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1.
RNA ; 27(4): 477-495, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33446492

RESUMO

tRNA-derived fragments (tRFs) have recently gained a lot of scientific interest due to their diverse regulatory roles in several cellular processes. However, their function in dynamic biological processes such as development and regeneration remains unexplored. Here, we show that tRFs are dynamically expressed during planarian regeneration, suggesting a possible role for these small RNAs in the regulation of regeneration. In order to characterize planarian tRFs, we first annotated 457 tRNAs in S. mediterranea combining two tRNA prediction algorithms. Annotation of tRNAs facilitated the identification of three main species of tRFs in planarians-the shorter tRF-5s and itRFs, and the abundantly expressed 5'-tsRNAs. Spatial profiling of tRFs in sequential transverse sections of planarians revealed diverse expression patterns of these small RNAs, including those that are enriched in the head and pharyngeal regions. Expression analysis of these tRF species revealed dynamic expression of these small RNAs over the course of regeneration suggesting an important role in planarian anterior and posterior regeneration. Finally, we show that 5'-tsRNA in planaria interact with all three SMEDWI proteins and an involvement of AGO1 in the processing of itRFs. In summary, our findings implicate a novel role for tRFs in planarian regeneration, highlighting their importance in regulating complex systemic processes. Our study adds to the catalog of posttranscriptional regulatory systems in planaria, providing valuable insights on the biogenesis and the function of tRFs in neoblasts and planarian regeneration.


Assuntos
Proteínas Argonautas/genética , Proteínas de Helminto/genética , Planárias/genética , RNA de Helmintos/genética , Pequeno RNA não Traduzido/genética , RNA de Transferência/genética , Regeneração/genética , Algoritmos , Animais , Proteínas Argonautas/metabolismo , Pareamento de Bases , Sequência de Bases , Regulação da Expressão Gênica , Proteínas de Helminto/metabolismo , Anotação de Sequência Molecular , Conformação de Ácido Nucleico , Planárias/metabolismo , RNA de Helmintos/química , RNA de Helmintos/classificação , RNA de Helmintos/metabolismo , Pequeno RNA não Traduzido/química , Pequeno RNA não Traduzido/classificação , Pequeno RNA não Traduzido/metabolismo , RNA de Transferência/química , RNA de Transferência/classificação , RNA de Transferência/metabolismo
2.
Nucleic Acids Res ; 47(16): 8720-8733, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31276587

RESUMO

Expression of human mitochondrial DNA is indispensable for proper function of the oxidative phosphorylation machinery. The mitochondrial genome encodes 22 tRNAs, 2 rRNAs and 11 mRNAs and their post-transcriptional modification constitutes one of the key regulatory steps during mitochondrial gene expression. Cytosine-5 methylation (m5C) has been detected in mitochondrial transcriptome, however its biogenesis has not been investigated in details. Mammalian NOP2/Sun RNA Methyltransferase Family Member 2 (NSUN2) has been characterized as an RNA methyltransferase introducing m5C in nuclear-encoded tRNAs, mRNAs and microRNAs and associated with cell proliferation and differentiation, with pathogenic variants in NSUN2 being linked to neurodevelopmental disorders. Here we employ spatially restricted proximity labelling and immunodetection to demonstrate that NSUN2 is imported into the matrix of mammalian mitochondria. Using three genetic models for NSUN2 inactivation-knockout mice, patient-derived fibroblasts and CRISPR/Cas9 knockout in human cells-we show that NSUN2 is necessary for the generation of m5C at positions 48, 49 and 50 of several mammalian mitochondrial tRNAs. Finally, we show that inactivation of NSUN2 does not have a profound effect on mitochondrial tRNA stability and oxidative phosphorylation in differentiated cells. We discuss the importance of the newly discovered function of NSUN2 in the context of human disease.


Assuntos
5-Metilcitosina/metabolismo , Eczema/genética , Transtornos do Crescimento/genética , Deficiência Intelectual/genética , Metiltransferases/genética , Microcefalia/genética , Processamento Pós-Transcricional do RNA , RNA Mitocondrial/genética , RNA de Transferência/genética , Animais , Sistemas CRISPR-Cas , Eczema/metabolismo , Eczema/patologia , Fácies , Fibroblastos/metabolismo , Fibroblastos/patologia , Edição de Genes , Técnicas de Inativação de Genes , Transtornos do Crescimento/metabolismo , Transtornos do Crescimento/patologia , Células HEK293 , Humanos , Deficiência Intelectual/metabolismo , Deficiência Intelectual/patologia , Metilação , Metiltransferases/deficiência , Camundongos , Camundongos Knockout , Microcefalia/metabolismo , Microcefalia/patologia , Mitocôndrias/genética , Mitocôndrias/metabolismo , Conformação de Ácido Nucleico , Fosforilação Oxidativa , Cultura Primária de Células , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Mitocondrial/metabolismo , RNA de Transferência/metabolismo
3.
Development ; 144(18): 3211-3223, 2017 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-28807895

RESUMO

Brain regeneration in planarians is mediated by precise spatiotemporal control of gene expression and is crucial for multiple aspects of neurogenesis. However, the mechanisms underpinning the gene regulation essential for brain regeneration are largely unknown. Here, we investigated the role of the miR-124 family of microRNAs in planarian brain regeneration. The miR-124 family (miR-124) is highly conserved in animals and regulates neurogenesis by facilitating neural differentiation, yet its role in neural wiring and brain organization is not known. We developed a novel method for delivering anti-miRs using liposomes for the functional knockdown of microRNAs. Smed-miR-124 knockdown revealed a key role for these microRNAs in neuronal organization during planarian brain regeneration. Our results also demonstrated an essential role for miR-124 in the generation of eye progenitors. Additionally, miR-124 regulates Smed-slit-1, which encodes an axon guidance protein, either by targeting slit-1 mRNA or, potentially, by modulating the canonical Notch pathway. Together, our results reveal a role for miR-124 in regulating the regeneration of a functional brain and visual system.


Assuntos
Encéfalo/fisiologia , MicroRNAs/metabolismo , Planárias/genética , Planárias/fisiologia , Regeneração , Vias Visuais/fisiologia , Animais , Fenômenos Biofísicos , Gânglios dos Invertebrados/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Lipossomos/química , Fusão de Membrana , MicroRNAs/genética , Modelos Biológicos , Neurônios/metabolismo , Penetrância , Fenótipo , Receptores Notch/metabolismo , Reprodutibilidade dos Testes , Transdução de Sinais , Vírus/metabolismo
4.
Development ; 144(17): 3066-3079, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28807897

RESUMO

Identifying key cellular events that facilitate stem cell function and tissue organization is crucial for understanding the process of regeneration. Planarians are powerful model system to study regeneration and stem cell (neoblast) function. Here, using planaria, we show that the initial events of regeneration, such as epithelialization and epidermal organization are critically regulated by a novel cytoplasmic poly A-binding protein, SMED-PABPC2. Knockdown of smed-pabpc2 leads to defects in epidermal lineage specification, disorganization of epidermis and ECM, and deregulated wound healing, resulting in the selective failure of neoblast proliferation near the wound region. Polysome profiling suggests that epidermal lineage transcripts, including zfp-1, are translationally regulated by SMED-PABPC2. Together, our results uncover a novel role for SMED-PABPC2 in the maintenance of epidermal and ECM integrity, critical for wound healing and subsequent processes for regeneration.


Assuntos
Citoplasma/metabolismo , Epiderme/metabolismo , Planárias/metabolismo , Proteína I de Ligação a Poli(A)/metabolismo , Animais , Linhagem da Célula , Proliferação de Células , Epitélio/metabolismo , Matriz Extracelular/metabolismo , Técnicas de Silenciamento de Genes , Homeostase , Modelos Biológicos , Planárias/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Regeneração , Cicatrização
5.
G3 (Bethesda) ; 6(10): 3035-3048, 2016 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-27489207

RESUMO

In eukaryotes, 3' untranslated regions (UTRs) play important roles in regulating posttranscriptional gene expression. The 3'UTR is defined by regulated cleavage/polyadenylation of the pre-mRNA. The advent of next-generation sequencing technology has now enabled us to identify these events on a genome-wide scale. In this study, we used poly(A)-position profiling by sequencing (3P-Seq) to capture all poly(A) sites across the genome of the freshwater planarian, Schmidtea mediterranea, an ideal model system for exploring the process of regeneration and stem cell function. We identified the 3'UTRs for ∼14,000 transcripts and thus improved the existing gene annotations. We found 97 transcripts, which are polyadenylated within an internal exon, resulting in the shrinking of the ORF and loss of a predicted protein domain. Around 40% of the transcripts in planaria were alternatively polyadenylated (ApA), resulting either in an altered 3'UTR or a change in coding sequence. We identified specific ApA transcript isoforms that were subjected to miRNA mediated gene regulation using degradome sequencing. In this study, we also confirmed a tissue-specific expression pattern for alternate polyadenylated transcripts. The insights from this study highlight the potential role of ApA in regulating the gene expression essential for planarian regeneration.


Assuntos
Genoma Helmíntico , Estudo de Associação Genômica Ampla , Platelmintos/genética , Poliadenilação , RNA Mensageiro/genética , Regiões 3' não Traduzidas , Animais , Biologia Computacional/métodos , Sequenciamento de Nucleotídeos em Larga Escala , MicroRNAs/genética , Anotação de Sequência Molecular , Poli A , Interferência de RNA , Processamento Pós-Transcricional do RNA , Reprodutibilidade dos Testes
6.
Sci Rep ; 4: 7357, 2014 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-25483805

RESUMO

Hedgehog (Hh) is a secreted morphogen that elicits differentiation and patterning in developing tissues. Multiple proposed mechanisms to regulate Hh dispersion includes lipoprotein particles and exosomes. Here we report that vertebrate Sonic Hedgehog (Shh) is secreted on two types of extracellular-vesicles/exosomes, from human cell lines and primary chick notochord cells. Although largely overlapping in size as estimated from electron micrographs, the two exosomal fractions exhibited distinct protein and RNA composition. We have probed the functional properties of these vesicles using cell-based assays of Hh-elicited gene expression. Our results suggest that while both Shh-containing exo-vesicular fractions can activate an ectopic Gli-luciferase construct, only exosomes co-expressing Integrins can activate endogenous Shh target genes HNF3ß and Olig2 during the differentiation of mouse ES cells to ventral neuronal progenitors. Taken together, our results demonstrate that primary vertebrate cells secrete Shh in distinct vesicular forms, and support a model where packaging of Shh along with other signaling proteins such as Integrins on exosomes modulates target gene activation. The existence of distinct classes of Shh-containing exosomes also suggests a previously unappreciated complexity for fine-tuning of Shh-mediated gradients and pattern formation.


Assuntos
Exossomos/metabolismo , Proteínas Hedgehog/metabolismo , Transdução de Sinais , Animais , Embrião de Galinha , Exossomos/genética , Espaço Extracelular , Células HEK293 , Humanos , MicroRNAs/metabolismo , Modelos Biológicos , Transporte Proteico , Vertebrados
7.
RNA ; 19(10): 1394-404, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23974438

RESUMO

In recent years, the planarian Schmidtea mediterranea has emerged as a tractable model system to study stem cell biology and regeneration. MicroRNAs are small RNA species that control gene expression by modulating translational repression and mRNA stability and have been implicated in the regulation of various cellular processes. Though recent studies have identified several miRNAs in S. mediterranea, their expression in neoblast subpopulations and during regeneration has not been examined. Here, we identify several miRNAs whose expression is enriched in different neoblast subpopulations and in regenerating tissue at different time points in S. mediterranea. Some of these miRNAs were enriched within 3 h post-amputation and may, therefore, play a role in wound healing and/or neoblast migration. Our results also revealed miRNAs, such as sme-miR-2d-3p and the sme-miR-124 family, whose expression is enriched in the cephalic ganglia, are also expressed in the brain primordium during CNS regeneration. These results provide new insight into the potential biological functions of miRNAs in neoblasts and regeneration in planarians.


Assuntos
MicroRNAs/genética , Planárias/fisiologia , Regeneração/fisiologia , Células-Tronco/fisiologia , Animais , Biomarcadores/metabolismo , Biologia Computacional , Perfilação da Expressão Gênica , Hibridização In Situ , Análise de Sequência com Séries de Oligonucleotídeos , Planárias/citologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células-Tronco/citologia
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