Project description: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)
Project description: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.
Project description:MicroRNAs (miRNAs) act in concert with Argonaute (AGO) proteins to broadly repress mRNA targets. After AGO loading, miRNAs generally exhibit slow turnover. An important exception occurs when miRNAs encounter targets with extensive complementarity, which can trigger a process termed target-directed microRNA degradation (TDMD). Prevailing models of TDMD invoke miRNA tailing and trimming as an essential step in the decay mechanism. Here, a genome-wide screen revealed a novel cullin-RING ubiquitin ligase, which we named the DECAY complex, that mediates TDMD. The DECAY complex interacts with AGO proteins, mediates TDMD induced by multiple transcripts, and does not require tailing and trimming to elicit miRNA turnover. Based upon these findings, we propose a model in which the DECAY complex mediates TDMD by promoting proteasomal decay of miRNA-containing complexes.
Project description:MicroRNAs (miRNAs) act in concert with Argonaute (AGO) proteins to broadly repress mRNA targets. After AGO loading, miRNAs generally exhibit slow turnover. An important exception occurs when miRNAs encounter targets with extensive complementarity, which can trigger a process termed target-directed microRNA degradation (TDMD). Prevailing models of TDMD invoke miRNA tailing and trimming as an essential step in the decay mechanism. Here, a genome-wide screen revealed a novel cullin-RING ubiquitin ligase, which we named the DECAY complex, that mediates TDMD. The DECAY complex interacts with AGO proteins, mediates TDMD induced by multiple transcripts, and does not require tailing and trimming to elicit miRNA turnover. Based upon these findings, we propose a model in which the DECAY complex mediates TDMD by promoting proteasomal decay of miRNA-containing complexes.
Project description:MicroRNAs (miRNAs) act in concert with Argonaute (AGO) proteins to broadly repress mRNA targets. After AGO loading, miRNAs generally exhibit slow turnover. An important exception occurs when miRNAs encounter targets with extensive complementarity, which can trigger a process termed target-directed microRNA degradation (TDMD). Prevailing models of TDMD invoke miRNA tailing and trimming as an essential step in the decay mechanism. Here, a genome-wide screen revealed a novel cullin-RING ubiquitin ligase, which we named the DECAY complex, that mediates TDMD. The DECAY complex interacts with AGO proteins, mediates TDMD induced by multiple transcripts, and does not require tailing and trimming to elicit miRNA turnover. Based upon these findings, we propose a model in which the DECAY complex mediates TDMD by promoting proteasomal decay of miRNA-containing complexes.
Project description:Next-generation sequencing experiments have shown that microRNAs are expressed in many different isoforms (isomiRs), whose biological relevance is often unclear. We found that mature miR-21, the most widely researched microRNA because of its importance in human disease, is produced in two prevalent isomiR forms that differ by one nucleotide at their 3’ end, and moreover that the 3’ end of miR-21 is post transcriptionally adenylated by the noncanonical poly(A) polymerase PAPD5. PAPD5 knockdown caused an increase in the miR-21 expression level, suggesting that PAPD5-mediated adenylation of miR-21 leads to its degradation. Exoribonuclease knockdown experiments followed by small RNA sequencing suggested that PARN degrades miR-21 in the 3’-to-5’ direction. In accordance with this model, microarray expression profiling demonstrated that PAPD5 knockdown results in a downregulation of miR-21 target mRNAs. We found that disruption of the miR-21 adenylation and degradation pathway is a general feature in tumors across a wide range of tissues, as evidenced by data from The Cancer Genome Atlas, as well as in the non-cancerous proliferative disease psoriasis. We conclude that PAPD5 and PARN mediate degradation of oncomiR miR-21 through a tailing and trimming process, and that this pathway is disrupted in cancer and other proliferative diseases.