Augmentation of nonsense mediated decay by rapamycin

Abstract

RNA surveillance by the Nonsense Mediated Decay (NMD) pathway eliminates potentially deleterious transcripts containing Premature Termination Codons (PTCs). The transition from a pioneering round of translation to steady state translation is hypothesized to be a major checkpoint in this process. One hallmark of mRNAs licensed for translation is the exchange of 7-methylguanosine cap binding proteins. However, mRNAs undergoing steady state translation are also NMD substrates, raising mechanistic questions about the NMD checkpoint. To test the role of cap binding proteins in NMD, we modulated the protein composition of cytoplasmic messenger ribonucleoprotein particles (mRNPs) with the naturally occurring macrolide rapamycin. We demonstrate that despite well-documented attenuation of cap-dependent mRNA translation, rapamycin can augment NMD. Rapamycin-treatment significantly reduces the levels of endogenous and exogenous PTC-containing mRNA isoforms in a dose-and UPF1-dependent manner. PTC-containing transcripts exhibit a shorter half-life upon rapamacyin-treatment as compared to non-PTC isoforms. Rapamycin also causes depletion of PTC-containing mRNA isoforms from polyribosomes, suggesting that actively translating ribosomes can transition between low and high NMD states. Importantly, mRNPs show depletion of eIF4E and retention of the nuclear Cap BindingComplex (CBC) in rapamycin-treated cells. Our data demonstrate that rapamycin potentiates pioneer-like mRNP context thereby decreasing NMD evasion.

An alternative UPF1 isoform drives conditional remodeling of nonsense‐mediated mRNA decay

“…We again compared published RNA‐seq datasets with our UPF1 RIP‐seq data to provide a preliminary test of this hypothesis. As we observed using published RNA‐seq data from cells undergoing ER stress, mRNAs preferentially bound by UPF1 LL were systematically down‐regulated upon treatment with moderate doses of the initiation inhibitor hippuristanol or the elongation inhibitors emetine and cycloheximide (Fig EV4A–C) (Data ref: Martinez‐Nunez & Sanford, 2016; Martinez‐Nunez et al , 2017; Kearse et al , 2019; Data ref: Kearse et al , 2019; Waldron et al , 2019; Data ref: Waldron et al , 2019). In the case of cycloheximide treatment, only 15 min of treatment was sufficient to observe significant loss of UPF1 LL ‐enriched transcripts, consistent with a decay‐driven process.…”