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1.
Nat Commun ; 12(1): 4498, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301931

RESUMO

In animal germlines, PIWI proteins and the associated PIWI-interacting RNAs (piRNAs) protect genome integrity by silencing transposons. Here we report the extensive sequence and quantitative correlations between 2',3'-cyclic phosphate-containing RNAs (cP-RNAs), identified using cP-RNA-seq, and piRNAs in the Bombyx germ cell line and mouse testes. The cP-RNAs containing 5'-phosphate (P-cP-RNAs) identified by P-cP-RNA-seq harbor highly consistent 5'-end positions as the piRNAs and are loaded onto PIWI protein, suggesting their direct utilization as piRNA precursors. We identified Bombyx RNase Kappa (BmRNase κ) as a mitochondria-associated endoribonuclease which produces cP-RNAs during piRNA biogenesis. BmRNase κ-depletion elevated transposon levels and disrupted a piRNA-mediated sex determination in Bombyx embryos, indicating the crucial roles of BmRNase κ in piRNA biogenesis and embryonic development. Our results reveal a BmRNase κ-engaged piRNA biogenesis pathway, in which the generation of cP-RNAs promotes robust piRNA production.


Assuntos
Endorribonucleases/genética , Perfilação da Expressão Gênica/métodos , Proteínas de Insetos/genética , RNA Interferente Pequeno/genética , RNA/genética , Animais , Sequência de Bases , Bombyx , Linhagem Celular , Endorribonucleases/metabolismo , Feminino , Proteínas de Insetos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Mutação , Ácidos Fosfatídicos/química , RNA/química , RNA/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , RNA-Seq/métodos , Testículo/metabolismo
2.
Methods ; 2021 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-33962012

RESUMO

RNA cleavages by many ribonucleases generate RNA molecules that contain a 2',3'-cyclic phosphate (cP) at their 3'-termini, and many cP-containing RNAs (cP-RNAs) are expressed as functional molecules in cells and tissues. 5'-tRNA half molecules are representative examples of functional cP-RNAs, playing important roles in various biological processes. We here show in vitro production of cP-containing 5'-tRNA half molecules that is able to prepare abundant synthetic cP-RNAs enough for functional analyses. Furthermore, we report a multiplex TaqMan RT-qPCR method which can simultaneously quantify multiple cP-containing 5'-tRNA half species. The method enabled us to efficiently quantify 5'-tRNA halves using samples with limited amounts, such as human plasma samples, revealing drastic enhancement of 5'-tRNA half levels at approximately 1,000-fold in patients infected with Mycobacterium tuberculosis. These in vitro production and multiplex quantification methods can be applied to any cP-RNAs, and they provide cost-effective, in-house techniques to accelerate expressional and functional characterizations of 5'-tRNA halves and other cP-RNAs.

3.
PLoS Biol ; 18(12): e3000982, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33332353

RESUMO

Toll-like receptors (TLRs) play a crucial role in the innate immune response. Although endosomal TLR7 recognizes single-stranded RNAs, their endogenous RNA ligands have not been fully explored. Here, we report 5'-tRNA half molecules as abundant activators of TLR7. Mycobacterial infection and accompanying surface TLR activation up-regulate the expression of 5'-tRNA half molecules in human monocyte-derived macrophages (HMDMs). The abundant accumulation of 5'-tRNA halves also occur in HMDM-secreted extracellular vehicles (EVs); the abundance of EV-5'-tRNAHisGUG half molecules is >200-fold higher than that of the most abundant EV-microRNA (miRNA). Sequence identification of the 5'-tRNA halves using cP-RNA-seq revealed abundant and selective packaging of specific 5'-tRNA half species into EVs. The EV-5'-tRNAHisGUG half was experimentally demonstrated to be delivered into endosomes in recipient cells and to activate endosomal TLR7. Up-regulation of the 5'-tRNA half molecules was also observed in the plasma of patients infected with Mycobacterium tuberculosis. These results unveil a novel tRNA-engaged pathway in the innate immune response and assign the role of "immune activators" to 5'-tRNA half molecules.


Assuntos
Vesículas Extracelulares/genética , RNA de Transferência de Histidina/metabolismo , Receptor 7 Toll-Like/metabolismo , Endossomos/metabolismo , Vesículas Extracelulares/metabolismo , Regulação da Expressão Gênica/genética , Humanos , Imunidade Inata/genética , Imunidade Inata/fisiologia , Macrófagos/metabolismo , RNA de Transferência/metabolismo , RNA de Transferência de Histidina/genética , RNA de Transferência de Histidina/fisiologia , Células THP-1 , Receptor 7 Toll-Like/fisiologia
4.
RNA Biol ; 17(8): 1060-1069, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32397797

RESUMO

Eukaryotic cells equip robust systems to respond to stress conditions. In stressed mammalian cells, angiogenin endoribonuclease cleaves anticodon-loops of tRNAs to generate tRNA halves termed tRNA-derived stress-induced RNAs (tiRNAs), which promote stress granule formation and regulate translation. The 5'-tiRNAs (5'-tRNA halves) contain a 2',3'-cyclic phosphate (cP) and thus belong to cP-containing RNAs (cP-RNAs). The cP-RNAs form a hidden layer of the transcriptome because standard RNA-seq cannot amplify and sequence them. In this study, we performed genome-wide analyses of short cP-RNA transcriptome in oxidative stress-exposed human cells. Using cP-RNA-seq that can specifically sequence cP-RNAs, we identified tiRNAs and numerous other cP-RNAs that are mainly derived from rRNAs and mRNAs. Although tiRNAs were produced from a wide variety of tRNA species, abundant species of tiRNAs were derived from a focal-specific subset of tRNAs. Regarding rRNA- and mRNA-derived cP-RNAs, determination of the processing sites of substrate RNAs revealed highly specific RNA cleavage events between pyrimidines and adenosine in generation of those cP-RNAs. Those cP-RNAs were derived from specific loci of substrate RNAs rather than from the overall region, implying that cP-RNAs are produced by regulated biogenesis pathways and not by random degradation events. We experimentally confirmed the identified sequences to be expressed as cP-RNAs in the cells, and their expressions were upregulated upon induction of oxidative stress. These analyses of the cP-RNA transcriptome unravel an abundant class of short ncRNAs that accumulate in cells under oxidative stress.

5.
PLoS Genet ; 15(11): e1008469, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31721758

RESUMO

RNA molecules generated by ribonuclease cleavage sometimes harbor a 2',3'-cyclic phosphate (cP) at their 3'-ends. Those cP-containing RNAs (cP-RNAs) form a hidden layer of transcriptome because standard RNA-seq cannot capture them as a result of cP's prevention of an adapter ligation reaction. Here we provide genome-wide analyses of short cP-RNA transcriptome across multiple mouse tissues. Using cP-RNA-seq that can exclusively sequence cP-RNAs, we identified numerous novel cP-RNA species which are mainly derived from cytoplasmic tRNAs, mRNAs, and rRNAs. Determination of the processing sites of substrate RNAs for cP-RNA generation revealed highly-specific RNA cleavage events between cytidine and adenosine in cP-RNA biogenesis. cP-RNAs were not evenly derived from the overall region of substrate RNAs but rather from specific sites, implying that cP-RNAs are not from random degradation but are produced through a regulated biogenesis pathway. The identified cP-RNAs were abundantly accumulated in mouse tissues, and the expression levels of cP-RNAs showed age-dependent reduction. These analyses of cP-RNA transcriptome unravel a novel, abundant class of non-coding RNAs whose expression could have physiological roles.


Assuntos
Envelhecimento/genética , Sequência de Bases/genética , RNA/genética , Transcriptoma/genética , Envelhecimento/patologia , Animais , Regulação da Expressão Gênica/genética , Genômica , Humanos , Camundongos , Fosfatos/química , Fosfatos/metabolismo , RNA/química , Clivagem do RNA/genética , RNA Ribossômico/genética , RNA Nucleolar Pequeno/genética , RNA de Transferência/genética , RNA não Traduzido/genética , Análise de Sequência de RNA
6.
RNA Biol ; 16(12): 1817-1825, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31512554

RESUMO

Post-transcriptional non-template additions of nucleotides to 3'-ends of RNAs play important roles in the stability and function of RNA molecules. Although tRNA nucleotidyltransferase (CCA-adding enzyme) is known to add CCA trinucleotides to 3'-ends of tRNAs, whether other RNA species can be endogenous substrates of CCA-adding enzyme has not been widely explored yet. Herein, we used YAMAT-seq to identify non-tRNA substrates of CCA-adding enzyme. YAMAT-seq captures RNA species that form secondary structures with 4-nt protruding 3'-ends of the sequence 5'-NCCA-3', which is the hallmark structure of RNAs that are generated by CCA-adding enzyme. By executing YAMAT-seq for human breast cancer cells and mining the sequence data, we identified novel candidate substrates of CCA-adding enzyme. These included fourteen 'CCA-RNAs' that only contain CCA as non-genomic sequences, and eleven 'NCCA-RNAs' that contain CCA and other nucleotides as non-genomic sequences. All newly-identified (N)CCA-RNAs were derived from the mitochondrial genome and were localized in mitochondria. Knockdown of CCA-adding enzyme severely reduced the expression levels of (N)CCA-RNAs, suggesting that the CCA-adding enzyme-catalyzed CCA additions stabilize the expression of (N)CCA-RNAs. Furthermore, expression levels of (N)CCA-RNAs were severely reduced by various cellular treatments, including UV irradiation, amino acid starvation, inhibition of mitochondrial respiratory complexes, and inhibition of the cell cycle. These results revealed a novel CCA-mediated regulatory pathway for the expression of mitochondrial non-coding RNAs.


Assuntos
Mitocôndrias/genética , Nucleotidiltransferases/genética , RNA Mitocondrial/genética , RNA de Transferência/genética , Pareamento de Bases , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/genética , Ciclo Celular/efeitos da radiação , Linhagem Celular Tumoral , Biologia Computacional/métodos , Meios de Cultura/química , Meios de Cultura/farmacologia , Células Epiteliais , Genoma Mitocondrial , Células HEK293 , Células HeLa , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Células MCF-7 , Mitocôndrias/metabolismo , Mitocôndrias/efeitos da radiação , Conformação de Ácido Nucleico , Motivos de Nucleotídeos , Nucleotidiltransferases/antagonistas & inibidores , Nucleotidiltransferases/metabolismo , RNA Mitocondrial/química , RNA Mitocondrial/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , RNA de Transferência/química , RNA de Transferência/metabolismo , Raios Ultravioleta
7.
Front Genet ; 9: 562, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30538719

RESUMO

Cellular RNA molecules contain phosphate or hydroxyl ends. A 2',3'-cyclic phosphate (cP) is one of the 3'-terminal forms of RNAs mainly generated from RNA cleavage by ribonucleases. Although transcriptome profiling using RNA-seq has become a ubiquitous tool in biological and medical research, cP-containing RNAs (cP-RNAs) form a hidden transcriptome layer, which is infrequently recognized and characterized, because standard RNA-seq is unable to capture them. Despite cP-RNAs' invisibility in RNA-seq data, increasing evidence indicates that they are not accumulated simply as non-functional degradation products; rather, they have physiological roles in various biological processes, designating them as noteworthy functional molecules. This review summarizes our current knowledge of cP-RNA biogenesis pathways and their catalytic enzymatic activities, discusses how the cP-RNA generation affects biological processes, and explores future directions to further investigate cP-RNA biology.

8.
Methods Mol Biol ; 1680: 65-73, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29030841

RESUMO

Cellular RNAs are often expressed as multiple isoforms of complex heterogeneity in both length and terminal sequences. IsomiRs, the isoforms of microRNAs, are such an example. Distinct quantification of each RNA variant is necessary to unravel the biogenesis mechanism and biological significance of heterogenetic RNA expression. Here we describe Dumbbell-PCR (Db-PCR), a TaqMan RT-PCR-based method that distinctively quantifies a specific small RNA variant with single-nucleotide resolution at terminal sequences. Db-PCR enables the quantitative analysis of RNA terminal heterogeneity without performing Next-Generation Sequencing.


Assuntos
Variação Genética , Reação em Cadeia da Polimerase , Pequeno RNA não Traduzido/genética , MicroRNAs/genética , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de RNA
9.
Nucleic Acids Res ; 46(D1): D152-D159, 2018 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-29186503

RESUMO

MINTbase is a repository that comprises nuclear and mitochondrial tRNA-derived fragments ('tRFs') found in multiple human tissues. The original version of MINTbase comprised tRFs obtained from 768 transcriptomic datasets. We used our deterministic and exhaustive tRF mining pipeline to process all of The Cancer Genome Atlas datasets (TCGA). We identified 23 413 tRFs with abundance of ≥ 1.0 reads-per-million (RPM). To facilitate further studies of tRFs by the community, we just released version 2.0 of MINTbase that contains information about 26 531 distinct human tRFs from 11 719 human datasets as of October 2017. Key new elements include: the ability to filter tRFs on-the-fly by minimum abundance thresholding; the ability to filter tRFs by tissue keywords; easy access to information about a tRF's maximum abundance and the datasets that contain it; the ability to generate relative abundance plots for tRFs across cancer types and convert them into embeddable figures; MODOMICS information about modifications of the parental tRNA, etc. Version 2.0 of MINTbase contains 15x more datasets and nearly 4x more distinct tRFs than the original version, yet continues to offer fast, interactive access to its contents. Version 2.0 is available freely at http://cm.jefferson.edu/MINTbase/.


Assuntos
Bases de Dados de Ácidos Nucleicos , Neoplasias/genética , RNA de Transferência/genética , Genoma Humano , Humanos , RNA Mitocondrial/genética , RNA Neoplásico/genética , RNA Nuclear/genética , Interface Usuário-Computador
10.
Sci Rep ; 7(1): 4110, 2017 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-28646211

RESUMO

Piwi proteins and their bound Piwi-interacting RNAs (piRNAs) are predominantly expressed in the germline and play crucial roles in germline development by silencing transposons and other targets. Bombyx mori BmN4 cells are culturable germ cells that equip the piRNA pathway. Because of the scarcity of piRNA-expressing culturable cells, BmN4 cells are being utilized for the analyses of piRNA biogenesis. We here report that the piRNA biogenesis in BmN4 cells is regulated by cell density. As cell density increased, the abundance of Piwi proteins and piRNA biogenesis factors was commonly upregulated, resulting in an increased number of perinuclear nuage-like granules where Piwi proteins localize. Along with these phenomena, the abundance of mature piRNAs also globally increased, whereas levels of long piRNA precursor and transposons decreased, suggesting that increasing cell density promotes piRNA biogenesis pathway and that the resultant accumulation of mature piRNAs is functionally significant for transposon silencing. Our study reveals a previously uncharacterized link between cell density and piRNA biogenesis, designates cell density as a critical variable in piRNA studies using BmN4 cell system, and suggests the alteration of cell density as a useful tool to monitor piRNA biogenesis and function.


Assuntos
Bombyx/genética , Células Germinativas/metabolismo , RNA Interferente Pequeno/genética , Animais , Contagem de Células , Linhagem Celular , Células Cultivadas , Biologia Computacional , Grânulos Citoplasmáticos/metabolismo , Elementos de DNA Transponíveis , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo
11.
Nucleic Acids Res ; 45(9): e70, 2017 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-28108659

RESUMO

Besides translation, transfer RNAs (tRNAs) play many non-canonical roles in various biological pathways and exhibit highly variable expression profiles. To unravel the emerging complexities of tRNA biology and molecular mechanisms underlying them, an efficient tRNA sequencing method is required. However, the rigid structure of tRNA has been presenting a challenge to the development of such methods. We report the development of Y-shaped Adapter-ligated MAture TRNA sequencing (YAMAT-seq), an efficient and convenient method for high-throughput sequencing of mature tRNAs. YAMAT-seq circumvents the issue of inefficient adapter ligation, a characteristic of conventional RNA sequencing methods for mature tRNAs, by employing the efficient and specific ligation of Y-shaped adapter to mature tRNAs using T4 RNA Ligase 2. Subsequent cDNA amplification and next-generation sequencing successfully yield numerous mature tRNA sequences. YAMAT-seq has high specificity for mature tRNAs and high sensitivity to detect most isoacceptors from minute amount of total RNA. Moreover, YAMAT-seq shows quantitative capability to estimate expression levels of mature tRNAs, and has high reproducibility and broad applicability for various cell lines. YAMAT-seq thus provides high-throughput technique for identifying tRNA profiles and their regulations in various transcriptomes, which could play important regulatory roles in translation and other biological processes.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA de Transferência/química , Análise de Sequência de RNA/métodos , Linhagem Celular Tumoral , Biologia Computacional , DNA Complementar , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
12.
RNA ; 23(2): 161-168, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27879434

RESUMO

Transfer RNAs (tRNAs) are fundamental adapter components of translational machinery. tRNAs can further serve as a source of tRNA-derived noncoding RNAs that play important roles in various biological processes beyond translation. Among all species of tRNAs, tRNAHisGUG has been known to uniquely contain an additional guanosine residue at the -1 position (G-1) of its 5'-end. To analyze this -1 nucleotide in detail, we developed a TaqMan qRT-PCR method that can distinctively quantify human mature cytoplasmic tRNAHisGUG containing G-1, U-1, A-1, or C-1 or lacking the -1 nucleotide (starting from G1). Application of this method to the mature tRNA fraction of BT-474 breast cancer cells revealed the presence of tRNAHisGUG containing U-1 as well as the one containing G-1 Moreover, tRNA lacking the -1 nucleotide was also detected, thus indicating the heterogeneous expression of 5'-tRNAHisGUG variants. A sequence library of sex hormone-induced 5'-tRNA halves (5'-SHOT-RNAs), identified via cP-RNA-seq of a BT-474 small RNA fraction, also demonstrated the expression of 5'-tRNAHisGUG halves containing G-1, U-1, or G1 as 5'-terminal nucleotides. Although the detected 5'-nucleotide species were identical, the relative abundances differed widely between mature tRNA and 5'-half from the same BT-474 cells. The majority of mature tRNAs contained the -1 nucleotide, whereas the majority of 5'-halves lacked this nucleotide, which was biochemically confirmed using a primer extension assay. These results reveal the novel identities of tRNAHisGUG molecules and provide insights into tRNAHisGUG maturation and the regulation of tRNA half production.


Assuntos
Anticódon/química , Células Epiteliais/metabolismo , Variação Genética , Nucleotídeos/química , RNA de Transferência de Histidina/química , Anticódon/metabolismo , Pareamento de Bases , Linhagem Celular Tumoral , Células Epiteliais/citologia , Humanos , Sequências Repetidas Invertidas , Conformação de Ácido Nucleico , Nucleotídeos/metabolismo , Reação em Cadeia da Polimerase/métodos , RNA de Transferência de Histidina/genética , RNA de Transferência de Histidina/metabolismo , Taq Polimerase/genética , Taq Polimerase/metabolismo
13.
RNA Biol ; 13(5): 477-85, 2016 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-26950678

RESUMO

We report a Caucasian boy with intractable epilepsy and global developmental delay. Whole-exome sequencing identified the likely genetic etiology as a novel p.K212E mutation in the X-linked gene HSD17B10 for mitochondrial short-chain dehydrogenase/reductase SDR5C1. Mutations in HSD17B10 cause the HSD10 disease, traditionally classified as a metabolic disorder due to the role of SDR5C1 in fatty and amino acid metabolism. However, SDR5C1 is also an essential subunit of human mitochondrial RNase P, the enzyme responsible for 5'-processing and methylation of purine-9 of mitochondrial tRNAs. Here we show that the p.K212E mutation impairs the SDR5C1-dependent mitochondrial RNase P activities, and suggest that the pathogenicity of p.K212E is due to a general mitochondrial dysfunction caused by reduction in SDR5C1-dependent maturation of mitochondrial tRNAs.


Assuntos
3-Hidroxiacil-CoA Desidrogenases/genética , Deficiências do Desenvolvimento/genética , Epilepsia Resistente a Medicamentos/genética , Mutação , Ribonuclease P/metabolismo , Análise de Sequência de DNA/métodos , Criança , Exoma , Genes Ligados ao Cromossomo X , Humanos , Masculino , Mitocôndrias/genética , Mitocôndrias/metabolismo , RNA de Transferência/metabolismo
14.
Gene Regul Syst Bio ; 9: 27-33, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26401098

RESUMO

The advent of next-generation sequencing technologies has not only accelerated findings on various novel non-coding RNA (ncRNA) species but also led to the revision of the biological significance and versatility of fundamental RNA species with canonical function, such as transfer RNAs (tRNAs). Although tRNAs are best known as adapter components of translational machinery, recent studies suggest that tRNAs are not always end products but can further serve as a source for short ncRNAs. In many organisms, various tRNA-derived ncRNA species are produced from mature tRNAs or their precursor transcripts as functional molecules involved in various biological processes beyond translation. In this review, we focus on the tRNA-derived ncRNAs associated with Argonaute proteins and summarize recent studies on their conceivable biogenesis factors and on their emerging roles in gene expression regulation as regulatory RNAs.

15.
Proc Natl Acad Sci U S A ; 112(29): E3816-25, 2015 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-26124144

RESUMO

Sex hormones and their receptors play critical roles in the development and progression of the breast and prostate cancers. Here we report that a novel type of transfer RNA (tRNA)-derived small RNA, termed Sex HOrmone-dependent TRNA-derived RNAs (SHOT-RNAs), are specifically and abundantly expressed in estrogen receptor (ER)-positive breast cancer and androgen receptor (AR)-positive prostate cancer cell lines. SHOT-RNAs are not abundantly present in ER(-) breast cancer, AR(-) prostate cancer, or other examined cancer cell lines from other tissues. ER-dependent accumulation of SHOT-RNAs is not limited to a cell culture system, but it also occurs in luminal-type breast cancer patient tissues. SHOT-RNAs are produced from aminoacylated mature tRNAs by angiogenin-mediated anticodon cleavage, which is promoted by sex hormones and their receptors. Resultant 5'- and 3'-SHOT-RNAs, corresponding to 5'- and 3'-tRNA halves, bear a cyclic phosphate (cP) and an amino acid at the 3'-end, respectively. By devising a "cP-RNA-seq" method that is able to exclusively amplify and sequence cP-containing RNAs, we identified the complete repertoire of 5'-SHOT-RNAs. Furthermore, 5'-SHOT-RNA, but not 3'-SHOT-RNA, has significant functional involvement in cell proliferation. These results have unveiled a novel tRNA-engaged pathway in tumorigenesis of hormone-dependent cancers and implicate SHOT-RNAs as potential candidates for biomarkers and therapeutic targets.


Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Hormônios Esteroides Gonadais/metabolismo , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , RNA de Transferência/metabolismo , Aminoácidos/metabolismo , Animais , Sequência de Bases , Bombyx , Linhagem Celular Tumoral , Proliferação de Células , Células Epiteliais/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Humanos , Hidroxilação , Masculino , Modelos Biológicos , Dados de Sequência Molecular , Fosfatos , RNA de Transferência/química , RNA de Transferência/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptores Androgênicos/metabolismo , Receptores de Estrogênio/metabolismo , Ribonuclease Pancreático/metabolismo , Análise de Sequência de RNA
16.
Nat Struct Mol Biol ; 22(6): 485-91, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25984970

RESUMO

The human APOBEC3G (A3G) DNA cytosine deaminase restricts and hypermutates DNA-based parasites including HIV-1. The viral infectivity factor (Vif) prevents restriction by triggering A3G degradation. Although the structure of the A3G catalytic domain is known, the structure of the N-terminal Vif-binding domain has proven more elusive. Here, we used evolution- and structure-guided mutagenesis to solubilize the Vif-binding domain of A3G, thus permitting structural determination by NMR spectroscopy. A smaller zinc-coordinating pocket and altered helical packing distinguish the structure from previous catalytic-domain structures and help to explain the reported inactivity of this domain. This soluble A3G N-terminal domain is bound by Vif; this enabled mutagenesis and biochemical experiments, which identified a unique Vif-interacting surface formed by the α1-ß1, ß2-α2 and ß4-α4 loops. This structure sheds new light on the Vif-A3G interaction and provides critical information for future drug development.


Assuntos
Citidina Desaminase/química , Citidina Desaminase/metabolismo , Produtos do Gene vif do Vírus da Imunodeficiência Humana/metabolismo , Desaminase APOBEC-3G , Citidina Desaminase/genética , Análise Mutacional de DNA , Humanos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Ligação Proteica , Conformação Proteica , Mapeamento de Interação de Proteínas
17.
RNA Biol ; 12(5): 501-8, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25833336

RESUMO

Transfer RNAs (tRNAs) play a central role in translation and also recently appear to have a variety of other functions in biological processes beyond translation. Here we report the development of Four-Leaf clover qRT-PCR (FL-PCR), a convenient PCR-based method, which can specifically quantify individual mature tRNA species. In FL-PCR, T4 RNA ligase 2 specifically ligates a stem-loop adapter to mature tRNAs but not to precursor tRNAs or tRNA fragments. Subsequent TaqMan qRT-PCR amplifies only unmodified regions of the tRNA-adapter ligation products; therefore, FL-PCR quantification is not influenced by tRNA post-transcriptional modifications. FL-PCR has broad applicability for the quantification of various tRNAs in different cell types, and thus provides a much-needed simple method for analyzing tRNA abundance and heterogeneity.


Assuntos
Conformação de Ácido Nucleico , RNA de Transferência/química , RNA de Transferência/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sequência de Bases , Linhagem Celular Tumoral , Humanos , Dados de Sequência Molecular
18.
Am J Hum Genet ; 96(4): 675-81, 2015 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-25817015

RESUMO

Mutations in genes encoding aminoacyl-tRNA synthetases are known to cause leukodystrophies and genetic leukoencephalopathies-heritable disorders that result in white matter abnormalities in the central nervous system. Here we report three individuals (two siblings and an unrelated individual) with severe infantile epileptic encephalopathy, clubfoot, absent deep tendon reflexes, extrapyramidal symptoms, and persistently deficient myelination on MRI. Analysis by whole exome sequencing identified mutations in the nuclear-encoded alanyl-tRNA synthetase (AARS) in these two unrelated families: the two affected siblings are compound heterozygous for p.Lys81Thr and p.Arg751Gly AARS, and the single affected child is homozygous for p.Arg751Gly AARS. The two identified mutations were found to result in a significant reduction in function. Mutations in AARS were previously associated with an autosomal-dominant inherited form of axonal neuropathy, Charcot-Marie-Tooth disease type 2N (CMT2N). The autosomal-recessive AARS mutations identified in the individuals described here, however, cause a severe infantile epileptic encephalopathy with a central myelin defect and peripheral neuropathy, demonstrating that defects of alanyl-tRNA charging can result in a wide spectrum of disease manifestations.


Assuntos
Anormalidades Múltiplas/genética , Alanina-tRNA Ligase/genética , Epilepsia/genética , Modelos Moleculares , Bainha de Mielina/patologia , Doenças do Sistema Nervoso Periférico/genética , Fenótipo , Anormalidades Múltiplas/patologia , Alanina-tRNA Ligase/química , Sequência de Aminoácidos , Sequência de Bases , Epilepsia/patologia , Genes Recessivos/genética , Humanos , Lactente , Recém-Nascido , Dados de Sequência Molecular , Mutação/genética , Doenças do Sistema Nervoso Periférico/patologia , Estudos Prospectivos , Análise de Sequência de DNA , Síndrome , Estados Unidos
19.
Artigo em Inglês | MEDLINE | ID: mdl-26389128

RESUMO

Since their discovery in the 1950s, transfer RNAs (tRNAs) have been best known as adapter molecules that play a central role in translating genetic information. However, recent biochemical and bioinformatic evidence has led to a previously unexpected conceptual consensus that tRNAs are not always end products; they further serve as a source of small functional RNAs. In many organisms, specific tRNA fragments are produced from mature tRNAs or their precursor transcripts not as random degradation products, but as functional molecules involved in many biological processes beyond translation. In this review, we summarize recent studies of tRNA fragments that have provided new insights into tRNA biology by examining the molecular functions of tRNA fragments and proteins with which they interact.

20.
PLoS One ; 8(9): e75512, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24069426

RESUMO

The killer yeast species Pichiaacaciae produces a heteromeric killer protein, PaT, that causes DNA damage and arrests the cell cycle of sensitive Saccharomyces cerevisiae in the S phase. However, the mechanism by which DNA damage occurs remains elusive. A previous study has indicated that Orf2p, a subunit of PaT, specifically cleaves an anticodon loop of an S. cerevisiae transfer RNA (tRNA(Gln)mcm5s2UUG). This finding raised a question about whether the DNA damage is a result of the tRNA cleavage or whether Orf2p directly associates with and cleaves the genomic DNA of sensitive yeast cells. We showed that Orf2p cleaves genomic DNA in addition to cleaving tRNA in vitro. This DNA cleavage requires the same Orf2p residue as that needed for tRNA cleavage, His299. The expression of Orf2p, in which His299 was substituted to alanine, abolished the cell cycle arrest of the host cell. Moreover, the translation impairment induced by tRNA cleavage enabled Orf2p to enter the nucleus, thereby inducing histone phosphorylation.


Assuntos
Clivagem do DNA , Fatores Matadores de Levedura/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , Pontos de Checagem do Ciclo Celular , Núcleo Celular/metabolismo , Dano ao DNA , Histonas/metabolismo , Fosforilação , Biossíntese de Proteínas , Transporte Proteico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo
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