AGO2 protects Drosophila siRNAs and microRNAs from target-directed degradation, even in the absence of 2′-O-methylation
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ABSTRACT:
PROVIDER: PRJNA688281 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR13314100.fastq.gz | Fastqsanger.gz | |||
| SRR13314101.fastq.gz | Fastqsanger.gz | |||
| SRR13314102.fastq.gz | Fastqsanger.gz | |||
| SRR13314103.fastq.gz | Fastqsanger.gz | |||
| SRR13314104.fastq.gz | Fastqsanger.gz |
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Project description:Target-directed degradation (TDD) of microRNAs (miRNAs) plays an important role in shaping miRNA abundances across bilateria. Some endogenous small interfering RNAs (siRNAs) of Drosophila cells have target sites resembling those that trigger miRNA TDD, raising the question as to whether they too might undergo such regulation. Here, we find that at least seven of these siRNAs are indeed sensitive to TDD when loaded into AGO1, the Argonaute paralog that preferentially associates with miRNAs. Despite this sensitivity when loaded into AGO1, these siRNAs are not detectably regulated by TDD because most molecules are loaded into AGO2, the Argonaute paralog that preferentially associates with siRNAs, and we find that siRNAs and miRNAs loaded into AGO2 are insensitive to TDD. One explanation for the protection of these small RNAs loaded into AGO2 is that these small RNAs are 2′-O-methylated at their 3′ termini. However, contrary to previous proposals, 2′-O-methylation does not protect these RNAs from TDD, which indicates that their protection is instead conferred by features of the AGO2 protein itself. Together, these observations clarify the requirements for regulation by TDD and revise our understanding of the role of 2′-O-methylation in small-RNA biology.
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Project description:Target-directed microRNA (miRNA) degradation (TDMD), which is mediated by the protein ZSWIM8, plays a widespread role in shaping miRNA abundances across bilateria. Some endogenous small interfering RNAs (siRNAs) of Drosophila cells have target sites resembling those that trigger TDMD, raising the question as to whether they too might undergo such regulation by Dora, the Drosophila ZSWIM8 homolog. Here, we find that some of these siRNAs are indeed sensitive to Dora when loaded into Ago1, the Argonaute paralog that preferentially associates with miRNAs. Despite this sensitivity when loaded into Ago1, these siRNAs are not detectably regulated by target-directed degradation because most molecules are loaded into Ago2, the Argonaute paralog that preferentially associates with siRNAs, and we find that siRNAs and miRNAs loaded into Ago2 are insensitive to Dora. One explanation for the protection of these small RNAs loaded into Ago2 is that these small RNAs are 2'-O-methylated at their 3' termini. However, 2'-O-methylation does not protect these RNAs from Dora-mediated target-directed degradation, which indicates that their protection is instead conferred by features of the Ago2 protein itself. Together, these observations clarify the requirements for regulation by target-directed degradation and expand our understanding of the role of 2'-O-methylation in small-RNA biology.
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Project description:Small interfering RNAs (siRNAs) direct RNA interference (RNAi) in eukaryotes. In flies, somatic cells produce siRNAs from exogenous double-stranded RNA as a defense against viral infection. Here, we identify 21-nt long, endogenous siRNAs (endo-siRNAs) corresponding to transposons and heterochromatic sequences in the somatic cells of Drosophila melanogaster. We also detected endo-siRNAs complementary to mRNAs: these siRNAs disproportionately mapped to the complementary regions of overlapping mRNAs predicted to form dsRNA in vivo. Normal accumulation of somatic endo-siRNAs requires the siRNA-generating ribonuclease, Dicer-2, and the RNAi effector protein, Ago2. We propose that endo-siRNAs generated by the fly RNAi pathway silence selfish genetic elements in the soma much as piRNAs do in the germ line. Keywords: Small RNA detection and quantification. Small RNAs (18-30 nt) from fly heads (WT, ago2 mutants, dcr-2 homozygous and heterozygous mutants, and WT expressing an inverted repeat directed against exon 3 of the gene "white") and S2 cells (transgenic for a construct expressing siRNAs against white and GFP) were sequenced using a Solexa Genome Analyzer instrument. Raw sequence data files for this study are available for download from the SRA FTP site at ftp://ftp.ncbi.nlm.nih.gov/sra/Studies/SRP000/SRP000181
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Project description:Diabetes is associated with cardiovascular complications. microRNAs translocate into subcellular organelles to modify genes involved in diabetic cardiomyopathy. However, functional properties of subcellular Ago2, a core microRNA, remain elusive. We elucidated the function and mechanism of subcellular localized Ago2 on mouse models for diabetes mellitus and diabetic cardiomyopathy. Ago2 decreased in cardiomyocyte mitochondria in both models. Overexpression of mitochondrial Ago2 attenuated diabetes-induced cardiac dysfunction. Ago2 recruited TUFM, a mitochondria translation elongation factor, to activate translation of electron transport chain (ETC) subunits and decrease reactive oxygen species. Malonylation, a post-translational modification of Ago2, reduced the importing of Ago2 into mitochondria in diabetic cardiomyopathy. Ago2 malonylation was regulated by a cytoplasmic-localized short isoform of SIRT3 through a previously unknown demalonylase function. Our results reveal that the SIRT3–Ago2–CYTB axis links glucotoxicity to cardiac ETC imbalance, providing new mechanistic insights and the basis to develop mitochondria targeting therapies for diabetic cardiomyopathy.
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Project description:Diabetes is associated with cardiovascular complications. microRNAs translocate into subcellular organelles to modify genes involved in diabetic cardiomyopathy. However, functional properties of subcellular Ago2, a core microRNA, remain elusive. We elucidated the function and mechanism of subcellular localized Ago2 on mouse models for diabetes mellitus and diabetic cardiomyopathy. Ago2 decreased in cardiomyocyte mitochondria in both models. Overexpression of mitochondrial Ago2 attenuated diabetes-induced cardiac dysfunction. Ago2 recruited TUFM, a mitochondria translation elongation factor, to activate translation of electron transport chain (ETC) subunits and decrease reactive oxygen species. Malonylation, a post-translational modification of Ago2, reduced the importing of Ago2 into mitochondria in diabetic cardiomyopathy. Ago2 malonylation was regulated by a cytoplasmic-localized short isoform of SIRT3 through a previously unknown demalonylase function. Our results reveal that the SIRT3–Ago2–CYTB axis links glucotoxicity to cardiac ETC imbalance, providing new mechanistic insights and the basis to develop mitochondria targeting therapies for diabetic cardiomyopathy.
2024-09-27 | GSE242024 | GEO
Project description:Diabetes is associated with cardiovascular complications. microRNAs translocate into subcellular organelles to modify genes involved in diabetic cardiomyopathy. However, functional properties of subcellular Ago2, a core microRNA, remain elusive. We elucidated the function and mechanism of subcellular localized Ago2 on mouse models for diabetes mellitus and diabetic cardiomyopathy. Ago2 decreased in cardiomyocyte mitochondria in both models. Overexpression of mitochondrial Ago2 attenuated diabetes-induced cardiac dysfunction. Ago2 recruited TUFM, a mitochondria translation elongation factor, to activate translation of electron transport chain (ETC) subunits and decrease reactive oxygen species. Malonylation, a post-translational modification of Ago2, reduced the importing of Ago2 into mitochondria in diabetic cardiomyopathy. Ago2 malonylation was regulated by a cytoplasmic-localized short isoform of SIRT3 through a previously unknown demalonylase function. Our results reveal that the SIRT3–Ago2–CYTB axis links glucotoxicity to cardiac ETC imbalance, providing new mechanistic insights and the basis to develop mitochondria targeting therapies for diabetic cardiomyopathy.
2024-09-27 | GSE242185 | GEO
Project description:Diabetes is associated with cardiovascular complications. microRNAs translocate into subcellular organelles to modify genes involved in diabetic cardiomyopathy. However, functional properties of subcellular Ago2, a core microRNA, remain elusive. We elucidated the function and mechanism of subcellular localized Ago2 on mouse models for diabetes mellitus and diabetic cardiomyopathy. Ago2 decreased in cardiomyocyte mitochondria in both models. Overexpression of mitochondrial Ago2 attenuated diabetes-induced cardiac dysfunction. Ago2 recruited TUFM, a mitochondria translation elongation factor, to activate translation of electron transport chain (ETC) subunits and decrease reactive oxygen species. Malonylation, a post-translational modification of Ago2, reduced the importing of Ago2 into mitochondria in diabetic cardiomyopathy. Ago2 malonylation was regulated by a cytoplasmic-localized short isoform of SIRT3 through a previously unknown demalonylase function. Our results reveal that the SIRT3–Ago2–CYTB axis links glucotoxicity to cardiac ETC imbalance, providing new mechanistic insights and the basis to develop mitochondria targeting therapies for diabetic cardiomyopathy.
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Project description:The RNase III enzyme DICER generates both microRNAs (miRNAs) and endogenous short interfering RNAs (endo-siRNAs). Dicer deletion in mouse oocytes leads to female infertility due to defects during meiosis I. Because miRNA function is suppressed in mouse oocytes, it has been proposed that endo-siRNAs may have a role during female meiosis. By utilizing a catalytically inactive knock-in allele of AGO2 specifically in oocytes, we disrupt the function of siRNAs and analyze the transcriptome of these oocytes in comparison with Ago2 null, and Dicer null oocytes.
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Project description:The RNase III enzyme DICER generates both microRNAs (miRNAs) and endogenous short interfering RNAs (endo-siRNAs). Dicer deletion in mouse oocytes leads to female infertility due to defects during meiosis I. Because miRNA function is suppressed in mouse oocytes, it has been proposed that endo-siRNAs may have a role during female meiosis. By utilizing a catalytically inactive knock-in allele of AGO2 specifically in oocytes, we disrupt the function of siRNAs and analyze the transcriptome of these oocytes in comparison with Ago2 null, and Dicer null oocytes. Germinal vesicle-intact, full-grown oocytes were collected from eCG-primed females expressing a catalytically inactive knock-in allele of Ago2 (Ago2 ADH) specifically in oocytes. Oocytes were also collected from Ago2 null and Ago2 fl/fl oocytes, as well as Dicer wild-type and null mice. Oocytes were freed of attached cumulus cells by pipetting. Twenty oocytes collected from 3 different mice were used for high-throughput RNA sequencing. Three replicates for all genotypes except Ago2 null (two). The mouse background strain is "mostly" C57BL6, i.e., it is a cross between 3 different transgenic mice of different strains.
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