RESUMO
Circular RNAs (circRNAs) produced from back-spliced exons are widely expressed, but individual circRNA functions remain poorly understood owing to the lack of adequate methods for distinguishing circRNAs from cognate messenger RNAs with overlapping exons. Here, we report that CRISPR-RfxCas13d can effectively discriminate circRNAs from mRNAs by using guide RNAs targeting sequences spanning back-splicing junction (BSJ) sites featured in RNA circles. Using a lentiviral library that targets sequences across BSJ sites of highly expressed human circRNAs, we show that a group of circRNAs are important for cell growth mostly in a cell-type-specific manner and that a common oncogenic circRNA, circFAM120A, promotes cell proliferation by preventing the mRNA for family with sequence similarity 120A (FAM120A) from binding the translation inhibitor IGF2BP2. Further application of RfxCas13d-BSJ-gRNA screening has uncovered circMan1a2, which has regulatory potential in mouse embryo preimplantation development. Together, these results establish CRISPR-RfxCas13d as a useful tool for the discovery and functional study of circRNAs at both individual and large-scale levels.
Assuntos
Sistemas CRISPR-Cas , Neoplasias do Colo/patologia , Regulação Neoplásica da Expressão Gênica , RNA Circular/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Processamento Alternativo , Animais , Apoptose , Proliferação de Células , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Sequences of circular RNAs (circRNAs) produced from back-splicing of exon(s) completely overlap with those from cognate linear RNAs transcribed from the same gene loci with the exception of their back-splicing junction (BSJ) sites. Therefore, examination of global circRNA expression from RNA-seq datasets generally relies on the detection of RNA-seq fragments spanning BSJ sites, which is different from the quantification of linear RNA expression by normalized RNA-seq fragments mapped to whole gene bodies. Thus, direct comparison of circular and linear RNA expression from the same gene loci in a genome-wide manner has remained challenging. Here, we update the previously-reported CIRCexplorer pipeline to version 3 for circular and linear RNA expression analysis from ribosomal-RNA depleted RNA-seq (CIRCexplorer3-CLEAR). A new quantitation parameter, fragments per billion mapped bases (FPB), is applied to evaluate circular and linear RNA expression individually by fragments mapped to circRNA-specific BSJ sites or to linear RNA-specific splicing junction (SJ) sites. Comparison of circular and linear RNA expression levels is directly achieved by dividing FPBcirc by FPBlinear to generate a CIRCscore, which indicates the relative circRNA expression level using linear RNA expression level as the background. Highly-expressed circRNAs with low cognate linear RNA expression background can be readily identified by CIRCexplorer3-CLEAR for further investigation. CIRCexplorer3-CLEAR is publically available at https://github.com/YangLab/CLEAR.
Assuntos
RNA Circular/metabolismo , RNA/metabolismo , Interface Usuário-Computador , Linhagem Celular Tumoral , Humanos , RNA/química , Splicing de RNA , RNA Circular/química , Análise de Sequência de RNA , TranscriptomaRESUMO
N6-methyladenosine (m6A) and adenosine-to-inosine (A-to-I) editing are two of the most abundant RNA modifications, both at adenosines. Yet, the interaction of these two types of adenosine modifications is largely unknown. Here we show a global A-to-I difference between m6A-positive and m6A-negative RNA populations. Both the presence and extent of A-to-I sites in m6A-negative RNA transcripts suggest a negative correlation between m6A and A-to-I. Suppression of m6A-catalyzing enzymes results in global A-to-I RNA editing changes. Further depletion of m6A modification increases the association of m6A-depleted transcripts with adenosine deaminase acting on RNA (ADAR) enzymes, resulting in upregulated A-to-I editing on the same m6A-depleted transcripts. Collectively, the effect of m6A on A-to-I suggests a previously underappreciated interplay between two distinct and abundant RNA modifications, highlighting a complex epitranscriptomic landscape.
Assuntos
Adenosina/análogos & derivados , Adenosina/química , Inosina/química , Edição de RNA/genética , RNA/genética , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Linhagem Celular Tumoral , Regulação da Expressão Gênica/genética , Células HEK293 , Células HeLa , Humanos , Metiltransferases/genética , Metiltransferases/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismoRESUMO
RNA is essential for all kingdoms of life and exerts important functions beyond transferring genetic information from DNA to protein. With the advent of the state-of-the-art deep sequencing technology, a large portion of noncoding transcripts in eukaryotic genomes has been broadly identified. Among them, long noncoding RNAs (lncRNAs) have been emerged as a new class of RNA molecules that have regulatory potential in a variety of physiological and pathological processes. Here we summarize recent research progresses that have been made by scientists in China on lncRNAs, including their biogenesis, functional implication and the underlying mechanism of action at the current stage. © 2016 IUBMB Life, 68(11):887-893, 2016.