interest. Targeting miRNAs on genomic level with CRISPR/dCas9-based methods is an attractive alternative to blocking mature miRNAs. Yet, so far no clear guidelines on the design of CRISPR inhibition (CRISPRi) experiments, specifically for miRNA repression, have been proposed. To address this need, here we propose a strategy for effective inhibition of miRNAs and miRNA clusters using CRISPRi. We provide clues on how to approach the challenges in using CRISPR/dCas in miRNA studies, which include prediction of miRNA transcription start sites (TSSs) and the design of single guide RNAs (sgRNAs). The strategy implements three TSS prediction online tools, dedicated specifically for miRNAs: miRStart, FANTOM 5 miRNA atlas, DIANA-miRGen, and CRISPOR tool for sgRNAs design; it includes testing and selection of optimal sgRNAs. We demonstrate that compared to siRNA/shRNA-based miRNA silencing, CRISPRi improves the repression specificity for miRNAs with highly similar sequence and contribute to higher uniformity of the effects of silencing the whole miRNA clusters. This strategy may be adapted for CRISPR-mediated activation (CRISPRa) of miRNA expression.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , MicroRNAs , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , MicroRNAs/genética , MicroRNAs/metabolismo , RNA Guia de Cinetoplastídeos/genética , RNA Guia de Cinetoplastídeos/metabolismo , Homologia de Sequência , Sítio de Iniciação de Transcrição
10.
J Immunol ; 208(5): 1128-1138, 2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-35173035

RESUMO

Since the publication of the first chicken genome sequence, we have encountered genes playing key roles in mammalian immunology, but being seemingly absent in birds. One of those was, until recently, Foxp3, the master transcription factor of regulatory T cells in mammals. Therefore, avian regulatory T cell research is still poorly standardized. In this study we identify a chicken ortholog of Foxp3 We prove sequence homology with known mammalian and sauropsid sequences, but also reveal differences in major domains. Expression profiling shows an association of Foxp3 and CD25 expression levels in CD4+CD25+ peripheral T cells and identifies a CD4-CD25+Foxp3high subset of thymic lymphocytes that likely represents yet undescribed avian regulatory T precursor cells. We conclude that Foxp3 is existent in chickens and that it shares certain functional characteristics with its mammalian ortholog. Nevertheless, pathways for regulatory T cell development and Foxp3 function are likely to differ between mammals and birds. The identification and characterization of chicken Foxp3 will help to define avian regulatory T cells and to analyze their functional properties and thereby advance the field of avian immunology.


Assuntos
Galinhas/genética , Galinhas/imunologia , Fatores de Transcrição Forkhead/genética , Linfócitos T Reguladores/imunologia , Sequência de Aminoácidos/genética , Animais , Sequência de Bases , Diferenciação Celular/imunologia , Perfilação da Expressão Gênica , Genoma/genética , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Ativação Linfocitária/imunologia , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência
11.
Int J Mol Sci ; 23(3)2022 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-35163119

RESUMO

Juvenile hormone and ecdysone are key regulators in the metamorphosis and development. Grocho (Gro) is a highly conserved protein required for metamorphosis and development. Brown planthopper (Nilaparvata lugens) is a major pest affecting rice production in China and many Asian countries. Although the molecular function of Gro has been investigated in holometabolous insects such as Aedes aegypti and Drosophila melanogaster, their role in the hemimetabolous insect, brown planthopper, and the relationship between NlGro/NlGro1-L and JH/ecdysone signaling pathway, remained unknown. In this study, NlGroucho (NlGro) and NlGroucho1-like (NlGro1-L) were cloned. An analysis of the predicted protein sequence showed that NlGro has highly conserved Q domain and WD40 domain, and NlGro1-L has a highly conserved WD40 domain. The expression profiles of both genes were studied by quantitative real-time PCR (qRT-PCR). Their relative expressions were high in egg, head, wing, ovary, and testis. NlGro and NlGro1-L were found to interact genetically with juvenile hormone and ecdysone signaling by hormone treatment and RNAi of JH/ecdysone signaling-related genes. Moreover, when NlGro or NlGro1-L was down-regulated alone, the survival rate was decreased, the ovarian development was delayed, and the oviposition was also affected. All defects were aggravated when NlGro and NlGro1-L were down-regulated together. This study will help to develop new pesticides on the basis of the function of NlGro and NlGro1-L, and provide new possibilities for the control of Nilaparvata lugens.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Hemípteros/crescimento & desenvolvimento , Proteínas de Insetos/metabolismo , Hormônios Juvenis/farmacologia , Metamorfose Biológica , Ovário/crescimento & desenvolvimento , Sequência de Aminoácidos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Feminino , Hemípteros/efeitos dos fármacos , Hemípteros/genética , Hemípteros/metabolismo , Proteínas de Insetos/genética , Ovário/efeitos dos fármacos , Ovário/metabolismo , Oviposição , Homologia de Sequência , Asas de Animais/efeitos dos fármacos , Asas de Animais/crescimento & desenvolvimento
12.
ACS Chem Neurosci ; 13(5): 537-539, 2022 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-35152697

RESUMO

Protein aggregation through homotypic interactions is a hallmark of neurodegenerative diseases. Recently, heterotypic amyloid interactions through cross-seeding were found to modify protein aggregation and are reported in the brain of Alzheimer's disease patients. However, whether amyloid-ß (Aß) assembly can be modulated by heterotypic interactions between Aß aggregation-prone regions (APRs) and short homologous segments in unrelated human proteins needs to be elucidated. A recent study revealed that the aggregation kinetics and fibril morphology of Aß is altered by heterotypic interactions between its APRs and homologous segments of unrelated proteins. The data provide novel insights into the structure, origins, and aggregation principles of the amyloid assembly process.


Assuntos
Doença de Alzheimer , Amiloide , Doença de Alzheimer/metabolismo , Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Proteínas Amiloidogênicas/química , Humanos , Cinética , Homologia de Sequência
13.
Talanta ; 240: 123188, 2022 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-34990986

RESUMO

Since novel nutrient sources with high protein content, such as yeast, fungi, bacteria, algae, and insects, are increasingly introduced in the consumer market, safety evaluation studies on their potentially allergenic proteins are required. A pipeline for in silico establishing the sequence-based homology between proteins of spirulina (Arthrospira platensis) and chlorella (Chlorella vulgaris) micro-algae and those included in the AllergenOnline (AO) database (AllergenOnline.org) is described. The extracted proteins were first identified through tryptic peptides analysis by reversed-phase liquid chromatography and high resolution/accuracy Fourier-transform tandem mass spectrometry (RPLC-ESI-FTMS/MS), followed by a quest on the UniProt database. The AO database was subsequently interrogated to assess sequence similarity between identified microalgal proteins and known allergens, based on criteria established by the World Health Organization (WHO) and Food and Agriculture Organization (FAO). A direct search for microalgal proteins already included in allergen databases was also performed using the Allergome database. Six proteins exhibiting a significant homology with food allergens were identified in spirulina extracts. Five of them, i.e., two thioredoxins (D4ZSU6, K1VP15), a superoxide dismutase (C3V3P3), a glyceraldehyde-3-phosphate dehydrogenase (K1W168), and a triosephosphate isomerase (D5A635), resulted from the search on AO. The sixth protein, C-phycocyanin beta subunit (P72508), was directly obtained after examining the Allergome database. Two proteins exhibiting significant sequence homology with food allergens were retrieved in chlorella extracts, viz. calmodulin (A0A2P6TFR8), which is related to troponin c (D7F1Q2), and fructose-bisphosphate aldolase (A0A2P6TDD0). Specific serum screenings based on immunochemical tests should be undertaken to confirm or rule out the allergenicity of the identified proteins.


Assuntos
Chlorella vulgaris , Microalgas , Spirulina , Alérgenos , Proteômica , Homologia de Sequência
14.
Biochem Biophys Res Commun ; 593: 116-121, 2022 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-35063766

RESUMO

Ribosome dimerization is one of the bacterial events that suppresses protein synthesis in the stationary phase. Protein factors responsible for ribosome dimerization in bacteria are well characterized, whereas no information is available for the corresponding factors in archaeal and eukaryotic cells. Here we describe a protein found among the ribosome-associated proteins which dimerizes the 30S ribosomal subunit of the archaeon Pyrococcus furiosus. The ribosome-associated proteins were prepared by high-salt wash of crude ribosomes, and analyzed by nanoflow liquid chromatography-tandem mass spectrometry (nano LC-MS/MS). Of the detected proteins we focused on a protein (PF0560) whose Protein Score was the highest of all of the function-unknown proteins. PF0560 protein had a pronounced effect on the sedimentation pattern of the 30S ribosomal subunit; addition of this protein to isolated 30S subunit reduced the 30S fraction and increased the amount of the 50S fraction. This increase presumably corresponds to the dimer of the 30S subunit. The PF0560-dependent 30S-dimerization, was also observed by gel electrophoretic analysis. This effect was not observed in EDTA-treated 30S subunit, with protein-free 16S rRNA or with bacterial/eukaryotic ribosomal small subunits. Furthermore, PF0560 protein suppressed the formation of functional 70S ribosomes. These results suggest that PF0560 is a novel 30S dimerization factor, which might participate in regulation of archaeal translation.


Assuntos
Proteínas Arqueais/metabolismo , Dimerização , Proteoma/metabolismo , Pyrococcus furiosus/metabolismo , RNA Ribossômico 16S/química , Proteínas Ribossômicas/metabolismo , Ribossomos/química , Sequência de Aminoácidos , Proteínas Arqueais/genética , Magnésio/química , Proteoma/análise , Pyrococcus furiosus/genética , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , Proteínas Ribossômicas/genética , Homologia de Sequência
15.
Mol Biol Rep ; 49(2): 1303-1320, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34807377

RESUMO

BACKGROUND: Transcription elongation is a dynamic and tightly regulated step of gene expression in eukaryotic cells. Eleven nineteen Lysine rich Leukemia (ELL) and ELL Associated Factors (EAF) family of conserved proteins are required for efficient RNA polymerase II-mediated transcription elongation. Orthologs of these proteins have been identified in different organisms, including fission yeast and humans. METHODS AND RESULTS: In the present study, we have examined the sequence, structural and functional conservation between the fission yeast and human ELL and EAF orthologs. Our computational analysis revealed that these proteins share some sequence characteristics, and were predominantly disordered in both organisms. Our functional complementation assays revealed that both human ELL and EAF proteins could complement the lack of ell1+ or eaf1+ in Schizosaccharomyces pombe respectively. Furthermore, our domain mapping experiments demonstrated that both the amino and carboxyl terminal domains of human EAF proteins could functionally complement the S. pombe eaf1 deletion phenotypes. However, only the carboxyl-terminus domain of human ELL was able to partially rescue the phenotypes associated with lack of ell1+ in S. pombe. CONCLUSIONS: Collectively, our work adds ELL-EAF to the increasing list of human-yeast complementation gene pairs, wherein the simpler fission yeast can be used to further enhance our understanding of the role of these proteins in transcription elongation and human disease.


Assuntos
Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismo , Sequência de Aminoácidos/genética , Humanos , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , RNA Polimerase II/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Homologia de Sequência , Fatores de Transcrição/genética , Transcrição Gênica/genética , Transcrição Gênica/fisiologia , Fatores de Elongação da Transcrição/fisiologia
16.
Mol Ecol Resour ; 22(2): 823-833, 2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34407282

RESUMO

Transposable elements (TEs) are significant genomic components which can be detected either through sequence homology against existing databases or de novo, with the latter potentially reducing the risk of underestimating TE abundance. Here, we describe the semi-automated generation of a de novo TE library using the newly developed EDTA pipeline and DeepTE classifier in a non-model teleost (Corydoras fulleri). Using both genomic and transcriptomic data, we assess this de novo pipeline's performance across four TE based metrics: (i) abundance, (ii) composition, (iii) fragmentation, and (iv) age distributions. We then compare the results to those found when using a curated teleost library (Danio rerio). We identify quantitative differences in these metrics and highlight how TE library choice can have major impacts on TE-based estimates in non-model species.


Assuntos
Elementos de DNA Transponíveis , Genômica , Ácido Edético , Homologia de Sequência
17.
Int J Mol Sci ; 22(23)2021 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-34884522

RESUMO

Leishmania parasites are digenetic protists that shuffle between sand fly vectors and mammalian hosts, transforming from flagellated extracellular promastigotes that reside within the intestinal tract of female sand flies to the obligatory intracellular and non-motile amastigotes within mammalian macrophages. Stage differentiation is regulated mainly by post-transcriptional mechanisms, including translation regulation. Leishmania parasites encode six different cap-binding proteins, LeishIF4E1-6, that show poor conservation with their counterparts from higher eukaryotes and among themselves. In view of the changing host milieu encountered throughout their life cycle, we propose that each LeishIF4E has a unique role, although these functions may be difficult to determine. Here we characterize LeishIF4E-6, a unique eIF4E ortholog that does not readily associate with m7GTP cap in either of the tested life forms of the parasite. We discuss the potential effect of substituting two essential tryptophan residues in the cap-binding pocket, expected to be involved in the cap-binding activity, as judged from structural studies in the mammalian eIF4E. LeishIF4E-6 binds to LeishIF4G-5, one of the five eIF4G candidates in Leishmania. However, despite this binding, LeishIF4E-6 does not appear to function as a translation factor. Its episomal overexpression causes a general reduction in the global activity of protein synthesis, which was not observed in the hemizygous deletion mutant generated by CRISPR-Cas9. This genetic profile suggests that LeishIF4E-6 has a repressive role. The interactome of LeishIF4E-6 highlights proteins involved in RNA metabolism such as the P-body marker DHH1, PUF1 and an mRNA-decapping enzyme that is homologous to the TbALPH1.


Assuntos
Fator de Iniciação 4F em Eucariotos/metabolismo , Leishmania/metabolismo , Proteínas de Protozoários/metabolismo , Análogos de Capuz de RNA/genética , Proteínas de Ligação ao Cap de RNA/metabolismo , Sequência de Aminoácidos , Fator de Iniciação 4F em Eucariotos/química , Fator de Iniciação 4F em Eucariotos/genética , Leishmania/genética , Leishmania/crescimento & desenvolvimento , Biossíntese de Proteínas , Conformação Proteica , Proteínas de Protozoários/química , Proteínas de Protozoários/genética , Análogos de Capuz de RNA/metabolismo , Proteínas de Ligação ao Cap de RNA/genética , Homologia de Sequência
18.
Int J Mol Sci ; 22(23)2021 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-34884537

RESUMO

The PIWI-interacting RNA (piRNA) pathway provides an RNA interference (RNAi) mechanism known from Drosophila studies to maintain the integrity of the germline genome by silencing transposable elements (TE). Aedes aegypti mosquitoes, which are the key vectors of several arthropod-borne viruses, exhibit an expanded repertoire of Piwi proteins involved in the piRNA pathway, suggesting functional divergence. Here, we investigate RNA-binding dynamics and subcellular localization of A. aegypti Piwi4 (AePiwi4), a Piwi protein involved in antiviral immunity and embryonic development, to better understand its function. We found that AePiwi4 PAZ (Piwi/Argonaute/Zwille), the domain that binds the 3' ends of piRNAs, bound to mature (3' 2' O-methylated) and unmethylated RNAs with similar micromolar affinities (KD = 1.7 ± 0.8 µM and KD of 5.0 ± 2.2 µM, respectively; p = 0.05) in a sequence independent manner. Through site-directed mutagenesis studies, we identified highly conserved residues involved in RNA binding and found that subtle changes in the amino acids flanking the binding pocket across PAZ proteins have significant impacts on binding behaviors, likely by impacting the protein secondary structure. We also analyzed AePiwi4 subcellular localization in mosquito tissues. We found that the protein is both cytoplasmic and nuclear, and we identified an AePiwi4 nuclear localization signal (NLS) in the N-terminal region of the protein. Taken together, these studies provide insights on the dynamic role of AePiwi4 in RNAi and pave the way for future studies aimed at understanding Piwi interactions with diverse RNA populations.


Assuntos
Proteínas Argonautas/química , Proteínas Argonautas/metabolismo , Núcleo Celular/metabolismo , Elementos de DNA Transponíveis , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , RNA Interferente Pequeno/metabolismo , Aedes , Sequência de Aminoácidos , Animais , Proteínas Argonautas/genética , Núcleo Celular/genética , Proteínas de Insetos/genética , Mosquitos Vetores , Conformação Proteica , RNA Interferente Pequeno/genética , Homologia de Sequência
19.
Proc Natl Acad Sci U S A ; 118(49)2021 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-34873061

RESUMO

Information derived from metagenome sequences through deep-learning techniques has significantly improved the accuracy of template free protein structure modeling. However, most of the deep learning-based modeling studies are based on blind sequence database searches and suffer from low efficiency in computational resource utilization and model construction, especially when the sequence library becomes prohibitively large. We proposed a MetaSource model built on 4.25 billion microbiome sequences from four major biomes (Gut, Lake, Soil, and Fermentor) to decode the inherent linkage of microbial niches with protein homologous families. Large-scale protein family folding experiments on 8,700 unknown Pfam families showed that a microbiome targeted approach with multiple sequence alignment constructed from individual MetaSource biomes requires more than threefold less computer memory and CPU (central processing unit) time but generates contact-map and three-dimensional structure models with a significantly higher accuracy, compared with that using combined metagenome datasets. These results demonstrate an avenue to bridge the gap between the rapidly increasing metagenome databases and the limited computing resources for efficient genome-wide database mining, which provides a useful bluebook to guide future microbiome sequence database and modeling development for high-accuracy protein structure and function prediction.


Assuntos
Microbiota/genética , Alinhamento de Sequência/métodos , Análise de Sequência de Proteína/métodos , Algoritmos , Biologia Computacional/métodos , Bases de Dados de Proteínas , Aprendizado Profundo , Ecossistema , Evolução Molecular , Humanos , Metagenoma/genética , Redes Neurais de Computação , Conformação Proteica , Dobramento de Proteína , Proteínas/química , Homologia de Sequência
20.
Int J Mol Sci ; 22(24)2021 Dec 07.
Artigo em Inglês | MEDLINE | ID: mdl-34947989

RESUMO

In the last few years, microRNA-mediated regulation has been shown to be important in viral infections. In fact, viral microRNAs can alter cell physiology and act on the immune system; moreover, cellular microRNAs can regulate the virus cycle, influencing positively or negatively viral replication. Accordingly, microRNAs can represent diagnostic and prognostic biomarkers of infectious processes and a promising approach for designing targeted therapies. In the past 18 months, the COVID-19 infection from SARS-CoV-2 has engaged many researchers in the search for diagnostic and prognostic markers and the development of therapies. Although some research suggests that the SARS-CoV-2 genome can produce microRNAs and that host microRNAs may be involved in the cellular response to the virus, to date, not enough evidence has been provided. In this paper, using a focused bioinformatic approach exploring the SARS-CoV-2 genome, we propose that SARS-CoV-2 is able to produce microRNAs sharing a strong sequence homology with the human ones and also that human microRNAs may target viral RNA regulating the virus life cycle inside human cells. Interestingly, all viral miRNA sequences and some human miRNA target sites are conserved in more recent SARS-CoV-2 variants of concern (VOCs). Even if experimental evidence will be needed, in silico analysis represents a valuable source of information useful to understand the sophisticated molecular mechanisms of disease and to sustain biomedical applications.


Assuntos
MicroRNAs/genética , SARS-CoV-2/genética , Replicação Viral/genética , COVID-19/genética , Biologia Computacional/métodos , Vírus de DNA/genética , Expressão Gênica/genética , Regulação Viral da Expressão Gênica/genética , Genoma Viral/genética , Interações Hospedeiro-Patógeno/genética , RNA Viral/genética , Homologia de Sequência
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