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Two-motif model illuminates DICER's cleavage preferences [DICER-rescue]

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE247114 | GEO | 2023/11/30

REPOSITORIES: GEO

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Two-motif model illuminates DICER's cleavage preferences [DICER]

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.

2023-09-06 | GSE226874 | GEO

Two-motif model illuminates DICER's cleavage preferences [DICER-mutants]

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.

2023-11-30 | GSE247113 | GEO

The high-throughput DICER cleavage assays on 23296 shRNA variants

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.

2022-02-21 | GSE182701 | GEO

The loop position of shRNAs and pre-miRNAs is critical for the accuracy of Dicer processing in vivo

Project description:Short-hairpin RNA (shRNA)-induced RNAi is used for biological discovery and therapeutics. Dicer, whose normal role is to liberate endogenous miRNAs from their precursors, processes shRNAs into different biologically active siRNAs, affecting their efficacy and potential for off-targeting. We found that in cells, Dicer induced imprecise cleavage events around the expected sites based on the previously described 5'/3'-counting rules. These promiscuous non-canonical cleavages were abrogated when the cleavage site was positioned 2 nt from a bulge or loop. Interestingly, we observed that the ~1/3 of mammalian endogenous pre-miRNAs that contained such structures were more precisely processed by Dicer. Implementing a new "loop-counting rule", we designed potent anti-HCV shRNAs with substantially reduced off-target effects. Our results suggest that Dicer recognizes the loop/bulge structure in addition to the ends of shRNAs/pre-miRNAs for accurate processing. This has important implications for both miRNA processing and future design of shRNAs for RNAi-based genetic screens and therapies.

2012-12-25 | GSE41292 | GEO

The loop position of shRNAs and pre-miRNAs is critical for the accuracy of Dicer processing in vivo

2012-12-25 | E-GEOD-41292 | biostudies-arrayexpress

Sequence determinants of dsRNA processing by DICER [Rescue Experiment]

Project description:RNA silencing relies on specific and efficient processing of dsRNA by DICER which produces microRNAs (miRNAs) and small interfering RNAs (siRNAs). However, our current knowledge of DICER’s specificity is restricted to the secondary structures of its substrates: a dsRNA than 22 bp with a 2-nt 3′ overhang. We recently found evidence pointing to additional sequence-dependent determinant(s) beyond these features. To systematically interrogate the features of precursor miRNAs (pre-miRNAs), we carried out massively parallel assays with over a million pre-miRNA variants. Our analyses revealed a highly conserved cis-acting element, termed the “GYM” motif (paired G, paired pyrimidine, and mismatched C or A) at the cleavage site, which strongly promotes processing at a specific position. We find that the C-terminal double-stranded RNA binding domain (dsRBD) of DICER recognizes the GYM motif. Mutation of the dsRBD alters pre-miRNA cleavage sites and impairs processing in a motif-dependent fashion, which in turn affects the miRNA repertoire in cells. Consistently, integrating the GYM motif into short hairpin RNA (shRNA) or Dicer substrate siRNA (DsiRNA) potentiates RNA interference.

2022-12-16 | GSE202533 | GEO

Sequence determinants of dsRNA processing by DICER [Massively Parallel Assay]

2022-12-16 | GSE202530 | GEO

The high-throughput human pri-miRNA processing assays

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.

2023-01-23 | GSE215790 | GEO

Small RNA sequencing of mouse muscle and heart samples after small hairpin RNA delivery

Project description:RNA interference (RNAi) is a promising gene therapy strategy for targeting dominant mutations. This therapy leverages small hairpin RNAs (shRNAs) to knock down gene expression; however, delivery of too much shRNA can disrupt the processing of endogenous microRNAs (miRNAs), and lead to toxicity. In this study, we sought to understand the effect that excessive shRNAs have on muscle miRNAs by transducing several constructs in mice and performing small RNA sequencing on their muscle and liver tissues. We found that shRNA expression was highest in the heart, and that when shRNAs accumulated to about 27% of total small RNAs, mice experienced substantial cardiomyopathy. At this level in the heart, shRNAs in other muscle tissues were only ~1.8-7.6% of total miRNAs. Regardless of treatment, the predominant heart microRNAs remained stable across samples, while the lower-expressed miR-451 – one of the only microRNAs processed in a Dicer-independent manner – changed in direct correlation with shRNA level and toxicity. Our data suggest that certain muscle miRNAs compete with exogenous shRNAs, leading to the observed cardiomyopathy, and that the mechanism of cardiotoxicity in these mice in response shRNA treatment was in contrast to what has been previously shown in the liver. Quantifying microRNA profiles after excessive shRNA delivery illuminates the host response to rAAV-shRNA, allowing for safer and more robust therapeutic gene knockdown, and gives us insight into the basic functions of muscle microRNAs.

2019-12-30 | GSE129896 | GEO

MicroRNA-seq of MDA-MB-231 cells transduced with anti-ELOVL5 and control shRNAs

Project description:MiRNA isoforms (isomiRs) are single stranded small RNAs originating from the same pri-miRNA hairpin as a result of cleavage by Drosha and Dicer enzymes. Variations at the 5’-end of a miRNA alter the seed region of the molecule, thus affecting the targetome of the miRNA. MDA-MB-231 cells were transduced with shRNAs against ELOVL5 and luciferase genes. Small RNA sequencing was used to study the cleavage profiles of shRNAs.

2021-05-03 | GSE173709 | GEO

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