Widespread Accumulation of Ribosome-Associated Isolated 3' UTRs in Neuronal Cell Populations of the Aging Brain

Abstract

Particular brain regions and cell populations exhibit increased susceptibility to aging-related stresses. Here, we describe the age-specific and brain-region-specific accumulation of ribosome-associated 3' UTR RNAs that lack the 5' UTR and open reading frame. Our study reveals that this phenomenon impacts hundreds of genes in aged D1 spiny projection neurons of the mouse striatum and also occurs in the aging human brain. Isolated 3' UTR accumulation is tightly correlated with mitochondrial gene expression and oxidative stress, with full-length mRNA expression that is reduced but not eliminated, and with production of short 3' UTR-encoded peptides. Depletion of the oxidation-sensitive Fe-S cluster ribosome recycling factor ABCE1 induces the accumulation of 3' UTRs, consistent with a model in which ribosome stalling and mRNA cleavage by No-Go decay yields isolated 3' UTR RNAs protected by ribosomes. Isolated 3' UTR accumulation is a hallmark of brain aging, likely reflecting regional differences in metabolism and oxidative stress.

Keywords: 3′ UTR; ABCE1; aging; brain; mRNA; oxidative stress; ribosome; ribosome recycling; translation.

Figure 1.. Isolated 3′ UTRs Accumulate in Aged D1 SPNs of the Mouse Striatum, but Not D2 SPNs or Young SPNs

(A) Overview of the TRAP protocol used to isolate cell-type-specific mRNA from D1 and D2 SPNs of the mouse striatum in young and aged mice. (B) Heatmap of normalized read coverage over the 250 most 3′ UTR-enriched genes(see below) in 2-year-old D1 SPNs centered on the stop codon and extending 5′ and 3′ to the TSS and poly(A) site, respectively. (C) Examples of the log₁₀ coverage of four biological replicates per condition stacked on top of each other across a control gene, Ppia, and two genes exhibiting isolated 3′ UTRs exclusively in aged D1 SPNs. Dotted line indicates the stop codon. (D) Schematic of the R_tc metric. (E) The cumulative distribution of R_tc values of genes among the mouse SPN samples reveals a long tail for aged D1 SPNs. (F) Counts of the number of genes at various R_tc cutoffs in aged D1 SPNs.

Figure 2.. Increased Levels of Oxidative Stress Accompany Isolated 3′ UTR Accumulation in Aging D1 SPNs

(A) Venn diagram depicting the number of genes differentially expressed between D1 and D2 SPNs at each time point and the log-fold intra-cell-type gene expression changes from PN42 to 2 years. (B) The number of core components of oxidative phosphorylation differentially expressed between young and old D1 and D2 SPNs. p values indicate enrichment over background. ***p < 0.001, hypergeometric test. (C) Representative images of DAPI, lipofuscin, GFP, and merged channels for 9-week and 19-month-old CP73 mice expressing EGFP-L10a in D1 SPNs. (D) The proportion of lipofuscin-positive cells in GFP ± cells in CP73 and CP101 mouse lines at 9 weeks. Each point represents a slide and the x-coordinate was jittered to improve readability. ***p < 0.001, chi-squared test. (E) The cumulative distribution of the proportional area of each cell positive for lipofuscin accumulation in 9-week and 19-month-old mice.

Figure 3.. Oxidative Stress and ABCE1 Depletion Induce 3′ UTR Enrichment and a Model for Isolated 3′ UTR Accumulation

(A) A model of isolated 3′ UTR RNA species formation. An excess of age- or drug-induced ROS impairs the activity of the iron-sulfur cluster protein ribosome recycling factor ABCE1. ABCE1 deficiency results in an increase in ribosomes stalled on the stop codon and reinitiating in the 3′ UTR. Transcripts with stalled ribosomes are endonucleolytically cleaved upstream of stalled ribosomes by the No-Go decay pathway. The 5’ cleavage product is degraded by the 3′-to-5’ exosome, but the 3′ fragment is protected from Xrn1 5’-to-3′ digestion by stalled ribosome(s) at the stop codon. (B) qPCR analysis of 3′ UTR abundance normalized to primer pairs in the coding region for i) cells treated for 3 days with siRNAs targeted to Abce1 compared to cells transfected with control siRNAs and ii) cells treated for 3 days with 100 μM paraquat compared to untreated cells. Error bars indicate 95% confidence interval, * indicates significance by bootstrap (p < 0.01). (C) Meta plot of the density (in average reads per million, RPM) of 5’ ends of 27- to 31-nt-long ribosome protected fragments (RPF) plotted as a function of distance from the stop codon. p values represent Wilcoxon Rank Sum test for the first 250 nt after the stop codon. (D) Mean RPM +SEM in the 3′ UTRs of genes, binned by R_tc in aged D1 SPNs. p values represent the Wilcoxon Rank Sum test between the highest (7^th) bin of the control condition (siC) and the highest bin of each of the test conditions. (E) Ribosome footprint density over the first 200 nt of the 3′ UTR from genes in the top and bottom 15% of aged D1 SPN R_tc values. (F) Schematic of cleavage upstream of a stalled ribosome and subsequent protection of a short cleavage fragment by an upstream translating ribosome and the proportion of short cleavage fragments sequenced from each experimental condition. (G) Smoothed meta plot of the density (in RPM) of short (14–16 nt) ribosome-protected fragments in relation to the stop codon for different experimental conditions. Insets are mean + SEM of RPM in the 200-nt upstream and downstream of the stop codon.

Figure 4.. 3′ UTR-Enriched Genes Accumulate in the Aging Human Brain and Are Associated with Translation of Short Peptides

(A) Stacked plot of the log₁₀ coverage of Sox9 in human astrocytes colored by age. Inset is the R_tc value plotted as a function of age (p value by F-test). (B) The relationship between age and the mean R_tc in 1380 GTEx samples colored by brain and non-brain tissues and the linear fit between age and R_tc across all genes for brain and non-brain tissues. For each individual sample, the mean R_tc and confidence intervals about the mean are plotted versus age. (C) The mean R_tc in neuronal GTEx samples plotted against expression of ABCE1 and boxplots of the distribution of ABCE1 gene expression in different neuronal tissues. Samples with decreased ABCE1 levels exhibit significantly higher average R_tc (p value by F-test). (D) The relationship between age and R_tc separated by brain region. (E) The top 100 genes most positively correlated with increased R_tc in neuronal GTEx samples by rank and p value. Insets are a donut plot demonstrating the distribution of functional classifications of genes and a histogram of the p value distribution of positive gene correlations.

Figure 5.. Evidence of 3′ UTR-Encoded Peptides Originating from Isolated 3′ UTR Genes

(A) Box plots of the distribution of distances from the stop codon to the next 3′ UTR stop codon (in any frame) plotted as a function of R_tc in 7 equal sized bins. (B) Example of translated 3′ UTR ORFs from the 3′ UTRs of PQLC1 and (c) SLC2A6. Red vertical dashes indicate stop codons in frames relative to the canonical stop codon. Red text indicates the peptide spectra that mapped to the 3′ ORF, which is shown in orange. (C) Example of translated 3′ UTR ORFs from the 3′ UTRs of SLC2A6. Red vertical dashes indicate stop codons in frames relative to the canonical stop codon. Red text indicates the peptide spectra that mapped to the 3′ ORF, which is shown in orange. (D) Box plots of the mean R_tc for all genes compared to those with 3′ UTR peptides (p = 4.9e–7, t test).