RESUMO
UV crosslinking and immunoprecipitation (CLIP) coupled with high-throughput sequencing is the most state-of-the-art technology to characterize protein-RNA interactions, yet only a small portion of RNA-binding proteins (RBPs) have been studied by CLIP due to its complex procedures and high level of false-positive signals. Herein, we report a SpyCLIP method that employs a covalent linkage formed between the RBP-fused SpyTag and SpyCatcher, which can withstand the harshest washing conditions for removing nonspecific interactions. Moreover, SpyCLIP circumvents the radioactive labeling and PAGE-membrane purification steps, and the whole procedure can be performed on beads and is readily amenable to automation. We investigated multiple RBPs by SpyCLIP and generated high-quality RNA binding maps with significantly improved reproductivity and accuracy. Therefore, the small tag size and convenient protocol of SpyCLIP provides a robust method for both routine characterization and high-throughput studies of protein-RNA interactions.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Análise de Sequência de RNA/métodos , Algoritmos , Sítios de Ligação , Reagentes de Ligações Cruzadas/química , Células HEK293 , Humanos , Imunoprecipitação , Ligação Proteica , Mapeamento de Interação de Proteínas , RNA/química , Proteínas de Ligação a RNA/química , Transcriptoma , Raios UltravioletaRESUMO
Recently, the US Food and Drug Administration (FDA) approved the first small interfering RNA (siRNA) drug, marking a significant milestone in the therapeutic use of RNA interference (RNAi) technology. However, off-target gene silencing by siRNA remains one of the major obstacles in siRNA therapy. Although siRNA off-target effects caused by a mechanism known for microRNA (miRNA)-mediated gene repression have been extensively discussed, whether RNAi can cause unintended cleavage through the effector protein AGO2 at sites harboring partially complementary sequences to the siRNA remains unknown. Here, we report a strategy to establish a comprehensive picture of siRNA cleaved and noncleaved off-targets by performing SpyCLIP using wild-type and catalytically inactive AGO2 mutants in parallel. Additionally, we investigated naturally occurring cleavage events mediated by endogenous miRNAs using the same strategy. Our results demonstrated that AGO2 SpyCLIP is a powerful method to identify both the cleaved and noncleaved targets of siRNAs, providing valuable information for improving siRNA design rules.