MicroRNP‐mediated translational activation of nonadenylated mRNAs in a mammalian cell‐free system

Abstract

MicroRNAs are small noncoding RNAs that regulate translation and mRNA stability by binding target mRNAs in complex with Argonaute (AGO) proteins. AGO interacts with a member of the TNRC6 family proteins to form a microRNP complex, which recruits the CCR4-NOT complex to accelerate deadenylation and inhibits translation. MicroRNAs primarily repress translation of target mRNAs but have been shown to enhance translation of a specific type of target reporter mRNAs in various experimental systems: G0 quiescent mammalian cells, Xenopus laevis oocytes, Drosophila embryo extracts, and HeLa cells. In all of the cases mentioned, a common feature of the activated target mRNAs is the lack of a poly(A) tail. Here, we show let-7-microRNP-mediated translational activation of nonadenylated target mRNAs in a mammalian cell-free system, which contains over-expressed AGO2, TNRC6B, and PAPD7 (TUTase5, TRF4-1). Importantly, translation of nonadenylated mRNAs was activated also by tethered TNRC6B silencing domain (SD), in the presence of PAPD7. Deletion of the poly(A)-binding protein (PABP) interacting motif (PAM2) from the TNRC6B-SD abolished the translational activation, suggesting the involvement of PABP in the process. Similar results were also obtained in cultured HEK293T cells. This work may provide novel insights into microRNP-mediated mRNA regulation.

ABCE1 Acts as a Positive Regulator of Exogenous RNA Decay

“…Forty-eight hours after siRNA-transfection, the cells were further transfected with 5×Flag-EGFP mRNA for 1 h, and then the cells were washed with phosphate-buffered saline to completely shut off supply of 5×Flag-EGFP mRNA. Subsequently, the cells were harvested at the specified time after PBS wash. Total RNA was isolated from the transfected cells using an acid guanidinium thiocyanate-phenol-chloroform extraction method and analyzed by northern blotting using DIG-labeled RNAs as described previously with minor modifications [25]. Total RNA was resolved in a 1.8% agarose/1×MOPS/2% formaldehyde gel.…”

Terminal nucleotidyl transferases (TENTs) in mammalian RNA metabolism

“…A recent report using a mammalian cell-free system based on HEK293F cell extracts suggests involvement of TENT4A in miRNP-mediated translational activation of non-adenylated mRNAs [ 55 ]. Surprisingly, solely the presence of TENT4A, rather than its poly(A) polymerase activity, seems necessary for this activation.…”

Utilizing functional cell‐free extracts to dissect ribonucleoprotein complex biology at single‐molecule resolution

“…Cell extracts have also been the cornerstone of miRNA research, enabling mechanistic interrogation of miRNA biogenesis (Betancur & Tomari, 2012;Förstemann et al, 2007;Miyoshi et al, 2010;O'Brien et al, 2018), miRISC assembly (Azuma-Mukai et al, 2008;Iwasaki et al, 2010;Kawamata et al, 2009;Kawamata & Tomari, 2011;Yoda et al, 2010) and miRISC-mediated gene regulation (Fukao et al, 2014;Mathonnet et al, 2007;Pillai et al, 2005;Wakiyama et al, 2006;Wakiyama et al, 2007;Wakiyama et al, 2018;B. Wang et al, 2006;B.…”

“…Most RISC-active cell extracts have been derived from Drosophila or Caenorhabditis elegans embryos and human cell culture, requiring the meticulous optimization of cell lysis (Fukao et al, 2014;Mathonnet et al, 2007;Wakiyama et al, 2006;Wakiyama et al, 2007;Wakiyama et al, 2018). While commercial cell-free extracts recapitulate gene silencing, they are incompetent in miRNA biogenesis and miRISC assembly (B.…”

“…Cell extracts have also been the cornerstone of miRNA research, enabling mechanistic interrogation of miRNA biogenesis (Betancur & Tomari, 2012; Förstemann et al, 2007; Miyoshi et al, 2010; O'Brien et al, 2018), miRISC assembly (Azuma‐Mukai et al, 2008; Iwasaki et al, 2010; Kawamata et al, 2009; Kawamata & Tomari, 2011; Yoda et al, 2010) and miRISC‐mediated gene regulation (Fukao et al, 2014; Mathonnet et al, 2007; Pillai et al, 2005; Wakiyama et al, 2006; Wakiyama et al, 2007; Wakiyama et al, 2018; B. Wang et al, 2006; B. Wang et al, 2007) at the ensemble level. miRNAs are evolutionarily conserved small (21–24 nucleotides) non‐coding RNAs which guide miRNA‐induced silencing complexes (miRISC) to fine tune gene expression through translational repression and/or mRNA degradation (Jonas & Izaurralde, 2015).…”