ABSTRACT
The Microprocessor complex is crucial in microRNA (miRNA) biogenesis, as it processes primary miRNAs (pri-miRNAs) into precursor miRNAs. Here, we present a high-throughput, radioisotope-free protocol for studying pri-miRNA processing using randomized sequences. We describe steps for randomized substrate preparation, protein purification, processing assays, and DNA library construction for sequencing. This technique explores pri-miRNA processing, uncovers key RNA elements, and illuminates gene expression regulation. However, its efficacy may be constrained by data analysis complexity and the requirement for specialized equipment. For complete details on the use and execution of this protocol, please refer to Nguyen et al. (2023).1.
Subject(s)
MicroRNAs , RNA Processing, Post-Transcriptional , MicroRNAs/genetics , MicroRNAs/metabolism , Gene Expression Regulation , Cell Nucleus/metabolism , Randomized Controlled Trials as TopicABSTRACT
The Microprocessor complex (MP) is a vital component in the biogenesis of microRNAs (miRNAs) in animals. It plays a crucial role in the biogenesis of microRNAs (miRNAs) in mammals as it cleaves primary miRNAs (pri-miRNAs) to initiate their production. The accurate enzymatic activity of MP is critical to ensuring proper sequencing and expression of miRNAs and their correct cellular functions. RNA elements in pri-miRNAs, including secondary structures and sequencing motifs, RNA editing and modifications, and cofactors, can impact MP cleavage and affect miRNA expression and sequence. To evaluate MP cleavage activity with various RNA substrates under different conditions, we set up an in vitro pri-miRNA cleavage assay. This involves purifying human MP from HEK293E cells, synthesizing pri-miRNAs using in vitro transcription, and performing pri-miRNA cleavage assays using basic laboratory equipment and reagents. These procedures can be performed in various labs and improved for high-throughput analysis of enzymatic activities with thousands of RNA substrates.
Subject(s)
MicroRNAs , RNA Processing, Post-Transcriptional , Animals , Humans , Ribonuclease III/chemistry , Ribonuclease III/genetics , Ribonuclease III/metabolism , MicroRNAs/chemistry , RNA Editing , Microcomputers , Mammals/geneticsABSTRACT
MicroRNAs (miRNAs) are small, non-coding RNA molecules that play a crucial role in gene silencing. The gene-silencing activity of miRNAs depends on their sequences and expression levels. The human RNase III enzyme DICER cleaves miRNA precursors (pre-miRNAs) to produce miRNAs, making it crucial for miRNA production and cellular miRNA functions. DICER is also critical for the gene silencing technology using short-hairpin RNAs (shRNAs), which are cleaved by DICER to generate siRNAs that knockdown target genes. The DICER cleavage assay is an important tool for investigating its molecular mechanisms, which are essential for understanding its functions in miRNA biogenesis and shRNA-based gene silencing technology. The assay involves DICER protein purification, preparation of pre-miRNA and shRNA substrates, and the cleavage assay, using common molecular biology equipment and commercialized reagents that can be applied to other RNA endonucleases.
Subject(s)
MicroRNAs , Humans , MicroRNAs/chemistry , Ribonuclease III/genetics , Ribonuclease III/chemistry , Ribonuclease III/metabolism , RNA, Small Interfering/genetics , RNA, Double-StrandedABSTRACT
Microprocessor (MP), DROSHA-DGCR8, processes primary miRNA transcripts (pri-miRNAs) to initiate miRNA biogenesis. The canonical cleavage mechanism of MP has been extensively investigated and comprehensively validated for two decades. However, this canonical mechanism cannot account for the processing of certain pri-miRNAs in animals. In this study, by conducting high-throughput pri-miRNA cleavage assays for approximately 260,000 pri-miRNA sequences, we discovered and comprehensively characterized a noncanonical cleavage mechanism of MP. This noncanonical mechanism does not need several RNA and protein elements essential for the canonical mechanism; instead, it utilizes previously unrecognized DROSHA dsRNA recognition sites (DRESs). Interestingly, the noncanonical mechanism is conserved across animals and plays a particularly significant role in C. elegans. Our established noncanonical mechanism elucidates MP cleavage in numerous RNA substrates unaccounted for by the canonical mechanism in animals. This study suggests a broader substrate repertoire of animal MPs and an expanded regulatory landscape for miRNA biogenesis.
Subject(s)
MicroRNAs , Animals , MicroRNAs/metabolism , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , RNA-Binding Proteins/metabolism , Ribonuclease III/metabolism , RNA, Double-Stranded , RNA Processing, Post-TranscriptionalABSTRACT
MicroRNAs (miRNAs) play critical roles in gene expression and numerous human diseases. The success of miRNA biogenesis is largely determined by the primary miRNA (pri-miRNA) processing by the DROSHA-DGCR8 complex, called Microprocessor. Here, we analysed the high-throughput pri-miRNA processing assays and secondary structures of pri-miRNAs to investigate the roles of bulges in the pri-miRNA processing. We found that bulges in multiple places control both the cleavage efficiency and accuracy of pri-miRNA processing. These bulges were shown to act on Microprocessor via its catalytic subunit, DROSHA, and function in a position and strand-dependent manner. Interestingly, we discovered that the enriched and conserved bulges, called midB, can correct DROSHA orientation on pri-miRNAs, thereby enhancing production of miRNAs. The revealed functions of the bulges help improve our understanding of pri-miRNA processing and suggest their potential roles in miRNA biogenesis regulation.
