Unknown,Transcriptomics,Genomics,Proteomics

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PARN mediates 3'-end trimming of Argonaute-cleaved precursor microRNAs

ABSTRACT: microRNAs (miRNAs) are typically generated as ~22-nucleotide double-stranded RNAs via processing of precursor hairpins by the RNase III enzyme Dicer, after which they are loaded into Argonaute (Ago) proteins to form RNA-induced silencing complex (RISC). However, the biogenesis of miR-451, an erythropoietic miRNA conserved in vertebrates, does not require Dicer processing. Instead, the short pre-miR-451 precursor hairpin is directly loaded into Ago, followed by cleavage of the 3' arm and trimming of the 3' end to the mature length by PARN. Here we show the in vivo activity of miR-430 Ago2-hairpin, a canonical microRNA engineered to fit the structure of miR-451 and hence become Ago2-dependent. Moreover, we test a modified miR-430 Ago2-haipin with 3x phoshorothioate bonds that impairs trimmng. Surprisingly, our data show that trimming of Ago-cleaved pre-miRNAs is not essential for target silencing, indicating that RISC is functional with miRNAs longer than 22-nucleotides. Rescue of MZdicer zebrafish mutant with the injection of trimmable and nontrimmable miR-430 Ago2 hairpins: Transcriptome of wild type, MZdicer mutant, and MZdicer mutant micoinjected with miR-430 duplex, miR-430 (Ago2-haripin), miR-430 (Ago2-haripin 3xPhosphorothioate)

ORGANISM(S): Danio rerio

SUBMITTER: Antonio Giraldez

PROVIDER: E-GEOD-51036 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Poly(A)-specific ribonuclease mediates 3'-end trimming of Argonaute2-cleaved precursor microRNAs.

Yoda Mayuko M Cifuentes Daniel D Izumi Natsuko N Sakaguchi Yuriko Y Suzuki Tsutomu T Giraldez Antonio J AJ Tomari Yukihide Y

Cell reports 20131024 3

MicroRNAs (miRNAs) are typically generated as ~22-nucleotide double-stranded RNAs via the processing of precursor hairpins by the ribonuclease III enzyme Dicer, after which they are loaded into Argonaute (Ago) proteins to form an RNA-induced silencing complex (RISC). However, the biogenesis of miR-451, an erythropoietic miRNA conserved in vertebrates, occurs independently of Dicer and instead requires cleavage of the 3' arm of the pre-miR-451 precursor hairpin by Ago2. The 3' end of the Ago2-cle ...[more]

PMID: 24209750

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Project description:miRNA-mediated gene expression silencing has previously been shown to be important for a variety of physiological and pathological processes. Here, we have explored the role of one bona fide human-specific miRNA (miR-941) in evolution of the human-specific expression and function. Using combination of high-throughput sequencing (GSE26545), miRNA transfection and large-scale PCR of various human populations, we have shown that emergence and rapid expansion of miR-941 might take place on the human evolutionary linage between six and one million years ago. Functionally, miR-941 could be associated with hedgehog and insulin signaling pathways, and thus might potentially play a role in evolution of human longevity. Human-specific effects of miR-941 regulation are detectable in human brain and affect genes involved in neurotransmitter signaling. Furthermore, emergence of miR-941 on the human evolutionary linage was accompanied by the accelerated loss of its binding sites. Taken together, these results strongly implicate the contribution of miR-941 in evolution of the human-specific phenotype. Ago2 Immunoprecipitation (Ago2-IP) experiments after miR-941 overexpression were conducted in 293T cell line. Briefly, All transfections were performed using human 293T cells cultured in 6-well tissue culture plates. Lipofectamine 2000 (Invitrogen) was used for a Synthetic miR-941 or a scrambled oligo transfection, at 30 nmol/l each (final concentration) per 1x106 cells/well of a 6-well plate using DharmaFECT (GE Healthcare). Total 5x106 cells were collected and subjected to Ago2 immunoprecipitation (Ago2-IP) using the RNA isolation kit Mouse Ago2 (Wako Chemicals) according to the manufacturer's instructions. For a negative control, immunoprecipitation was performed using nonimmune IgG beads prepared with the antibody immobilization bead kit (Wako Chemicals). The IP pull down RNA was used as template for an “in vitro” transcription reaction generating biotin-labeled antisense cRNA. The cRNA was analyzed on affymetrix Human Genome U133 Plus 2.0 arrays following the manufacturer’s instructions. R RMA package was used to quantify gene expression levels.

Dataset Information

PARN mediates 3'-end trimming of Argonaute-cleaved precursor microRNAs

Publications

Poly(A)-specific ribonuclease mediates 3'-end trimming of Argonaute2-cleaved precursor microRNAs.

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