Fraction of genic or intronic transposons and of transposons downstream (ds) or upstream (us) of genes in two different datasets.

A concerted increase in transposon and intron expression with aging and senescence.

Transposon (A) and intron (B) expression is increased in skin fibroblasts isolated from aged individuals. What is more, expression of both transposons and introns shows a significant correlation (C). Similarly, transposon (D) and intron (E) expression is increased in MDAH041 cells induced into senescence (sen) by treatment with drugs or via passaging. Normalized reads from the top 1000 differentially expressed genes, transposons and introns were used in this analysis.

A) Transposon reads normalized by the expression of adjacent genes plotted across five age groups (adolescent n=32, young n=31, middle-aged n=22, old n=37 and very old n=21).

B) Intron reads normalized by the expression of adjacent genes plotted across five age groups (adolescent n=32, young n=31, middle-aged n=22, old n=37 and very old n=21).

C) Scatterplot between transposon and intron expression (normalized as in A and B) for all 143 samples. Each sample is colored by age.

D) Transposon reads normalized by the expression of adjacent genes plotted across four senescent conditions (H2O2, 5-azacytidine, adriamycin, replicative senescence) and four other conditions (serum-starved, immortalized, intermediate passage and early passage). N=3 per group.

E) Intron reads normalized by the expression of adjacent genes plotted across four senescent conditions (H2O2, 5-azacytidine, adriamycin, replicative senescence) and four other conditions (serum-starved, immortalized, intermediate passage and early passage). N=3 per group.

F) Scatterplot between transposon and intron expression (normalized as in E and F) for all 24 samples. Each sample is colored by senescence status.

A schematic model of transposon expression.

In our model, transcription can give rise to mRNAs and pre-mRNAs that contain retained introns when co-transcriptional splicing is impaired, as seen during aging and senescence, and these often contain transposon sequences. In addition, transcription can give rise to mRNAs and pre-mRNAs that contain transposon sequences towards the 3’-end of the mRNA when co-transcriptional termination at the polyadenylation signal (PAS) is impaired as seen with aging and senescence. Some of these RNAs may be successfully polyadenylated whereas others will be subject to nonsense mediated decay. Image created by Biorender.

© 2024, BioRender Inc. Any parts of this image created with BioRender are not made available under the same license as the Reviewed Preprint, and are © 2024, BioRender Inc.

Biased distribution of extragenic transposons relative to genes.

(A) Extragenic transposons with significantly (sig.) changed expression during aging or cellular senescence show a biased distribution, when mapped back onto the genome, with a preference towards a 5 kb region at the 3’-end of genes when compared to all annotated transposons (all) or randomly permuted transposons (random). (B) Extragenic transposons whose expression changes during cellular senescence are spread out further from genes compared to aging-associated transposons.

Increased readthrough levels with aging and senescence.

Readthrough transcription is increased in fibroblasts isolated from the very old (A, B) and after induction of senescence in vitro (D, E). Readthrough transcription is correlated with transposon expression (C, F).

A) Readthrough was determined in a region 10 to 20 kb downstream of genes for a subset of genes that were at least 20 kb away from the nearest neighboring gene (n=206 regions). The log2 ratio of readthrough to gene expression is plotted across five age groups (adolescent n=32, young n=31, middle-aged n=22, old n=37 and very old n=21).

B) As in (A) but data is plotted on a per sample basis.

C) Scatterplot between transposon expression and readthrough levels (normalized as in A and Fig. 1) for all 143 samples. Each sample is colored by age.

D) Readthrough was determined in a region 10 to 20 kb downstream of genes for a subset of genes that were at least 20 kb away from the nearest neighboring gene (n=299 regions). The log2 ratio of readthrough to gene expression is plotted across four senescent conditions (H2O2, 5-Aza, Adriamycin, replicative senescence) and for early passage cells. N=3 per group.

E) As in (D) but data is plotted on a per sample basis and for additional control datasets (serum-starved, immortalized, intermediate passage and early passage). N=3 per group.

F) Scatterplot between transposon expression and readthrough levels (normalized as in A and Fig. 1) for all 24 samples. Each sample is colored by senescence status.

Elevated transposon expression after induced readthrough.

Readthrough transcription is increased after KCl treatment of HEK293 cells (A, D) and to a lesser extent during influenza infection (D). These readthrough inducing treatments also promote increased transposon expression (B, E). Giving rise to a correlation between readthrough and transposon expression on a per sample basis (C, F). Data in (A-C) is from Rosa-Mercado et al. (2021). In this study HEK293 cells were subjected to 1 hour of hyperosmotic stress (KCl) after which RNA-seq and TTLseq were performed. Data in (D-F) is from Bauer et al. (2018). In this study HEK293 cells were subjected to 1 hour of hyperosmotic stress (KCl) after which mNETseq was performed.

A) Readthrough was determined in a region 10 to 20 kb downstream of genes for a subset of genes that was at least 20 kb away from the nearest neighbouring gene (n=1420 regions).

B) To normalize transposon expression counts for each transposon were corrected for the expression of the nearest gene. Normalized transposon counts for each sample are shown as box-whisker plot.

C) Normalized transposon counts (as in B) and readthrough levels are plotted for each sample (n=12).

D) Readthrough was determined in a region 10 to 20 kb downstream of genes for a subset of genes that was at least 20 kb away from the nearest neighbouring gene (n=2222 regions).

E) To normalize transposon expression counts for each transposon were corrected for the expression of the nearest gene. Normalized transposon levels for each sample are shown as box-whisker plot.

F) Normalized transposon counts (as in B) and readthrough counts are plotted for each sample (n=22). The shapes indicate the cell type (circles=HEK293, triangles=A549).

Increased expression of active LINE-1 elements with senescence but not aging.

A scatterplot between the log2-fold change of every transposon (p<0.05) differentially expressed with age or senescence and the log2-fold change of the nearest gene. The percentage of active LINE-1 elements among transposons with higher-than-expected expression levels is unchanged in aging (A) and increased during senescence (B). For the tables, we compared the percentage of active LINE-1 elements among all LINE-1 elements for 250 transposons with higher-than-expected expression levels (in orange; labeled “outliers”), all significantly changed transposons (blue plus orange; labeled “significant”) and all transposons passing read filtering (not plotted; labeled “all expressed”). The p-value is based on the comparison with “all expressed”.