Protein products of nonstop mRNA disrupt nucleolar homeostasis

Abstract

Stalled mRNA translation results in the production of incompletely synthesized proteins that are targeted for degradation by ribosome-associated quality control (RQC). Here we investigated the fate of defective proteins translated from stall-inducing, nonstop mRNA that escape ubiquitylation by the RQC protein LTN1. We found that nonstop protein products accumulated in nucleoli and this localization was driven by polylysine tracts produced by translation of the poly(A) tails of nonstop mRNA. Nucleolar sequestration increased the solubility of invading proteins but disrupted nucleoli, altering their dynamics, morphology, and resistance to stress in cell culture and intact flies. Our work elucidates how stalled translation may affect distal cellular processes and may inform studies on the pathology of diseases caused by failures in RQC and characterized by nucleolar stress.

Keywords: LTN1; Nonstop mRNA; Nucleolus; Phase separation; Protein quality control; Ribosome-associated quality control (RQC).

Fig. 1

PolyK tracts drive the protein products of nonstop mRNA into nucleoli. a Schematic of RQC pathway acting on GFP nonstop substrate. Translation proceeds through the UTR into the poly(A) tail, creating a polyK tract. Ribosome stalling then leads to dissociation of the 60S subunit, ubiquitination of the nascent chain by LTN1, and subsequent degradation. b Localization of GFP-nonstop and GFP-stop. HeLa cells were transfected with GFP reporter constructs, then fixed and immunostained with nucleolin (NCL). White dashed lines mark transfected cell outlines. Scale bar = 10 μm. c Effect of C-terminal cleavage on nucleolar localization. HeLa cells were transfected with GFP-TEV-containing constructs (shown in diagram) with or without a plasmid encoding the Tobacco Etch Virus (TEV) protease. Scale bar = 10 μm. d Localization of GFP reporters in HeLa cells immunostained with NCL. Scale bar = 10 μm

Fig. 2

Nucleolar targeting increases solubility of amyloidogenic sequences. a, b 24 h post-transfection HeLa cells expressing GFP-UTR were assessed for nucleolar and perinuclear amyloid by Amylo-glo staining. Microscopy is quantified in b. The mean Amylo-glo and GFP intensities were measured in 100 ROIs inside nucleoli and 100 perinuclear aggregates, in three independent experiments. Scale bar = 10 μm. c Fluorescence recovery after photobleaching (FRAP) was performed on the nucleolar (orange) and perinuclear (gray) compartments of HeLa cells expressing GFP-UTR. d Cells expressing GFP constructs for 24 h were assessed for amyloid by Amylo-glo staining. Scale bar = 10 μm. e FRAP was performed on the nucleoli of cells expressing the indicated constructs. f Anti-GFP western blot was performed on solubility-fractionated HeLa cell lysates. Lysate loading volumes were normalized so the soluble and insoluble fractions represent equivalent numbers of cells

Fig. 3

Accumulating nonstop proteins disrupt nucleoli. a, b siRNA-treated HeLa cells were treated with 10 μM MG132 for 13 h and allowed to recover for 5 h. Cells were assessed for amyloid by Amylo-glo staining. Scale bar = 10 μm. Microscopy is quantified in b: p = 0.003 for three independent experiments, ± SEM. c, d FRAP was performed using GFP-FBL in HeLa cells treated with siLTN1 and siSCR (c) or RFP-nonstop and RFP-stop (d). Data is presented as means ± the standard error (SE) of three independent experiments (5–20 cells each). p values are of the mobile fractions. e–g siLTN1-treated and GFP nonstop-expressing HUVEC cells have smaller, rounder nucleoli. Results of automated image analysis quantifying changes in nucleolar morphology of siLTN1 (f) and GFP-nonstop (g) HUVEC cells. Data are presented as means −1 ± SE of the control distribution (siSCR (f) and GFP-stop (g)) for >three independent experiments (>500 cells each). Red dots represent replicates. *p < 0.05; **p < 0.054. n.s., not significant. h–j Muscle cells from flies expressing one or two copies of an RNAi transgene targeting Ltn1 were immunostained for FBL without (h) or with (i) 37°C heat shock. Scale bar = 5 μm. Data are quantified in j. *p < 0.05. n.s., not significant

Fig. 4

Invading nucleolar proteins experience greater solubility but can also disrupt nucleoli. Model for the effect of nonstop proteins on cells: translation of the poly(A) tail of nonstop mRNAs produces a polyK tract. This causes the ribosome to stall, and in the case of insufficient LTN1, cells accumulate nonstop proteins. The polyK tail localizes the nonstop protein to nucleoli, bringing the aggregation-prone, hydrophobic translated 3′ UTR with it. Interactions between nucleolar components and the infiltrating aggregation-prone protein increase solubility of defective protein but alter nucleolar dynamics and morphology, and interfere with the ability to recover from stress (depicted in the model with the transition from black to red nucleoli)