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1.
Nucleic Acids Res ; 47(18): 9888-9901, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31504775

RESUMO

tRNA-derived RNA fragments (tRFs) have emerged as a new class of functional RNAs implicated in cancer, metabolic and neurological disorders, and viral infection. Yet our understanding of their biogenesis and functions remains limited. In the present study, through analysis of small RNA profile we have identified a distinct set of tRFs derived from pre-tRNA 3' trailers in the hepatocellular carcinoma cell line Huh7. Among those tRFs, tRF_U3_1, which is a 19-nucleotide-long chr10.tRNA2-Ser(TGA)-derived trailer, was expressed most abundantly in both Huh7 and cancerous liver tissues, being present primarily in the cytoplasm. We show that genetic loss of tRF_U3_1 does not affect cell growth and it is not involved in Ago2-mediated gene silencing. Using La/SSB knockout Huh7 cell lines, we demonstrate that this nuclear-cytoplasmic shuttling protein directly binds to the 3' U-tail of tRF_U3_1 and other abundantly expressed trailers and plays a critical role in their stable cytoplasmic accumulation. The pre-tRNA trailer-derived tRFs capable of sequestering the limiting amounts of La/SSB in the cytoplasm rendered cells resistant to various RNA viruses, which usurp La/SSB with RNA chaperone activity for their gene expression. Collectively, our results establish the trailer-derived tRF-La/SSB interface, regulating viral gene expression.


Assuntos
Proliferação de Células/genética , Citoplasma/genética , Precursores de RNA/genética , RNA de Transferência/genética , Linhagem Celular Tumoral , Regulação Viral da Expressão Gênica/genética , Humanos , Chaperonas Moleculares/genética
2.
Plant Sci ; 287: 110203, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31481208

RESUMO

Embryo and endosperm originate from the double fertilization, but they have different developmental fates and biological functions. We identified a previously undescribed maize seed mutant, wherein the embryo appears to be more severely affected than the endosperm (embryo-specific, emb). In the W22 background, the emb embryo arrests at the transition stage whereas its endosperm appears nearly normal in size. At maturity, the embryo in W22-emb is apparently small or even invisible. In contrast, the emb endosperm develops into a relative normal size. We cloned the mutant gene on the Chromosome 7L and designated it emb-7L. This gene is generally expressed, but it has a relatively higher expression level in leaves. Emb-7L encodes a chloroplast-localized P-type pentatricopeptide repeat (PPR) protein, consistent with the severe chloroplast deficiency in emb-7L albino seedling leaves. Full transcriptome analysis of the leaves of WT and emb-7L seedlings reveals that transcription of chloroplast protein-encoding genes are dramatically variable with pre-mRNA intron splicing apparently affected in a tissue-dependent pattern and the chloroplast structure and activity were dramatically affected including chloroplast membrane and photosynthesis machinery component and synthesis of metabolic products (e.g., fatty acids, amino acids, starch).


Assuntos
Proteínas de Plantas/genética , Processamento de RNA , Transcriptoma , Zea mays/genética , Cloroplastos/genética , Cloroplastos/ultraestrutura , Endosperma/embriologia , Endosperma/genética , Endosperma/crescimento & desenvolvimento , Endosperma/ultraestrutura , Regulação da Expressão Gênica de Plantas , Genes de Cloroplastos/genética , Íntrons/genética , Mutação , Fotossíntese , Folhas de Planta/embriologia , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/ultraestrutura , Precursores de RNA/genética , Plântula/embriologia , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/ultraestrutura , Zea mays/embriologia , Zea mays/crescimento & desenvolvimento , Zea mays/ultraestrutura
3.
Nat Commun ; 10(1): 3639, 2019 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-31409787

RESUMO

Human pre-catalytic spliceosomes contain several proteins that associate transiently just prior to spliceosome activation and are absent in yeast, suggesting that this critical step is more complex in higher eukaryotes. We demonstrate via RNAi coupled with RNA-Seq that two of these human-specific proteins, Smu1 and RED, function both as alternative splicing regulators and as general splicing factors and are required predominantly for efficient splicing of short introns. In vitro splicing assays reveal that Smu1 and RED promote spliceosome activation, and are essential for this step when the distance between the pre-mRNA's 5' splice site (SS) and branch site (BS) is sufficiently short. This Smu1-RED requirement can be bypassed when the 5' and 3' regions of short introns are physically separated. Our observations suggest that Smu1 and RED relieve physical constraints arising from a short 5'SS-BS distance, thereby enabling spliceosomes to overcome structural challenges associated with the splicing of short introns.


Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Citocinas/metabolismo , Íntrons , Processamento de RNA , Spliceossomos/metabolismo , Proteínas Cromossômicas não Histona/genética , Citocinas/genética , Humanos , Precursores de RNA/genética , Precursores de RNA/metabolismo , Sítios de Splice de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Spliceossomos/genética
4.
Plant Cell Physiol ; 60(9): 1974-1985, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31368506

RESUMO

Root hairs protruding from epidermal cells increase the surface area for water absorption and nutrient uptake. Various environmental factors including light, oxygen concentration, carbon dioxide concentration, calcium and mycorrhizal associations promote root hair formation in Arabidopsis thaliana. Light regulates the expression of a large number of genes at the transcriptional and post-transcriptional levels; however, there is little information linking the light response to root hair development. In this study, we describe a novel mutant, light-sensitive root-hair development 1 (lrh1), that displays enhanced root hair development in response to light. Hypocotyl and root elongation was inhibited in the lrh1 mutant, which had a late flowering phenotype. We identified the gene encoding the p14 protein, a putative component of the splicing factor 3b complex essential for pre-mRNA splicing, as being responsible for the lrh1 phenotype. Indeed, regulation of alternative splicing was affected in lrh1 mutants and treatment with a splicing inhibitor mimicked the lrh1 phenotype. Genome-wide alterations in pre-mRNA splicing patterns including differential splicing events of light signaling- and circadian clock-related genes were found in lrh1 as well as a difference in transcriptional regulation of multiple genes including upregulation of essential genes for root hair development. These results suggest that pre-mRNA splicing is the key mechanism regulating root hair development in response to light signals.


Assuntos
Processamento Alternativo , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Precursores de RNA/genética , Processamento de RNA , Arabidopsis/crescimento & desenvolvimento , Relógios Circadianos/genética , Hipocótilo/genética , Hipocótilo/crescimento & desenvolvimento , Mutação , Fenótipo , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , RNA de Plantas/genética , Transdução de Sinais
5.
Mol Biol (Mosk) ; 53(3): 524-528, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31184618

RESUMO

Proteins with homo-repeats of more than 4 amino acid residues in length were examined to understand whether some splicing sites in pre-mRNA may be attributed to homo-repeats in human proteins. The human proteome was found to contain a total of 404 proteins with homo-repeats that account for at least one splicing site in pre-mRNA. Pre-mRNA splicing sites were more often found in the C-terminal part (67%) than in the middle orN-terminal part of a homo-repeat. Ten homo-repeats were identified to have two splicing sites per repeat. The repeats were lysine homo-repeats in all but one case.


Assuntos
Proteínas/análise , Proteínas/química , Precursores de RNA/genética , Sítios de Splice de RNA/genética , Sequências Repetitivas de Aminoácidos/genética , Humanos , Lisina/genética , Lisina/metabolismo , Proteínas/genética , Proteoma/análise , Proteoma/química , Proteoma/genética , Processamento de RNA/genética
6.
PLoS Pathog ; 15(6): e1007884, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31206552

RESUMO

In contrast to human cells, very few HSV-1 genes are known to be spliced, although the same pre-mRNA processing machinery is shared. Here, through global analysis of splice junctions in cells infected with HSV-1 and an HSV-1 mutant virus with deletion of infectious cell culture protein 27 (ICP27), one of two viral immediate early (IE) genes essential for viral replication, we identify hundreds of novel alternative splice junctions mapping to both previously known HSV-1 spliced genes and previously unknown spliced genes, the majority of which alter the coding potential of viral genes. Quantitative and qualitative splicing efficiency analysis of these novel alternatively spliced genes based on RNA-Seq and RT-PCR reveals that splicing at these novel splice sites is efficient only when ICP27 is absent; while in wildtype HSV-1 infected cells, the splicing of these novel splice junctions is largely silenced in a gene/sequence specific manner, suggesting that ICP27 not only promotes accumulation of ICP27 targeted transcripts but also ensures correctness of the functional coding sequences through inhibition of alternative splicing. Furthermore, ICP27 toggles expression of ICP34.5, the major viral neurovirulence factor, through inhibition of splicing and activation of a proximal polyadenylation signal (PAS) in the newly identified intron, revealing a novel regulatory mechanism for expression of a viral gene. Thus, through the viral IE protein ICP27, HSV-1 co-opts both splicing and polyadenylation machinery to achieve optimal viral gene expression during lytic infection. On the other hand, during latent infection when ICP27 is absent, HSV-1 likely takes advantages of host splicing machinery to restrict expression of randomly activated antigenic viral genes to achieve immune evasion.


Assuntos
Regulação Viral da Expressão Gênica , Herpesvirus Humano 1/fisiologia , Proteínas Imediatamente Precoces/biossíntese , Poliadenilação , Precursores de RNA/metabolismo , Processamento de RNA , RNA Viral/metabolismo , Latência Viral/fisiologia , Células HEK293 , Humanos , Proteínas Imediatamente Precoces/genética , Precursores de RNA/genética , RNA Viral/genética , Proteínas Virais/biossíntese , Proteínas Virais/genética
7.
RNA ; 25(9): 1164-1176, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31217256

RESUMO

Even though the RNAs contained in the small (40S) and large (60S) ribosomal subunits are cotranscribed, their assembly proceeds largely separately, involving entirely distinct machineries. Nevertheless, separation of the two subunits, an event that is critical for assembly of the small subunit, is delayed until domain I of the large subunit is transcribed, indicating crosstalk between the two assembly pathways. Here we show that this crosstalk is mediated by the assembly factor Rrp5, one of only three proteins required for assembly of both ribosomal subunits. Quantitative RNA binding and cleavage data demonstrate that early on, Rrp5 blocks separation of the two subunits, and thus 40S maturation by inhibiting the access of Rcl1 to promote cleavage of the nascent rRNA. Upon transcription of domain I of 25S rRNA, the 60S assembly factors Noc1/Noc2 bind both this RNA and Rrp5 to change the Rrp5 RNA binding mode to enable pre-40S rRNA processing. Mutants in the HEAT-repeat domain of Noc1 are deficient in the separation of the subunits, which is rescued by overexpression of wild-type but not inactive Rcl1 in vivo. Thus, Rrp5 establishes a checkpoint for 60S assembly during 40S maturation to ensure balanced levels of the two subunits.


Assuntos
Proteínas Nucleares/genética , Proteínas de Ligação a RNA/genética , Subunidades Ribossômicas Maiores de Eucariotos/genética , Subunidades Ribossômicas Menores de Eucariotos/genética , Proteínas de Saccharomyces cerevisiae/genética , Sítios de Ligação/genética , DNA Espaçador Ribossômico/genética , Precursores de RNA/genética , Processamento Pós-Transcricional do RNA/genética , RNA Fúngico/genética , RNA Ribossômico/genética , Proteínas Ribossômicas/genética , Saccharomyces cerevisiae/genética
8.
Cell Biol Int ; 43(8): 954-959, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31070279

RESUMO

Apoptotic chromatin condensation inducer in the nucleus (Acinus) is an RNA-binding protein that has a functional role in inducing apoptotic chromatin condensation and regulating messenger RNA (mRNA) processing. Acinus interacts with the spliceosomal machinery and is a member of the ASAP (apoptosis and splicing-associated protein complex) as well as the EJC (exon junction complex), which gets deposited onto mRNA during splicing. In this study, we have used in vivo splicing assays to characterize the function of Acinus in pre-mRNA splicing more closely. We show that full-length Acinus-S', an isoform of Acinus, does not have a role in modulating splice site selection in human immunodeficiency virus 1 minigene reporter system. In contrast, we observed that the tethering of arginine/serine (RS) and RNPS1-SAP18-binding (RSB) domains of Acinus could regulate the selection of alternative splice sites, thereby revealing the potential of Acinus in stimulating alternative splicing. Altogether, our data suggest that the RS and RSB domains play a critical role in regulating splicing activity via selection of distinct splice sites during pre-mRNA splicing.


Assuntos
Processamento Alternativo , HIV-1/genética , Proteínas Nucleares/fisiologia , Domínios Proteicos , Precursores de RNA/genética , Arginina/metabolismo , Genes Reporter , Células HeLa , Humanos , Isoformas de Proteínas/fisiologia , RNA Mensageiro/genética , Proteínas de Ligação a RNA/fisiologia , Ribonucleoproteínas/metabolismo , Serina/metabolismo
9.
PLoS Genet ; 15(5): e1008157, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-31136569

RESUMO

Most transcriptional activity of exponentially growing cells is carried out by the RNA Polymerase I (Pol I), which produces a ribosomal RNA (rRNA) precursor. In budding yeast, Pol I is a multimeric enzyme with 14 subunits. Among them, Rpa49 forms with Rpa34 a Pol I-specific heterodimer (homologous to PAF53/CAST heterodimer in human Pol I), which might be responsible for the specific functions of the Pol I. Previous studies provided insight in the involvement of Rpa49 in initiation, elongation, docking and releasing of Rrn3, an essential Pol I transcription factor. Here, we took advantage of the spontaneous occurrence of extragenic suppressors of the growth defect of the rpa49 null mutant to better understand the activity of Pol I. Combining genetic approaches, biochemical analysis of rRNA synthesis and investigation of the transcription rate at the individual gene scale, we characterized mutated residues of the Pol I as novel extragenic suppressors of the growth defect caused by the absence of Rpa49. When mapped on the Pol I structure, most of these mutations cluster within the jaw-lobe module, at an interface formed by the lobe in Rpa135 and the jaw made up of regions of Rpa190 and Rpa12. In vivo, the suppressor allele RPA135-F301S restores normal rRNA synthesis and increases Pol I density on rDNA genes when Rpa49 is absent. Growth of the Rpa135-F301S mutant is impaired when combined with exosome mutation rrp6Δ and it massively accumulates pre-rRNA. Moreover, Pol I bearing Rpa135-F301S is a hyper-active RNA polymerase in an in vitro tailed-template assay. We conclude that RNA polymerase I can be engineered to produce more rRNA in vivo and in vitro. We propose that the mutated area undergoes a conformational change that supports the DNA insertion into the cleft of the enzyme resulting in a super-active form of Pol I.


Assuntos
Proteínas Pol1 do Complexo de Iniciação de Transcrição/genética , RNA Polimerase I/genética , DNA Ribossômico/genética , Proteínas Pol1 do Complexo de Iniciação de Transcrição/metabolismo , Precursores de RNA/genética , RNA Ribossômico , Ribossomos/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética , Transcrição Genética
10.
Nucleic Acids Res ; 47(9): 4751-4764, 2019 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-30949699

RESUMO

The DXO family of proteins participates in eukaryotic mRNA 5'-end quality control, removal of non-canonical NAD+ cap and maturation of fungal rRNA precursors. In this work, we characterize the Arabidopsis thaliana DXO homolog, DXO1. We demonstrate that the plant-specific modification within the active site negatively affects 5'-end capping surveillance properties of DXO1, but has only a minor impact on its strong deNADding activity. Unexpectedly, catalytic activity does not contribute to striking morphological and molecular aberrations observed upon DXO1 knockout in plants, which include growth and pigmentation deficiency, global transcriptomic changes and accumulation of RNA quality control siRNAs. Conversely, these phenotypes depend on the plant-specific N-terminal extension of DXO1. Pale-green coloration of DXO1-deficient plants and our RNA-seq data reveal that DXO1 affects chloroplast-localized processes. We propose that DXO1 mediates the connection between RNA turnover and retrograde chloroplast-to-nucleus signaling independently of its deNADding properties.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Cloroplastos/genética , Exorribonucleases/genética , Precursores de RNA/genética , RNA/genética , Arabidopsis/enzimologia , Proteínas de Arabidopsis/química , Proteínas de Cloroplastos/química , Cloroplastos/genética , Exorribonucleases/química , Técnicas de Inativação de Genes , Mutação , NAD/genética , RNA/química , Precursores de RNA/química , Processamento Pós-Transcricional do RNA , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Interferente Pequeno/genética
11.
Methods Mol Biol ; 1933: 33-48, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30945177

RESUMO

Noncoding RNAs perform diverse regulatory functions in living cells. In plants, two RNA polymerase II-related enzymes, RNA polymerases IV and V (Pol IV and V), specialize in the synthesis of noncoding RNAs that silence a subset of transposable elements and genes via RNA-directed DNA methylation (RdDM). In this process, Pol IV partners with RNA-dependent RNA polymerase 2 (RDR2) to produce double-stranded RNAs that are then cut by an RNase III enzyme, Dicer-like 3 (DCL3), into 24 nt small interfering RNAs (siRNAs). The siRNAs are loaded into an Argonaute family protein, primarily AGO4, and guide the complex to complementary DNA target sequences where RdDM and repressive chromatin modifications ensue. The dependence of 24 nt siRNA biogenesis on Pol IV and RDR2 has been known for more than a decade, but the elusive pre-siRNA transcripts synthesized by Pol IV and RDR2 have only recently been identified. This chapter describes the approaches that enabled our identification of Pol IV/RDR2-dependent RNAs (P4R2 RNAs) in Arabidopsis thaliana. These included the use of a triple Dicer mutant (dcl2 dcl3 dcl4) to cause P4R2 RNAs to accumulate, genome-wide identification and mapping of P4R2 RNAs using a modified Illumina small RNA-Seq protocol, and multiple bioinformatic pipelines for data analysis and displaying results.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA Polimerase II/genética , Precursores de RNA/genética , RNA de Plantas/genética , RNA Interferente Pequeno/genética , Proteínas de Arabidopsis/antagonistas & inibidores , Biologia Computacional/métodos , Regulação da Expressão Gênica de Plantas , RNA Polimerase II/antagonistas & inibidores
12.
Int J Mol Sci ; 20(5)2019 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-30813567

RESUMO

A set of tissue-specific splicing factors are thought to govern alternative splicing events during neural progenitor cell (NPC)-to-neuron transition by regulating neuron-specific exons. Here, we propose one such factor, RNA-binding protein Quaking 5 (Qki5), which is specifically expressed in the early embryonic neural stem cells. We performed mRNA-SEQ (Sequence) analysis using mRNAs obtained by developing cerebral cortices in Qk (Quaking) conditional knockout (cKO) mice. As expected, we found a large number of alternative splicing changes between control and conditional knockouts relative to changes in transcript levels. DAVID (The Database for Annotation, Visualization and Integrated Discovery) and Metascape analyses suggested that the affected spliced genes are involved in axon development and microtubule-based processes. Among these, the mRNA coding for the Ninein protein is listed as one of Qki protein-dependent alternative splicing targets. Interestingly, this exon encodes a very long polypeptide (2121 nt), and has been previously defined as a dynamic RNA switch during the NPC-to-neuron transition. Additionally, we validated that the regulation of this large exon is consistent with the Qki5-dependent alternative exon inclusion mode suggested by our previous Qki5 HITS-CLIP (high throughput sequencing-cross linking immunoprecipitation) analysis. Taken together, these data suggest that Qki5 is an important factor for alternative splicing in the NPC-to-neuron transition.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Éxons/genética , Regulação da Expressão Gênica , Células-Tronco Neurais/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA/genética , Processamento Alternativo/genética , Animais , Citoesqueleto/metabolismo , Ontologia Genética , Camundongos Transgênicos , RNA/metabolismo , Precursores de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de RNA
13.
Proc Natl Acad Sci U S A ; 116(14): 6868-6877, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30886105

RESUMO

Arginine methylation is a ubiquitous posttranslational modification that regulates critical cellular processes including signal transduction and pre-mRNA splicing. Here, we report that the tumor-suppressor PTEN is methylated by protein arginine methyltransferase 6 (PRMT6). Mass-spectrometry analysis reveals that PTEN is dimethylated at arginine 159 (R159). We found that PTEN is mutated at R159 in cancers, and the PTEN mutant R159K loses its capability to inhibit the PI3K-AKT cascade. Furthermore, PRMT6 is physically associated with PTEN, promotes asymmetrical dimethylation of PTEN, and regulates the PI3K-AKT cascade through PTEN R159 methylation. In addition, using transcriptome analyses, we found that PTEN R159 methylation is involved in modulation of pre-mRNA alternative splicing. Our results demonstrate that PTEN is functionally regulated by arginine methylation. We propose that PTEN arginine methylation modulates pre-mRNA alternative splicing and influences diverse physiologic processes.


Assuntos
Processamento Alternativo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Precursores de RNA/metabolismo , RNA Neoplásico/metabolismo , Transdução de Sinais , Linhagem Celular Tumoral , Células HEK293 , Humanos , Metilação , Proteínas de Neoplasias/genética , Neoplasias/genética , Proteínas Nucleares/genética , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/genética , Proteína-Arginina N-Metiltransferases/genética , Proteínas Proto-Oncogênicas c-akt/genética , Precursores de RNA/genética , RNA Neoplásico/genética
14.
Planta ; 249(6): 1997-2014, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30904945

RESUMO

MAIN CONCLUSION: The work offers a comprehensive evaluation on the phylogenetics and conservation of splicing patterns of the plant SPF30 splicing factor gene family. In eukaryotes, one pre-mRNA can generate multiple mRNA transcripts by alternative splicing (AS), which expands transcriptome and proteome diversity. Splicing factor 30 (SPF30), also known as survival motor neuron domain containing protein 1 (SMNDC1), is a spliceosomal protein that plays an essential role in spliceosomal assembly. Although SPF30 genes have been well characterised in human and yeast, little is known about their homologues in plants. Here, we report the genome-wide identification and phylogenetic analysis of SPF30 genes in the plant kingdom. In total, 82 SPF30 genes were found in 64 plant species from algae to land plants. Alternative transcripts were found in many SPF30 genes and splicing profile analysis revealed that the second intron in SPF30 genome is frequently associated with AS events and contributed to the birth of novel exons in a few SPF30 members. In addition, different conserved sequences were observed at these putative splice sites among moss, monocots and dicots, respectively. Our findings will facilitate further functional characterization of plant SPF30 genes as putative splicing factors.


Assuntos
Processamento Alternativo/genética , Plantas/genética , Precursores de RNA/genética , Fatores de Processamento de RNA/genética , Evolução Biológica , Sequência Conservada , Éxons/genética , Íntrons/genética , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fatores de Processamento de RNA/metabolismo , RNA Mensageiro/genética , RNA de Plantas/genética , Spliceossomos/genética , Spliceossomos/metabolismo
15.
J Integr Plant Biol ; 61(6): 728-748, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30839161

RESUMO

RNA-binding proteins (RBPs) play an important role in post-transcriptional gene regulation. However, the functions of RBPs in plants remain poorly understood. Maize kernel mutant dek42 has small defective kernels and lethal seedlings. Dek42 was cloned by Mutator tag isolation and further confirmed by an independent mutant allele and clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 materials. Dek42 encodes an RRM_RBM48 type RNA-binding protein that localizes to the nucleus. Dek42 is constitutively expressed in various maize tissues. The dek42 mutation caused a significant reduction in the accumulation of DEK42 protein in mutant kernels. RNA-seq analysis showed that the dek42 mutation significantly disturbed the expression of thousands of genes during maize kernel development. Sequence analysis also showed that the dek42 mutation significantly changed alternative splicing in expressed genes, which were especially enriched for the U12-type intron-retained type. Yeast two-hybrid screening identified SF3a1 as a DEK42-interacting protein. DEK42 also interacts with the spliceosome component U1-70K. These results suggested that DEK42 participates in the regulation of pre-messenger RNA splicing through its interaction with other spliceosome components. This study showed the function of a newly identified RBP and provided insights into alternative splicing regulation during maize kernel development.


Assuntos
Proteínas de Plantas/metabolismo , Precursores de RNA/genética , Processamento de RNA/genética , Proteínas de Ligação a RNA/metabolismo , Sementes/embriologia , Sementes/genética , Zea mays/embriologia , Zea mays/genética , Alelos , Regulação da Expressão Gênica de Plantas , Mutação/genética , Fenótipo , Filogenia , Spliceossomos/metabolismo , Transcriptoma/genética
16.
Nucleic Acids Res ; 47(7): 3353-3364, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30820533

RESUMO

While the number of human miRNA candidates continuously increases, only a few of them are completely characterized and experimentally validated. Toward determining the total number of true miRNAs, we employed a combined in silico high- and experimental low-throughput validation strategy. We collected 28 866 human small RNA sequencing data sets containing 363.7 billion sequencing reads and excluded falsely annotated and low quality data. Our high-throughput analysis identified 65% of 24 127 mature miRNA candidates as likely false-positives. Using northern blotting, we experimentally validated miRBase entries and novel miRNA candidates. By exogenous overexpression of 108 precursors that encode 205 mature miRNAs, we confirmed 68.5% of the miRBase entries with the confirmation rate going up to 94.4% for the high-confidence entries and 18.3% of the novel miRNA candidates. Analyzing endogenous miRNAs, we verified the expression of 8 miRNAs in 12 different human cell lines. In total, we extrapolated 2300 true human mature miRNAs, 1115 of which are currently annotated in miRBase V22. The experimentally validated miRNAs will contribute to revising targetomes hypothesized by utilizing falsely annotated miRNAs.


Assuntos
Simulação por Computador , MicroRNAs/análise , MicroRNAs/genética , Análise de Sequência de RNA , Northern Blotting , Linhagem Celular , Conjuntos de Dados como Assunto , Reações Falso-Positivas , Humanos , MicroRNAs/isolamento & purificação , Anotação de Sequência Molecular , Precursores de RNA/análise , Precursores de RNA/genética , Reprodutibilidade dos Testes
17.
BMC Evol Biol ; 19(1): 55, 2019 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-30764768

RESUMO

BACKGROUND: Micro RNAs (miRNAs), a class of small non-coding RNAs, have been implicated in various aspects of plant development. miR394 is required for shoot apical meristem organization, stem cell maintenance and abiotic stress responses in Arabidopsis, where it functions by negatively regulating the transcript level of target LEAF CURLING RESPONSIVENESS (LCR), which is an F-box protein-coding gene. The evolutionary conservation of stem cell regulatory miR394-LCR module among plants remains elusive. RESULTS: Our study has identified 79 miR394 and 43 target sequences across 40 plant species using various homology based search tools and databases, and analysed their co-evolution pattern. We customised an annotation workflow which computationally validates 20 novel miR394s from 14 plant species. Independent phylogenetic trees were reconstructed with precursor MIR394s, mature miR394s, and their target sequences along with complementary miR394 binding sites. The phylogeny revealed that mature sequences of miR394s as well as their targets belonging to the F-box protein encoding gene families, were highly conserved. Though, miR394-3p were complementary to miR394s/miR394-5p, they clustered separately. CONCLUSION: The existence and separate clustering of miR394-3p and miR394s/miR394-5p indicate their independent regulation. The phylogeny also suggests that miR394s had evolved at the beginning of gymnosperm-angiosperm divergence. Despite strong conservation, some level of sequence variation in miR394s and the complementary binding sites of their targets suggests possible functional diversification of miR394-LCR mediated stem cell regulation in plants.


Assuntos
Evolução Molecular , MicroRNAs/genética , Plantas/genética , RNA de Plantas/genética , Arabidopsis/genética , Sequência de Bases , Sequência Conservada/genética , Regulação da Expressão Gênica de Plantas , Genoma de Planta , MicroRNAs/metabolismo , Anotação de Sequência Molecular , Família Multigênica , Filogenia , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Plantas/metabolismo , Especificidade da Espécie , Estresse Fisiológico/genética
18.
J Vis Exp ; (143)2019 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-30774126

RESUMO

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences is desired. We herein provide a detailed in vitro and in-cell selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) protocol to study the RNA structural change in the presence of an experimental drug for spinal muscular atrophy (SMA), survival of motor neuron (SMN)-C2, and in exon 7 of the pre-mRNA of the SMN2 gene. In in vitro SHAPE, an RNA sequence of 140 nucleotides containing SMN2 exon 7 is transcribed by T7 RNA polymerase, folded in the presence of SMN-C2, and subsequently modified by a mild 2'-OH acylation reagent, 2-methylnicotinic acid imidazolide (NAI). This 2'-OH-NAI adduct is further probed by a 32P-labeled primer extension and resolved by polyacrylamide gel electrophoresis (PAGE). Conversely, 2'-OH acylation in in-cell SHAPE takes place in situ with SMN-C2 bound cellular RNA in living cells. The pre-mRNA sequence of exon 7 in the SMN2 gene, along with SHAPE-induced mutations in the primer extension, was then amplified by PCR and subject to next-generation sequencing. Comparing the two methodologies, in vitro SHAPE is a more cost-effective method and does not require computational power to visualize results. However, the in vitro SHAPE-derived RNA model sometimes deviates from the secondary structure in a cellular context, likely due to the loss of all interactions with RNA-binding proteins. In-cell SHAPE does not need a radioactive material workplace and yields a more accurate RNA secondary structure in the cellular context. Furthermore, in-cell SHAPE is usually applicable for a larger range of RNA sequences (~1,000 nucleotides) by utilizing next-generation sequencing, compared to in vitro SHAPE (~200 nucleotides) that usually relies on PAGE analysis. In case of exon 7 in SMN2 pre-mRNA, the in vitro and in-cell SHAPE derived RNA models are similar to each other.


Assuntos
Precursores de RNA/química , RNA/metabolismo , Acilação , Sequência de Bases , Éxons/genética , Células HEK293 , Humanos , Precursores de RNA/genética , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Transcrição Genética
19.
Yi Chuan ; 41(2): 89-97, 2019 Feb 20.
Artigo em Chinês | MEDLINE | ID: mdl-30803940

RESUMO

Recursive splicing refers to the biological process that long introns are removed in multiple steps during pre-mRNA splicing. In comparison to large introns (>10 kb), most introns in higher eukaryotic genomes are removed in one step during transcription. Previous studies have revealed that recursive splicing events play important roles in many biological processes, including the pathogenesis and development of diseases. In recent years, more researchers have focused on recursive splicing events and found that recursive splicing occurs in Drosophila and many other vertebrates. Multiple recursive splicing sites have been predicted by different bioinformatics methods and verified by experiments. Current researches focus on the process of recursive splicing, recursive splicing site recognition and its influence on biological processes. In this review, we summarize the molecular mechanism of recursive splicing events in eukaryotic genomes and the present development in this field, aiming to lay the basis for further understanding of the mechanisms of RNA splicing.


Assuntos
Eucariotos/genética , Íntrons/genética , Processamento de RNA , Animais , Biologia Computacional , Drosophila , Precursores de RNA/genética
20.
Methods Mol Biol ; 1932: 89-97, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30701493

RESUMO

microRNAs (miRNAs) are short, noncoding regulatory RNAs derived from hairpin precursors (pre-miRNAs). In synergy with experimental approaches, computational approaches have become an invaluable tool for identifying miRNAs at the genome scale. We have recently reported a method called miRLocator, which applies machine learning algorithms to accurately predict the localization of most likely miRNAs within their pre-miRNAs. One major strength of miRLocator is the fact that the machine learning-based miRNA prediction model can be automatically trained using a set of miRNAs of particular interest, with informative features extracted from miRNA-miRNA* duplexes and the optimized ratio between positive and negative samples. Here, we present a detailed protocol for miRLocator that performs the training and prediction processes using a python implementation and web interface. The source codes, web interface, and manual documents are freely available to academic users at https://github.com/cma2015/miRLocator .


Assuntos
Biologia Computacional/métodos , MicroRNAs/genética , Precursores de RNA/genética , Computadores , Genoma/genética , Aprendizado de Máquina , Software
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