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Time-resolved small RNA sequencing unravels molecular principles of microRNA homeostasis

ABSTRACT: Time-resolved small RNA sequencing unravels molecular principles of microRNA homeostasis

PROVIDER: PRJNA531916 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR8872260.fastq.gz	Fastqsanger.gz
	SRR8872270.fastq.gz	Fastqsanger.gz
	SRR8872280.fastq.gz	Fastqsanger.gz
	SRR8872290.fastq.gz	Fastqsanger.gz
	SRR8877880.fastq.gz	Fastqsanger.gz

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Project description:Argonaute-bound microRNAs silence mRNA expression in a dynamic and regulated manner to control organismal development, physiology, and disease. We employed metabolic small RNA sequencing for a comprehensive view on intracellular microRNA kinetics in Drosophila. Based on absolute rate of biogenesis and decay, microRNAs rank among the fastest produced and longest-lived cellular transcripts, disposing up to 105 copies per cell at steady-state. Mature microRNAs are produced within minutes, revealing tight intracellular coupling of biogenesis that is selectively disrupted by pre-miRNA-uridylation. Control over Argonaute protein homeostasis generates a kinetic bottleneck that cooperates with non-coding RNA surveillance to ensure faithful microRNA loading. Finally, regulated small RNA decay enables the selective rapid turnover of Ago1-bound microRNAs, but not of Ago2-bound small interfering RNAs (siRNAs), reflecting key differences in the robustness of small RNA silencing pathways. Time-resolved small RNA sequencing opens new experimental avenues to deconvolute the timescales, molecular features, and regulation of small RNA silencing pathways in living cells.

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Time-resolved small RNA sequencing unravels molecular principles of microRNA homeostasis [Ago1OE + TailorKO]

Project description:Argonaute (Ago)-bound microRNAs (miRNAs) silence mRNA expression in a dynamic and regulated manner to control organismal development, physiology and disease. Here, we employed metabolic sequencing of small RNAs for a comprehensive view on intracellular miRNA kinetics in Drosophila. Based on absolute biogenesis and decay rates, miRNAs rank among the fastest produced and most long-lived cellular transcripts, enabling them to reach >105 copies per cell at steady-state. Tight coupling of steps in biogenesis produces mature miRNAs within minutes and is effectively disrupted by pre-miRNA uridylation. In contrast, control over Ago protein homeostasis generates a kinetic bottleneck that cooperates with ncRNA surveillance to ensure faithful miRNA loading. Finally, regulated small RNA decay enables the rapid turnover of specific Ago1-bound miRNAs but not of Ago2-bound siRNAs, reflecting key differences in the robustness of small RNA silencing pathways. Our work opens new experimental avenues to deconvolute the timescales, molecular features, and regulation of small RNA silencing pathways in living cells.

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