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1.
Nucleic Acids Res ; 43(14): 6730-8, 2015 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-26089388

RESUMO

In eucaryotes, gene expression is regulated by microRNAs (miRNAs) which bind to messenger RNAs (mRNAs) and interfere with their translation into proteins, either by promoting their degradation or inducing their repression. We study the effect of miRNA interference on each gene using experimental methods, such as microarrays and RNA-seq at the mRNA level, or luciferase reporter assays and variations of SILAC at the protein level. Alternatively, computational predictions would provide clear benefits. However, no algorithm toward this task has ever been proposed. Here, we introduce a new algorithm to predict genome-wide expression data from initial transcriptome abundance. The algorithm simulates the miRNA and mRNA hybridization competition that occurs in given cellular conditions, and derives the whole set of miRNA::mRNA interactions at equilibrium (microtargetome). Interestingly, solving the competition improves the accuracy of miRNA target predictions. Furthermore, this model implements a previously reported and fundamental property of the microtargetome: the binding between a miRNA and a mRNA depends on their sequence complementarity, but also on the abundance of all RNAs expressed in the cell, i.e. the stoichiometry of all the miRNA sites and all the miRNAs given their respective abundance. This model generalizes the miRNA-induced synchronistic silencing previously observed, and described as sponges and competitive endogenous RNAs.


Assuntos
Algoritmos , Inativação Gênica , MicroRNAs/metabolismo , Linhagem Celular , Humanos , MicroRNAs/química , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Transcriptoma
2.
Methods Mol Biol ; 809: 303-20, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22113285

RESUMO

Cyclobutane pyrimidine dimers (CPDs) and (6,4) pyrimidine-pyrimidone dimers are the major DNA lesions (or photoproducts) induced by ultraviolet light and are removed by the nucleotide excision repair (NER) pathway. If not repaired, DNA damage can lead to genome instability. The genome is organized into nuclear domains with distinct functions and chromatin structures. Although studies on NER in all chromosomal contexts are important to understand the mechanisms of genome maintenance, we focused on NER in the nucleolus. The attractive feature of the rDNA locus is its chromatin structure; not all rRNA genes are transcribed and both active (no nucleosomes) and inactive (nucleosomes) rRNA genes coexist in the nucleolus. These characteristics allow for direct comparison of NER in two very different chromatin structures. Yeast is used as a model system and the methods employed are as follows: nuclei isolation, restriction enzyme digestion of chromatin to release active rRNA genes, psoralen cross-linking, T4-endonuclease-V enzyme to detect CPDs and CPDs repair over relatively large stretches of DNA, and primer extension to follow DNA damage and repair at nucleotide level. Using this approach, we have shown that NER is faster in nonnucleosomes vs. nucleosomes rDNA, that the formation of CPDs promotes changes in the active rDNA chromatin, and that NER is coupled to rRNA genes transcription.


Assuntos
Dano ao DNA/efeitos da radiação , Reparo do DNA/fisiologia , DNA Ribossômico/genética , Raios Ultravioleta , Southern Blotting , Cromatina/metabolismo , Dano ao DNA/genética , Reparo do DNA/genética , Eletroforese em Gel de Ágar , Ficusina/química , RNA Polimerase I/metabolismo
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