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

PLoS Genet. 2021 Dec 16;17(12):e1009934. doi: 10.1371/journal.pgen.1009934. eCollection 2021 Dec.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Activating Transcription Factor 4 / genetics*
  • Antagomirs / genetics
  • Argonaute Proteins / genetics
  • Calnexin / genetics
  • Cell Movement / genetics
  • Cell Proliferation / genetics
  • Colorectal Neoplasms / genetics*
  • Colorectal Neoplasms / pathology
  • DEAD-box RNA Helicases / genetics
  • Endoplasmic Reticulum / genetics
  • Endoplasmic Reticulum Stress / genetics
  • Eukaryotic Initiation Factor-2 / genetics
  • Gene Knockout Techniques
  • HCT116 Cells
  • Humans
  • MicroRNAs / genetics*
  • Ribonuclease III / genetics*
  • Signal Transduction / genetics
  • Transcription Initiation Site


  • ATF4 protein, human
  • Antagomirs
  • Argonaute Proteins
  • Eukaryotic Initiation Factor-2
  • MIRN320 microRNA, human
  • MicroRNAs
  • Calnexin
  • Activating Transcription Factor 4
  • DICER1 protein, human
  • DROSHA protein, human
  • Ribonuclease III
  • DEAD-box RNA Helicases