Sequencing of Argonaute-bound microRNA/mRNA hybrids reveals regulation of the unfolded protein response by microRNA-320a.

Christopher J Fields,Lauren Gay,Tongjun Gu,Jiang Bian,Rolf Renne,Mingyi Xie,Tianqi Li,Peike Sheng,Nicholas M Hiers,Lu Li,Michael S Kilberg,Jixiu Shan,Taha Huda,Aleksandra Filipovska

doi:10.1371/journal.pgen.1009934

Christopher J Fields, Lauren Gay + Show 12 more

Open Access

https://doi.org/10.1371/journal.pgen.1009934

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Abstract

MicroRNAs (miRNA) are short non-coding RNAs widely implicated in gene regulation. Most metazoan miRNAs utilize the RNase III enzymes Drosha and Dicer for biogenesis. One notable exception is the RNA polymerase II transcription start sites (TSS) miRNAs whose biogenesis does not require Drosha. The functional importance of the TSS-miRNA biogenesis is uncertain. To better understand the function of TSS-miRNAs, we applied a modified Crosslinking, Ligation, and Sequencing of Hybrids on Argonaute (AGO-qCLASH) to identify the targets for TSS-miRNAs in HCT116 colorectal cancer cells with or without DROSHA knockout. We observed that miR-320a hybrids dominate in TSS-miRNA hybrids identified by AGO-qCLASH. Targets for miR-320a are enriched for the eIF2 signaling pathway, a downstream component of the unfolded protein response. Consistently, in miR-320a mimic- and antagomir- transfected cells, differentially expressed gene products are associated with eIF2 signaling. Within the AGO-qCLASH data, we identified the endoplasmic reticulum (ER) chaperone calnexin as a direct miR-320a down-regulated target, thus connecting miR-320a to the unfolded protein response. During ER stress, but not amino acid deprivation, miR-320a up-regulates ATF4, a critical transcription factor for resolving ER stress. In summary, our study investigates the targetome of the TSS-miRNAs in colorectal cancer cells and establishes miR-320a as a regulator of unfolded protein response.

Highlights

MicroRNAs are ~18–22 nucleotide non-coding RNAs that target the majority of messenger RNAs for post-transcriptional regulation in a tissue-specific manner [1]
We used CLASH and its variant quick CLASH (qCLASH) to elucidate the miRNA targetomes in HCT116 cells, including WT, DROSHA KO, and DICER KO cells (Fig 1A and 1B)
We concluded that the efficacy of the 4F9 antibody rivals the widely used 2A8 antibody, and both are suitable for CLASH experiments

Summary

Introduction

MicroRNAs (miRNAs) are ~18–22 nucleotide non-coding RNAs that target the majority of messenger RNAs (mRNAs) for post-transcriptional regulation in a tissue-specific manner [1]. The Microprocessor complex comprises two RNA-binding proteins (DGCR8) and one RNase III enzyme (Drosha) [4]. Following Drosha cleavage, Exportin-5 shuttles the resulting precursor miRNA (pre-miRNA) into the cytoplasm, where Dicer removes the loop to form a ~22 base pairs double-stranded RNA [5]. Mature miRNA is loaded onto the effector protein Argonaute (AGO), forming the RNA-induced silencing complex (RISC) [5,6]. One strand is retained as part of the RISC and guides the complex to target mRNAs. Canonically, miRNAs target mRNA’s 30 untranslated region (UTR) using their seed sequence (nucleotides 2–8) by Watson-Crick base-pairing [7]. The binding of the RISC complex leads to the recruitment of the CCR4-NOT deadenylase and DCP1-DCP2 decapping complexes for mRNA degradation [8]

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