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1.
Essays Biochem ; 2021 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-34623427

RESUMO

RNA molecules have emerged as a new class of promising therapeutics to expand the range of druggable targets in the genome. In addition to 'canonical' protein-coding mRNAs, the emerging richness of sense and antisense long non-coding RNAs (lncRNAs) provides a new reservoir of molecular tools for RNA-based drugs. LncRNAs are composed of modular structural domains with specific activities involving the recruitment of protein cofactors or directly interacting with nucleic acids. A single therapeutic RNA transcript can then be assembled combining domains with defined secondary structures and functions, and antisense sequences specific for the RNA/DNA target of interest. As the first representative molecules of this new pharmacology, we have identified SINEUPs, a new functional class of natural antisense lncRNAs that increase the translation of partially overlapping mRNAs. Their activity is based on the combination of two domains: an embedded mouse inverted SINEB2 element that enhances mRNA translation (effector domain) and an overlapping antisense region that provides specificity for the target sense transcript (binding domain). By genetic engineering, synthetic SINEUPs can potentially target any mRNA of interest increasing translation and therefore the endogenous level of the encoded protein. In this review, we describe the state-of-the-art knowledge of SINEUPs and discuss recent publications showing their potential application in diseases where a physiological increase of endogenous protein expression can be therapeutic.

2.
Methods Mol Biol ; 2351: 67-90, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34382184

RESUMO

The Cap Analysis of Gene Expression (CAGE) is a powerful method to identify Transcription Start Sites (TSSs) of capped RNAs while simultaneously measuring transcripts expression level. CAGE allows mapping at single nucleotide resolution at all active promoters and enhancers. Large CAGE datasets have been produced over the years from individual laboratories and consortia, including the Encyclopedia of DNA Elements (ENCODE) and Functional Annotation of the Mammalian Genome (FANTOM) consortia. These datasets constitute open resource for TSS annotations and gene expression analysis. Here, we provide an experimental protocol for the most recent CAGE method called Low Quantity (LQ) single strand (ss) CAGE "LQ-ssCAGE", which enables cost-effective profiling of low quantity RNA samples. LQ-ssCAGE is especially useful for samples derived from cells cultured in small volumes, cellular compartments such as nuclear RNAs or for samples from developmental stages. We demonstrate the reproducibility and effectiveness of the method by constructing 240 LQ-ssCAGE libraries from 50 ng of THP-1 cell extracted RNAs and discover lowly expressed novel enhancer and promoter-derived lncRNAs.


Assuntos
Biologia Computacional/métodos , Elementos Facilitadores Genéticos , Regiões Promotoras Genéticas , Capuzes de RNA , Sítio de Iniciação de Transcrição , Bases de Dados Genéticas , Regulação da Expressão Gênica , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Anotação de Sequência Molecular , Sequências Reguladoras de Ácido Nucleico , Reprodutibilidade dos Testes , Fluxo de Trabalho
3.
Genome Res ; 31(6): 995-1010, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33795334

RESUMO

Long noncoding RNAs or lncRNAs are a class of non-protein-coding RNAs that are >200 nt in length. Almost 50% of lncRNAs during zebrafish development are transcribed in an antisense direction to a protein-coding gene. However, the role of these natural antisense transcripts (NATs) during development remains enigmatic. To understand NATs in early vertebrate development, we took a computational biology approach and analyzed existing as well as novel data sets. Our analysis indicates that zebrafish NATs can be divided into two major classes based on their coexpression patterns with respect to the overlapping protein-coding genes. Group 1 NATs have characteristics similar to maternally deposited RNAs in that their levels decrease as development progresses. Group 1 NAT levels are negatively correlated with that of overlapping sense-strand protein-coding genes. Conversely, Group 2 NATs are coexpressed with overlapping protein-coding genes. In contrast to Group 1, which is enriched in genes involved in developmental pathways, Group 2 protein-coding genes are enriched in housekeeping functions. Group 1 NATs also show larger overlap and higher complementarity with the sense-strand mRNAs compared to other NATs. In addition, our transcriptomics data, quantifying RNA levels from cytoplasmic and nuclear compartments, indicates that Group 1 NATs are more abundant in the cytosol. Based on their expression pattern, cytosolic nature, and their higher complementarity to the overlapping developmental mRNAs, we speculate that Group 1 NATs function post-transcriptionally to silence spurious expression of developmental genes.

4.
Nucleic Acids Res ; 48(20): 11626-11644, 2020 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-33130894

RESUMO

SINEUPs are long non-coding RNAs (lncRNAs) that contain a SINE element, and which up-regulate the translation of target mRNA. They have been studied in a wide range of applications, as both biological and therapeutic tools, although the underpinning molecular mechanism is unclear. Here, we focused on the sub-cellular distribution of target mRNAs and SINEUP RNAs, performing co-transfection of expression vectors for these transcripts into human embryonic kidney cells (HEK293T/17), to investigate the network of translational regulation. The results showed that co-localization of target mRNAs and SINEUP RNAs in the cytoplasm was a key phenomenon. We identified PTBP1 and HNRNPK as essential RNA binding proteins. These proteins contributed to SINEUP RNA sub-cellular distribution and to assembly of translational initiation complexes, leading to enhanced target mRNA translation. These findings will promote a better understanding of the mechanisms employed by regulatory RNAs implicated in efficient protein translation.


Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Iniciação Traducional da Cadeia Peptídica , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , RNA Longo não Codificante/metabolismo , Citoplasma/metabolismo , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , Proteínas de Ligação a RNA/metabolismo
5.
FEBS Lett ; 594(24): 4357-4369, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33012004

RESUMO

Chemically modified mRNAs are extensively studied with a view toward their clinical application. In particular, long noncoding RNAs (lncRNAs) containing SINE elements, which enhance the translation of their target mRNAs (i.e., SINEUPs), have potential as RNA therapies for various diseases, such as haploinsufficiencies. To establish a SINEUP-based system for efficient protein expression, we directly transfected chemically modified in vitro transcribed (mIVT) SINEUP RNAs to examine their effects on target mRNA translation. mIVT SINEUP RNAs enhanced translation of EGFP mRNA and endogenous target Sox9 mRNA in both cultured cells and a cell-free translation system. Our findings reveal the functional role of RNA modifications in SINEUPs and suggest several broad clinical applications of such an RNA regulatory system.


Assuntos
Biossíntese de Proteínas , RNA Longo não Codificante/química , RNA Longo não Codificante/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Fluorescência Verde/biossíntese , Proteínas de Fluorescência Verde/genética , Células HEK293 , Células Hep G2 , Humanos , Técnicas In Vitro , Estabilidade de RNA , RNA Longo não Codificante/síntese química , RNA Longo não Codificante/genética , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição SOX9/biossíntese , Fatores de Transcrição SOX9/genética , Regulação para Cima
6.
Nucleic Acids Res ; 48(16): 9346-9360, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32697302

RESUMO

Long non-coding RNAs (lncRNAs) are attracting widespread attention for their emerging regulatory, transcriptional, epigenetic, structural and various other functions. Comprehensive transcriptome analysis has revealed that retrotransposon elements (REs) are transcribed and enriched in lncRNA sequences. However, the functions of lncRNAs and the molecular roles of the embedded REs are largely unknown. The secondary and tertiary structures of lncRNAs and their embedded REs are likely to have essential functional roles, but experimental determination and reliable computational prediction of large RNA structures have been extremely challenging. We report here the nuclear magnetic resonance (NMR)-based secondary structure determination of the 167-nt inverted short interspersed nuclear element (SINE) B2, which is embedded in antisense Uchl1 lncRNA and upregulates the translation of sense Uchl1 mRNAs. By using NMR 'fingerprints' as a sensitive probe in the domain survey, we successfully divided the full-length inverted SINE B2 into minimal units made of two discrete structured domains and one dynamic domain without altering their original structures after careful boundary adjustments. This approach allowed us to identify a structured domain in nucleotides 31-119 of the inverted SINE B2. This approach will be applicable to determining the structures of other regulatory lncRNAs.


Assuntos
Conformação de Ácido Nucleico , RNA Longo não Codificante/ultraestrutura , Retroelementos/genética , Elementos Nucleotídeos Curtos e Dispersos/genética , Biologia Computacional , Humanos , Espectroscopia de Ressonância Magnética , RNA Antissenso/genética , RNA Antissenso/ultraestrutura , RNA Longo não Codificante/genética , Transcriptoma/genética , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/ultraestrutura
7.
J Vis Exp ; (144)2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30774120

RESUMO

Targeted-protein enhancement is of importance not only for the study of biological processes but also for therapeutic and biotechnological applications. Here, we present a method to selectively up-regulate protein expression of desired genes in cultured cells by means of synthetic antisense non-coding RNAs known as SINEUPs. This positive control of gene expression is at the post-transcriptional level and exerted by an inverted short interspersed nuclear element (SINE) repeat at the 3' end of SINEUPs that comprises its effector domain (ED). SINEUPs can specifically bind to any protein-coding mRNA of choice through its binding domain (BD), a region designed to complement the sequence within the 5' untranslated region (5' UTR) and around the start codon of the mRNA. Target-specific SINEUPs designed in this manner are transfected to cultured cells, and protein and RNA are extracted for downstream analyses, generally 24-48 h post-transfection. SINEUP-induced protein up-regulation is detected by Western-blot analysis and RNA expression is measured using real-time quantitative reverse transcription PCR. We have observed that BD design is critical for achieving optimum SINEUP activity and that testing different BD sizes and positions with respect to the start codon of the target mRNA is recommended. Therefore, we describe here a semi-automated high-throughput imaging method based on fluorescence detection that can be implemented to target mRNA fused with green fluorescent protein (GFP). SINEUPs specifically enhance translation within normal physiological range of the cell, without altering the target transcript level. This method has been successfully employed against a range of endogenous and exogenous targets, in a wide variety of human, mouse, and insect cell lines along with in vivo systems. Moreover, SINEUPs have been reported to increase antibody production and work as an RNA therapeutic against haploinsufficient genes. The versatile and modular nature of SINEUPs makes them a suitable tool for gene-specific translational control.


Assuntos
Biossíntese de Proteínas/genética , RNA Longo não Codificante/genética , RNA Mensageiro/genética , Humanos , Transfecção
8.
PLoS One ; 13(2): e0183229, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29414979

RESUMO

SINEUPs are antisense long noncoding RNAs, in which an embedded SINE B2 element UP-regulates translation of partially overlapping target sense mRNAs. SINEUPs contain two functional domains. First, the binding domain (BD) is located in the region antisense to the target, providing specific targeting to the overlapping mRNA. Second, the inverted SINE B2 represents the effector domain (ED) and enhances translation. To adapt SINEUP technology to a broader number of targets, we took advantage of a high-throughput, semi-automated imaging system to optimize synthetic SINEUP BD and ED design in HEK293T cell lines. Using SINEUP-GFP as a model SINEUP, we extensively screened variants of the BD to map features needed for optimal design. We found that most active SINEUPs overlap an AUG-Kozak sequence. Moreover, we report our screening of the inverted SINE B2 sequence to identify active sub-domains and map the length of the minimal active ED. Our synthetic SINEUP-GFP screening of both BDs and EDs constitutes a broad test with flexible applications to any target gene of interest.


Assuntos
Biossíntese de Proteínas/genética , Proteínas/genética , RNA Longo não Codificante/genética , Animais , Linhagem Celular Tumoral , Células HEK293 , Humanos , Camundongos , Fosforilação
9.
Gan To Kagaku Ryoho ; 43(Suppl 1): 44-46, 2016 Dec.
Artigo em Japonês | MEDLINE | ID: mdl-28028277

RESUMO

In 2006, with the admission of a new batch of students, pharmaceutical education became a 6-year course. This was a result of the urgent need to train a new generation of pharmacists to respond to increasingly advanced and intricate medical care as well as the specific need to coordinate with multiple occupational categories. Meanwhile, with Japan becoming an aged society, medical care has undergone functional differentiation, and home care is now being promoted. As part of an 11- week practical course for 5th-year practical training, students attended visits to home care patients from an early stage, making it possible for them to be present at multiple visits. This was highly significant because it allowed students to experience various disease states of different patients and increase their practical knowledge of pharmaceuticals. This study explores the case example of proposals made by pharmacy students for improving medication-related problems in home care patients during 5th-year practical training.


Assuntos
Educação em Farmácia , Serviços de Assistência Domiciliar , Estudantes de Farmácia , Idoso , Feminino , Humanos , Cooperação do Paciente , Estudantes de Farmácia/legislação & jurisprudência
10.
Sci Rep ; 6: 25039, 2016 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-27112104

RESUMO

PIWI-interacting RNAs (piRNAs) are responsible for maintaining the genome stability by silencing retrotransposons in germline tissues- where piRNAs were first discovered and thought to be restricted. Recently, novel functions were reported for piRNAs in germline and somatic cells. Using deep sequencing of small RNAs and CAGE of postnatal development of mouse brain, we identified piRNAs only in adult mouse brain. These piRNAs have similar sequence length as those of MILI-bound piRNAs. In addition, we predicted novel candidate regulators and putative targets of adult brain piRNAs.


Assuntos
Encéfalo/crescimento & desenvolvimento , Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA Interferente Pequeno/genética , Análise de Sequência de RNA/métodos , Animais , Animais Recém-Nascidos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Masculino , Camundongos
11.
Artigo em Inglês | MEDLINE | ID: mdl-26673794

RESUMO

BACKGROUND: The capacity for plasticity in the adult brain is limited by the anatomical traces laid down during early postnatal life. Removing certain molecular brakes, such as histone deacetylases (HDACs), has proven to be effective in recapitulating juvenile plasticity in the mature visual cortex (V1). We investigated the chromatin structure and transcriptional control by genome-wide sequencing of DNase I hypersensitive sites (DHSS) and cap analysis of gene expression (CAGE) libraries after HDAC inhibition by valproic acid (VPA) in adult V1. RESULTS: We found that VPA reliably reactivates the critical period plasticity and induces a dramatic change of chromatin organization in V1 yielding significantly greater accessibility distant from promoters, including at enhancer regions. VPA also induces nucleosome eviction specifically from retrotransposon (in particular SINE) elements. The transiently accessible SINE elements overlap with transcription factor-binding sites of the Fox family. Mapping of transcription start site activity using CAGE revealed transcription of epigenetic and neural plasticity-regulating genes following VPA treatment, which may help to re-program the genomic landscape and reactivate plasticity in the adult cortex. CONCLUSIONS: Treatment with HDAC inhibitors increases accessibility to enhancers and repetitive elements underlying brain-specific gene expression and reactivation of visual cortical plasticity.

12.
Gene ; 569(2): 287-93, 2015 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-26045368

RESUMO

Whenever the function of a recombinant protein depends on post-translational processing, mammalian cells become an indispensable tool for their production. This is particularly true for biologics and therapeutic monoclonal antibodies (MAbs). Despite some drawbacks, Chinese Hamster Ovary (CHO) cells are the workhorse for MAbs production in academia and industry. Several methodologies have been adopted to improve expression and stability, including methods based on selective pressure or cell engineering. We have previously identified SINEUPs as a new functional class of natural and synthetic long non-coding RNAs that through the activity of an inverted SINEB2 element are able to promote translation of partially overlapping sense coding mRNAs. Here we show that by taking advantage of their modular structure, synthetic SINEUPs can be designed to increase production of secreted proteins. Furthermore, by experimentally validating antisense to elastin (AS-eln) RNA as a natural SINEUP, we show that SINEUP-mediated control may target extracellular proteins. These results lead us to propose synthetic SINEUPs as new versatile tools to optimize production of secreted proteins in manufacturing pipelines and natural SINEUPs as new regulatory RNAs in the secretory pathways.


Assuntos
Engenharia Celular , Biossíntese de Proteínas , RNA Antissenso/metabolismo , RNA Longo não Codificante/metabolismo , Proteínas Recombinantes/metabolismo , Animais , Sequência de Bases , Células CHO , Moléculas de Adesão Celular/genética , Cricetulus , Elastina/genética , Humanos , Dados de Sequência Molecular , RNA Antissenso/química , RNA Antissenso/genética , RNA Longo não Codificante/química , RNA Longo não Codificante/genética , Via Secretória
13.
Front Cell Neurosci ; 9: 174, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26029048

RESUMO

Despite recent efforts in discovering novel long non-coding RNAs (lncRNAs) and unveiling their functions in a wide range of biological processes their applications as biotechnological or therapeutic tools are still at their infancy. We have recently shown that AS Uchl1, a natural lncRNA antisense to the Parkinson's disease-associated gene Ubiquitin carboxyl-terminal esterase L1 (Uchl1), is able to increase UchL1 protein synthesis at post-transcriptional level. Its activity requires two RNA elements: an embedded inverted SINEB2 sequence to increase translation and the overlapping region to target its sense mRNA. This functional organization is shared with several mouse lncRNAs antisense to protein coding genes. The potential use of AS Uchl1-derived lncRNAs as enhancers of target mRNA translation remains unexplored. Here we define AS Uchl1 as the representative member of a new functional class of natural and synthetic antisense lncRNAs that activate translation. We named this class of RNAs SINEUPs for their requirement of the inverted SINEB2 sequence to UP-regulate translation in a gene-specific manner. The overlapping region is indicated as the Binding Doman (BD) while the embedded inverted SINEB2 element is the Effector Domain (ED). By swapping BD, synthetic SINEUPs are designed targeting mRNAs of interest. SINEUPs function in an array of cell lines and can be efficiently directed toward N-terminally tagged proteins. Their biological activity is retained in a miniaturized version within the range of small RNAs length. Its modular structure was exploited to successfully design synthetic SINEUPs targeting endogenous Parkinson's disease-associated DJ-1 and proved to be active in different neuronal cell lines. In summary, SINEUPs represent the first scalable tool to increase synthesis of proteins of interest. We propose SINEUPs as reagents for molecular biology experiments, in protein manufacturing as well as in therapy of haploinsufficiencies.

14.
Biochem Biophys Res Commun ; 452(2): 294-301, 2014 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-25193698

RESUMO

Comprehensive analysis of mammalian transcriptomes has surprisingly revealed that a major fraction of the RNAs produced by mammalian cells and tissues is comprised of long non-coding RNAs (lncRNAs). Such RNAs were previously disregarded as useless, but recent functional studies have revealed that they have multiple regulatory functions. A large subset of these lncRNAs are antisense to protein-coding genes; such RNAs are particularly attractive to researchers because their functions are better understood than other lncRNAs and their action can be easily modulated and engineered by modifying the antisense region. We discuss various aspects of regulation by antisense RNAs and other small nucleic acids and the challenges to bring these technologies to gene therapy. Despite several remaining issues related to delivery, RNA stability, side effects, and toxicity, the field is moving quickly towards future biotechnological and health applications. Therapies based on lncRNAs may be the key to increased cell-specificity of future gene therapies.


Assuntos
Terapia Genética/métodos , Genoma Humano , RNA Longo não Codificante/genética , Transcrição Genética , Marcação de Genes , Vetores Genéticos , Humanos
15.
Biochemistry ; 53(37): 5923-9, 2014 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-25162914

RESUMO

Photoactivation of attractant phototaxis receptor sensory rhodopsin I (SRI) in Halobacterium salinarum entails transfer of a proton from the retinylidene chromophore's Schiff base (SB) to an unidentified acceptor residue on the cytoplasmic half-channel, in sharp contrast to other microbial rhodopsins, including the closely related repellent phototaxis receptor SRII and the outward proton pump bacteriorhodopsin, in which the SB proton acceptor is an aspartate residue salt-bridged to the SB in the extracellular (EC) half-channel. His166 on the cytoplasmic side of the SB in SRI has been implicated in the SB proton transfer reaction by mutation studies, and mutants of His166 result in an inverted SB proton release to the EC as well as inversion of the protein's normally attractant phototaxis signal to repellent. Here we found by difference Fourier transform infrared spectroscopy the appearance of Fermi-resonant X-H stretch modes in light-minus-dark difference spectra; their assignment with (15)N labeling and site-directed mutagenesis demonstrates that His166 is the SB proton acceptor during the photochemical reaction cycle of the wild-type SRI-HtrI complex.


Assuntos
Halorrodopsinas/química , Histidina/química , Rodopsinas Sensoriais/química , Halobacterium salinarum/metabolismo , Halorrodopsinas/genética , Halorrodopsinas/metabolismo , Mutagênese Sítio-Dirigida , Isótopos de Nitrogênio , Prótons , Bases de Schiff/química , Rodopsinas Sensoriais/genética , Rodopsinas Sensoriais/metabolismo , Espectroscopia de Infravermelho com Transformada de Fourier
16.
Genome Res ; 23(11): 1938-50, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24002785

RESUMO

Spatiotemporal control of gene expression is central to animal development. Core promoters represent a previously unanticipated regulatory level by interacting with cis-regulatory elements and transcription initiation in different physiological and developmental contexts. Here, we provide a first and comprehensive description of the core promoter repertoire and its dynamic use during the development of a vertebrate embryo. By using cap analysis of gene expression (CAGE), we mapped transcription initiation events at single nucleotide resolution across 12 stages of zebrafish development. These CAGE-based transcriptome maps reveal genome-wide rules of core promoter usage, structure, and dynamics, key to understanding the control of gene regulation during vertebrate ontogeny. They revealed the existence of multiple classes of pervasive intra- and intergenic post-transcriptionally processed RNA products and their developmental dynamics. Among these RNAs, we report splice donor site-associated intronic RNA (sRNA) to be specific to genes of the splicing machinery. For the identification of conserved features, we compared the zebrafish data sets to the first CAGE promoter map of Tetraodon and the existing human CAGE data. We show that a number of features, such as promoter type, newly discovered promoter properties such as a specialized purine-rich initiator motif, as well as sRNAs and the genes in which they are detected, are conserved in mammalian and Tetraodon CAGE-defined promoter maps. The zebrafish developmental promoterome represents a powerful resource for studying developmental gene regulation and revealing promoter features shared across vertebrates.


Assuntos
Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Purinas/metabolismo , Sítio de Iniciação de Transcrição , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Evolução Molecular , Perfilação da Expressão Gênica , Genes , Genoma , Filogenia , Regiões Promotoras Genéticas , RNA/genética , RNA/metabolismo , Capuzes de RNA/genética , Splicing de RNA , Transcriptoma , Vertebrados/genética
17.
Neurobiol Aging ; 34(7): 1825-36, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23428183

RESUMO

To characterize the promoterome of caudate and putamen regions (striatum), frontal and temporal cortices, and hippocampi from aged human brains, we used high-throughput cap analysis of gene expression to profile the transcription start sites and to quantify the differences in gene expression across the 5 brain regions. We also analyzed the extent to which methylation influenced the observed expression profiles. We sequenced more than 71 million cap analysis of gene expression tags corresponding to 70,202 promoter regions and 16,888 genes. More than 7000 transcripts were differentially expressed, mainly because of differential alternative promoter usage. Unexpectedly, 7% of differentially expressed genes were neurodevelopmental transcription factors. Functional pathway analysis on the differentially expressed genes revealed an overrepresentation of several signaling pathways (e.g., fibroblast growth factor and wnt signaling) in hippocampus and striatum. We also found that although 73% of methylation signals mapped within genes, the influence of methylation on the expression profile was small. Our study underscores alternative promoter usage as an important mechanism for determining the regional differences in gene expression at old age.


Assuntos
Envelhecimento/genética , Envelhecimento/patologia , Encéfalo/patologia , Encéfalo/fisiologia , Regulação da Expressão Gênica/fisiologia , Regiões Promotoras Genéticas/fisiologia , Idoso , Idoso de 80 Anos ou mais , Núcleo Caudado/patologia , Núcleo Caudado/fisiologia , Feminino , Lobo Frontal/patologia , Lobo Frontal/fisiologia , Hipocampo/patologia , Hipocampo/fisiologia , Humanos , Masculino , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologia , Putamen/patologia , Putamen/fisiologia , Lobo Temporal/patologia , Lobo Temporal/fisiologia
18.
Nat Struct Mol Biol ; 20(3): 332-8, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23353788

RESUMO

How a more plastic chromatin state is maintained and reversed during development is unknown. Heterochromatin-mediated silencing of repetitive elements occurs in differentiated cells. Here, we used repetitive elements, including retrotransposons, as model loci to address how and when heterochromatin forms during development. RNA sequencing throughout early mouse embryogenesis revealed that repetitive-element expression is dynamic and stage specific, with most repetitive elements becoming repressed before implantation. We show that LINE-1 and IAP retrotransposons become reactivated from both parental genomes after fertilization. Chromatin immunoprecipitation for H3K4me3 and H3K9me3 in 2- and 8-cell embryos indicates that their developmental silencing follows loss of activating marks rather than acquisition of conventional heterochromatic marks. Furthermore, short LINE-1 RNAs regulate LINE-1 transcription in vivo. Our data indicate that reprogramming after mammalian fertilization comprises a robust transcriptional activation of retrotransposons and that repetitive elements are initially regulated through RNA.


Assuntos
Blastocisto/fisiologia , Metilação de DNA , Regulação da Expressão Gênica no Desenvolvimento , Heterocromatina/genética , Elementos Nucleotídeos Longos e Dispersos , Animais , Imunoprecipitação da Cromatina , Desenvolvimento Embrionário/genética , Feminino , Histonas/genética , Histonas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Conformação de Ácido Nucleico , Capuzes de RNA , Retroelementos , Transcrição Genética
19.
Nat Protoc ; 7(3): 542-61, 2012 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-22362160

RESUMO

Cap-analysis gene expression (CAGE) provides accurate high-throughput measurement of RNA expression. CAGE allows mapping of all the initiation sites of both capped coding and noncoding RNAs. In addition, transcriptional start sites within promoters are characterized at single-nucleotide resolution. The latter allows the regulatory inputs driving gene expression to be studied, which in turn enables the construction of transcriptional networks. Here we provide an optimized protocol for the construction of CAGE libraries on the basis of the preparation of 27-nt-long tags corresponding to initial bases at the 5' ends of capped RNAs. We have optimized the methods using simple steps based on filtration, which altogether takes 4 d to complete. The CAGE tags can be readily sequenced with Illumina sequencers, and upon modification they are also amenable to sequencing using other platforms.


Assuntos
Perfilação da Expressão Gênica/métodos , Biblioteca Gênica , Genômica/métodos , Capuzes de RNA/metabolismo , Análise de Sequência de DNA/métodos , Capuzes de RNA/genética
20.
Methods Mol Biol ; 786: 181-200, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-21938627

RESUMO

We provide here a protocol for the preparation of cap-analysis gene expression (CAGE) libraries, which allows for measuring the expression of eukaryotic capped RNAs and simultaneously map the promoter regions. The presented protocol simplifies the previously published ones and moreover produces tags that are 27 nucleotides long, which facilitates mapping to the genome. The protocol takes less than 5 days to complete and presents a notable improvement compared to previously published versions.


Assuntos
Perfilação da Expressão Gênica/métodos , Redes Reguladoras de Genes/genética , Regiões Promotoras Genéticas/genética , Capuzes de RNA/análise , Capuzes de RNA/genética , Análise de Sequência de RNA/métodos , Transcrição Genética/genética , Regulação da Expressão Gênica/genética , Biblioteca Gênica
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