Sequestration of microRNA-mediated target repression by the Ago2-associated RNA-binding protein FAM120A

• Published in: RNA (Cambridge) Vol. 25; no. 10; pp. 1291 - 1297
• Main Authors: Kelly, Timothy J., Suzuki, Hiroshi I., Zamudio, Jesse R., Suzuki, Megumu, Sharp, Phillip A.
• Format: Journal Article
• Language: English
• Published: United States Cold Spring Harbor Laboratory Press 01.10.2019
• Subjects: 3' Untranslated Regions; Animals; Argonaute 2 protein; Argonaute Proteins - metabolism; Cells, Cultured; Embryo cells; Embryonic Stem Cells - metabolism; Immunoprecipitation; Mass spectroscopy; Mice; MicroRNAs; MicroRNAs - metabolism; miRNA; Nerve Tissue Proteins - metabolism; Proteins; RNA-binding protein; RNA-Binding Proteins - metabolism; Stem cell transplantation; Stem cells; microRNA; Ago2; iCLIP; FAM120A
• ISSN: 1355-8382; 1469-9001; 1469-9001
• DOI: 10.1261/rna.071621.119

Argonaute (Ago) proteins interact with various binding partners and play a pivotal role in microRNA (miRNA)-mediated silencing pathways. By utilizing immunoprecipitation followed by mass spectrometry to determine cytoplasmic Ago2 protein complexes in mouse embryonic stem cells (mESCs), we identified a putative RNA-binding protein FAM120A (also known as OSSA/C9ORF10) as an Ago2 interacting protein. Individual nucleotide resolution cross-linking and immunoprecipitation (iCLIP) analysis revealed that FAM120A binds to homopolymeric tracts in 3′-UTRs of about 2000 mRNAs, particularly poly(G) sequences. Comparison of FAM120A iCLIP and Ago2 iCLIP reveals that greater than one-third of mRNAs bound by Ago2 in mESCs are co-bound by FAM120A. Furthermore, such FAM120A-bound Ago2 target genes are not subject to Ago2-mediated target degradation. Reporter assays suggest that the 3′-UTRs of several FAM120A-bound miRNA target genes are less sensitive to Ago2-mediated target repression than those of FAM120A-unbound miRNA targets and FAM120A modulates them via its G-rich target sites. These findings suggest that Ago2 may exist in multiple protein complexes with varying degrees of functionality.