Scientific Papers

A concerted increase in readthrough and intron retention drives transposon expression during aging and senescence

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Repetitive DNA makes up at least 50% of the human genome (de Koning et al., 2011) and has been linked to genomic instability, cancer and – somewhat less consistently – species longevity (Pabis, 2021; Khristich and Mirkin, 2020). Transposons are one particularly abundant class of repetitive sequences found in the nuclear genome of eukaryotes and are strongly associated with cancer-causing structural variants like DNA deletions, insertions, or inversions (Rodriguez-Martin et al., 2020). Although transposons are often considered parasitic, harmful or, at best, neutral recent evidence suggests they may contribute to genomic diversification on evolutionary timescales and to the cellular stress-response by providing transcription factor-binding sites (Villanueva-Cañas et al., 2019).

The major transposon families in the human genome are long interspersed nuclear element (LINE), short interspersed nuclear element (SINE), long terminal repeat (LTR) transposons, and DNA transposons comprising 21%, 11%, 8%, and 3% of the human genome, respectively (Kazazian and Moran, 2017).

In recent years, transposon expression, particularly of elements belonging to the LINE-1 family, has been hypothesized to be a near universal marker of aging. Several lines of evidence support a link between transposon reactivation and aging (Gorbunova et al., 2021).

Using RNA-seq, for example, to quantify reads mapping to transposable elements it was shown that expression of these was higher in 10- vs 1-day-old nematodes (LaRocca et al., 2020), 40- vs 10-day-old flies (Wood et al., 2016) and in muscle and liver of aged mice (De Cecco et al., 2019). Transposon expression also increases during in vitro senescence (Colombo et al., 2018) and in fibroblasts isolated from people between the ages of 1 and 96 years (LaRocca et al., 2020), whereas lifespan extending mutations and interventions in mice reduce transposon expression (Wahl et al., 2021).

However, not all studies report such consistent age-related increases across all transposon classes (Ghanam et al., 2019). Moreover, it would be a mistake to conflate changes in RNA-seq-, RNA-, DNA-, and protein-based measurements of transposon expression as evidence for one and the same phenomenon.

Quantification of transposon expression using RNA-seq or PCR techniques remains challenging given their repetitive nature and the fact that 98–99% of all transposons are co-expressed with neighboring transcriptional units (Deininger et al., 2017; Stow et al., 2021). In fact, only a small fraction of LINE-1 and SINE elements, the latter relying on co-transposition by LINEs, are expressed from a functional promoter. It is believed that such ‘hot’ LINE-1 loci drive most transposition events and genomic instability (Deininger et al., 2017; Rodriguez-Martin et al., 2020).

The large number of co-expressed transposons would likely mask any signal from autonomous elements in standard RNA-seq experiments. Therefore, alternative explanations are needed for increased transposon expression in such datasets and these could include age-related changes in transposon adjacent genes for example intron retention and transcriptional readthrough.

Most eukaryotic genes contain introns which need to be removed during a complex co-transcriptional process called splicing. Defects in splicing are the cause of many, often neurologic, hereditary conditions (Scotti and Swanson, 2016). One such type of splicing defect is intron retention. Although basal levels of intron retention may be benign or even physiologic, high levels are considered harmful (Zheng et al., 2020). Aging is accompanied by many changes to splicing patterns (Wang et al., 2018), including intron retention, which is increased during cellular senescence (Yao et al., 2020) and in aging Drosophila, mouse hippocampus and human prefrontal cortex. In addition, levels in human AD brain tissues are elevated even further than in the aged brain (Adusumalli et al., 2019).

Transcription is terminated by the cleavage and polyadenylation machinery when the polymerase transcribes through the polyadenylation signal leading to pre-mRNA cleavage, polymerase pausing, conformational change, and eventually polymerase detachment (Rosa-Mercado et al., 2021). However, this termination may fail leading to so-called readthrough transcription which increases during stress conditions (Rosa-Mercado and Steitz, 2022) and during cellular senescence, at least for a subset of genes (Muniz et al., 2017).

Emerging evidence suggests that intron retention, readthrough, and transposon expression are linked (Rosa-Mercado et al., 2021; Hadar et al., 2022), but this has not been studied in the context of aging. In this manuscript, we show that intron retention and readthrough taken together can explain most of the apparent increase in transposon expression seen in RNA-seq datasets of aging.

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