Project description:The high-throughput DICER cleavage assays were conducted shRNA variants containing different sequences. We showcase a comprehensive cleavage activity of DICER on different shRNAs containing different secondary structures.
Project description:DICER-LIKE1 (DCL1), a plant-specific RNase III enzyme, is fundamental to post-transcriptional gene regulation mediated by microRNAs (miRNAs). DCL1 processes precursor miRNAs into mature miRNAs, typically 20–22 nucleotides in length. Despite its crucial role, the RNA elements that guide DCL1's cleavage site selection have remained largely uncharacterized. In this study, we employed a high-throughput sequencing approach to analyze Arabidopsis thaliana DCL1 cleavage patterns on over 46,000 short-hairpin RNA (shRNA) sequences previously studied with human DICER. Our analyses revealed that DCL1 cleavage preferences are governed by specific secondary RNA structures and sequence motifs, among which a particular RNA element, designated the GHR motif, emerged as pivotal. This motif significantly influences cleavage site selection independently of the dsRBD and helicase domains of DCL1, operating primarily through the RNase IIIDa domain. Notably, the GHR motif is evolutionarily conserved across plant species and is essential for the precise cleavage of various plant pre-miRNAs. Our findings also suggest a novel role for the GHR motif in the biogenesis of non-canonical 22-nt miRNAs, expanding its functional impact. These insights deepen our understanding of the molecular mechanisms underlying DCL1's specificity and highlight its integral role in miRNA maturation and gene regulatory networks in plants.
Project description: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 (DRES). 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.
Project description:DICER is a key regulator of gene expression in animals through its production of miRNAs and siRNAs by processing shRNA. To advance our understanding of this process, we employed high-throughput assays using various shRNA variants and both wild-type and mutant DICER (DICERΔdsRBD). Our analysis revealed that DICER predominantly cleaves shRNAs at two positions, specifically at 21 (DC21) and 22 (DC22) nucleotides from the 5'-end. Our investigation identified two independent motifs, mAHG and YSR, that determine whether DICER cleaves at DC21 or DC22, depending on their location in shRNA/pre-miRNAs. These motifs can work together or independently to determine the cleavage sites of DICER. Furthermore, our findings indicate that dsRBD enhances cleavage at DC21, and mAHG strengthens the interaction between dsRBD and RNA, leading to an even greater enhancement of DC21 cleavage. Conversely, YSR functions independently of dsRBD. Our study proposes a two-motif model that sheds light on the intricate regulatory mechanisms involved in gene expression by elucidating how DICER recognizes its substrate, providing valuable insights into this critical biological process.
Project description:Human Microprocessor cleaves pri-miRNAs to initiate miRNA biogenesis. The accuracy and efficiency of Microprocessor cleavage ensure appropriate miRNA sequence and expression and thus its proper gene regulation. However, Microprocessor cleaves many pri-miRNAs incorrectly, so it requires assistance from its many cofactors. For example, SRSF3 enhances Microprocessor cleavage by interacting with the CNNC motif in pri-miRNAs. However, whether SRSF3 can function with other motifs and/or requires the motifs in a certain secondary structure is unknown. In addition, the function of SRSF7 (a paralog of SRSF3) in miRNA biogenesis still needs to be discovered. Here, we demonstrated that SRSF7 could stimulate Microprocessor cleavage. In addition, by conducting high-throughput pri-miRNA cleavage assays for Microprocessor and SRSF7 or SRSF3, we demonstrated that SRSF7 and SRSF3 function with the CRC and CNNC motifs, adopting certain secondary structures. In addition, SRSF7 and SRSF3 affect the Microprocessor cleavage sites in human cells. Our findings demonstrate the roles of SRSF7 in miRNA biogenesis and provide a comprehensive view of the molecular mechanism of SRSF7 and SRSF3 in enhancing Microprocessor cleavage.
Project description:The in vitro high-throughput human pri-miRNA processing assays were conducted to check whether mismatches and wobble base pairs in the upper stem of pri-miRNAs affects the DROSHA cleavage.
Project description:In humans, DICER is a key regulator of gene expression in animals through its production of miRNAs and siRNAs by processing miRNA precursors (pre-miRNAs), short-hairpin RNAs (shRNAs), and long double-stranded RNAs (dsRNAs). To advance our understanding of this process, we employed high-throughput assays using various shRNA variants and both wild-type and mutant DICER (DICERΔdsRBD). Our analysis revealed that DICER predominantly cleaves shRNAs at two positions, specifically at 21 (DC21) and 22 (DC22) nucleotides from the 5'-end. Our investigation identified two independent motifs, mWCU and YCR, that determine whether DICER cleaves at DC21 or DC22, depending on their locations in shRNA/pre-miRNAs. These motifs can work together or independently to determine the cleavage sites of DICER. Furthermore, our findings indicate that dsRBD enhances cleavage at DC21, and mWCU strengthens the interaction between dsRBD and RNA, leading to an even greater enhancement of DC21 cleavage. Conversely, YCR functions independently of dsRBD. Our study proposes a two-motif model that sheds light on the intricate regulatory mechanisms involved in gene expression by elucidating how DICER recognizes its substrate, providing valuable insights into this critical biological process.