RESUMO
Circular RNAs (circRNAs) produced from back-splicing of exons of pre-mRNAs are widely expressed, but current understanding of their functions is limited. These RNAs are stable in general and are thought to have unique structural conformations distinct from their linear RNA cognates. Here, we show that endogenous circRNAs tend to form 16-26 bp imperfect RNA duplexes and act as inhibitors of double-stranded RNA (dsRNA)-activated protein kinase (PKR) related to innate immunity. Upon poly(I:C) stimulation or viral infection, circRNAs are globally degraded by RNase L, a process required for PKR activation in early cellular innate immune responses. Augmented PKR phosphorylation and circRNA reduction are found in peripheral blood mononuclear cells (PBMCs) derived from patients with autoimmune disease systemic lupus erythematosus (SLE). Importantly, overexpression of the dsRNA-containing circRNA in PBMCs or T cells derived from SLE can alleviate the aberrant PKR activation cascade, thus providing a connection between circRNAs and SLE.
Assuntos
RNA Circular/metabolismo , RNA Circular/fisiologia , eIF-2 Quinase/metabolismo , Adolescente , Adulto , Doenças Autoimunes/genética , Linhagem Celular , Endorribonucleases/metabolismo , Feminino , Humanos , Imunidade Inata/genética , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Lúpus Eritematoso Sistêmico/genética , Pessoa de Meia-Idade , Fosforilação , RNA/metabolismo , Splicing de RNA/genética , Estabilidade de RNA/fisiologia , RNA Circular/genética , RNA de Cadeia Dupla/metabolismo , Viroses/metabolismo , eIF-2 Quinase/imunologiaRESUMO
Exon back-splicing-generated circular RNAs, as a group, can suppress double-stranded RNA (dsRNA)-activated protein kinase R (PKR) in cells. We have sought to synthesize immunogenicity-free, short dsRNA-containing RNA circles as PKR inhibitors. Here, we report that RNA circles synthesized by permuted self-splicing thymidylate synthase (td) introns from T4 bacteriophage or by Anabaena pre-tRNA group I intron could induce an immune response. Autocatalytic splicing introduces â¼74 nt td or â¼186 nt Anabaena extraneous fragments that can distort the folding status of original circular RNAs or form structures themselves to provoke innate immune responses. In contrast, synthesized RNA circles produced by T4 RNA ligase without extraneous fragments exhibit minimized immunogenicity. Importantly, directly ligated circular RNAs that form short dsRNA regions efficiently suppress PKR activation 103- to 106-fold higher than reported chemical compounds C16 and 2-AP, highlighting the future use of circular RNAs as potent inhibitors for diseases related to PKR overreaction.
Assuntos
Inibidores de Proteínas Quinases/farmacologia , RNA Circular/farmacologia , eIF-2 Quinase/antagonistas & inibidores , Células A549 , Bacteriófago T4/enzimologia , Bacteriófago T4/genética , Células HEK293 , Células HeLa , Humanos , Imunidade Inata/efeitos dos fármacos , Íntrons , Conformação de Ácido Nucleico , Inibidores de Proteínas Quinases/imunologia , RNA Ligase (ATP)/genética , RNA Ligase (ATP)/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA Circular/genética , RNA Circular/imunologia , Timidilato Sintase/genética , Timidilato Sintase/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , eIF-2 Quinase/metabolismoRESUMO
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
Circular RNAs are produced from back-splicing of exons of precursor mRNAs (pre-mRNAs). The sequences of exons in circular RNAs are identical to their linear cognate mRNAs, but the circular format may confer constraints on their folding and conformation, leading to potentially different functions from their linear RNA cognates. Here, we describe experimental and computational steps that optimize the selective 2'-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) to probe circular RNA secondary structure at single-nucleotide resolution in living cells.
Assuntos
RNA Circular , RNA , Conformação de Ácido Nucleico , RNA/química , Precursores de RNA , Splicing de RNA/genética , RNA Circular/genéticaRESUMO
Efforts to advance RNA aptamers as a new therapeutic modality have been limited by their susceptibility to degradation and immunogenicity. In a previous study, we demonstrated synthesized short double-stranded region-containing circular RNAs (ds-cRNAs) with minimal immunogenicity targeted to dsRNA-activated protein kinase R (PKR). Here we test the therapeutic potential of ds-cRNAs in a mouse model of imiquimod-induced psoriasis. We find that genetic supplementation of ds-cRNAs leads to inhibition of PKR, resulting in alleviation of downstream interferon-α and dsRNA signals and attenuation of psoriasis phenotypes. Delivery of ds-cRNAs by lipid nanoparticles to the spleen attenuates PKR activity in examined splenocytes, resulting in reduced epidermal thickness. These findings suggest that ds-cRNAs represent a promising approach to mitigate excessive PKR activation for therapeutic purposes.