RNA Polymerase II elongation speed increases with age
didascalia copertina
Abstract
Physiological homeostasis becomes compromised during aging because of impairment of cellular processes, including transcription and RNA splicing. In a recent study, Debes et al.1 investigated the molecular mechanisms leading to the decline in transcriptional fidelity during aging and examined potential preventive measures. Their findings showed a consistent increase in the average transcriptional elongation speed with age across five species and revealed an association of Pol II speed with genome-wide changes in transcript structure and chromatin organization. Notably, dietary restriction and lowered insulin–IGF signaling reversed most of these aging-related changes, paving the way for new possible lifespan-extending interventions.
Review
Introduction
This study1 focused on understanding how aging impacts transcriptional processes and the subsequent effects on messenger RNAs (mRNAs) biosynthesis and organismal function. Aging is a time-dependent physiological decline of biological processes2, many of which affect the quality and concentration of proteins. Among these processes, transcription is particularly important, because it is a main regulator of protein levels. Proper mRNA synthesis depends on transcriptional elongation, which, if dysregulated, can lead to erroneous transcripts and diseases3.
As organisms age, their transcriptomes undergo significant changes, impacting genes related to signaling, DNA damage response, protein homeostasis, immune responses, and stem cell plasticity2. Previous works have highlighted an increased variability in gene expression with age, but the extent to which transcription itself is affected by aging remains unclear4. Using high-throughput transcriptome profiling, the researchers aimed to elucidate the kinetics of transcription during aging, its impact on mRNA biosynthesis, and the role of these changes in age-related functional decline.
Discussion
The translocation speed of elongating Pol II (RNA polymerase II) can be measured using RNA sequencing (RNA-seq) coverage in introns. Read coverage generally decreases 5′ to 3′ along an intron, and the magnitude of this decrease depends on Pol II speed: the faster the elongation, the shallower the slope. Thus, by quantifying the gradient of read coverage along an intron, it is possible to determine the elongation speeds of Pol II at individual introns.
To monitor how the kinetics of transcription changes during aging, Debes et al. quantified the distribution of intronic reads resulting from RNA-seq in five species – the worm Caenorhabditis elegans, the fruitfly Drosophila melanogaster, the mouse Mus musculus, the rat Rattus norvegicus and the human Homo sapiens – at different adult ages and using diverse mammalian tissues, fly brains and whole worms. Human samples originated from whole blood of healthy donors and from two primary human cell lines (the fetal lung fibroblasts IMR90 and the human umbilical vein endothelial cells HUVECs) driven into replicative senescence. They observed an increase with age of the average Pol II elongation speed in all five species and all tissue types examined. This increase was, in most cases, reverted under lifespan-extending conditions. To determine whether changes in Pol II speed are causally involved in the aging process, they used genetically modified worm and fly strains carrying point mutations in the large subunit of Pol II, RBP1, that reduce its elongation speed. They observed that slowing down Pol II increased lifespan in both worms and fruit flies.
Optimal elongation rates are required for splicing fidelity5. Slow elongation favors weak splice sites that lead to exon inclusion, whereas these exons are skipped if elongation is faster6. To check whether splicing fidelity is affected by changes in Pol II speed, they quantified changes in splicing and observed that faster Pol II increased splicing efficiency. Additionally, the average fraction of rare splicing events increased during aging in flies and worms. This effect was reversed under most lifespan-extending conditions.
Increased speeds of Pol II can lead to more transcriptional errors because the proofreading capacity of Pol II is challenged7. To assess the potential effect of accelerated elongation on transcript quality beyond splicing, they then measured the number of mismatches in aligned reads for each gene, observing that the average fraction of mismatches increased with age, but decreased under most lifespan-extending treatments.
Subsequently, they explored alterations in chromatin structure as a possible cause of the age-associated changes in Pol II speeds. Nucleosome positioning along DNA is known to affect both Pol II elongation and splicing8. Thus, age-associated changes in chromatin structure could contribute to the changes in Pol II speed and splicing efficiency that they observed.
To test this, they performed micrococcal nuclease (MNase) digestion of chromatin from early (proliferating) and late-passage (senescent) human IMR90 cells followed by pair-end sequencing of mono-nucleosomal DNA. Following mapping, they examined nucleosome occupancy and nucleosome sharpness. Both measures were significantly, but moderately, altered in senescent cells. Average sharpness was slightly decreased (along both exons and introns) and average internucleosomal distances slightly increased in introns. In conclusion, the transition from a proliferating cell state to replicative senescence was associated with small but significant changes in chromatin structure. The organization of nucleosomes is severely influenced by histone availability. A global loss of histones constitutes a hallmark of aging and senescence2.
To assess whether Pol II elongation speed and senescence entry in human cells are causally affected by changes in nucleosomal density, they then generated IMR90 cell populations homogeneously overexpressing GFP-tagged H3 or H4. Overexpression of either histone resulted in significant reduction of Pol II speed, confirming the causal connection between chromatin structure and transcriptional elongation. Furthermore, H3 overexpression in glial cells significantly increased fruit fly lifespan. These in vivo results are consistent with in vitro data from IMR90 cells, demonstrating that H3 overexpression partially reverts the aging effects on chromatin density and promotes longevity in flies.
Conclusions
In this study1, the authors demonstrated a link between Pol II elongation speed and age across five metazoan species. They also documented aging-related changes in splicing and transcript quality, such as increase in splicing efficiency and increased numbers of mismatches, which probably contribute to age-associated phenotypes. Although average speed changes were significant, they remained small in absolute terms. This is expected, as drastic genome-wide changes of RNA biosynthesis would quickly be detrimental for cellular functions and would probably lead to early death. Thus, despite being small in magnitude, these effects are clearly relevant for organismal lifespan, enabling the authors to increase lifespan in two species by decelerating Pol II under dietary restriction and lowered insulin–IGF signaling.This pioneer work establishes a crucial link between the speed of Pol II elongation and the dysregulation of transcription, which negatively impacts cellular and organismal fitness, potentially influencing age-related phenotypes.
Limitations & Future perspectives
This study, while comprehensive in its approach, has some limitations that must be addressed in future research. First, the induction of senescence using replicative stress may not fully capture the in vivo conditions because senescence is associated not only with telomere attrition but also with an increased reactive oxygen species (ROS) concentrations2. Future studies should consider inducing cellular senescence through oxidative stress to better mimic physiological aging processes9. Additionally, although there is a correlation between age and the number of mismatches, a causal relationship between Pol II elongation speed and the number of mismatches cannot be definitively established. The increased number of mismatches could also be influenced by other aging-related mechanisms, such as the accumulation of reactive oxygen species2. Moreover, although the study spans multiple species, it does not explore the specific molecular mechanisms underlying changes in Pol II elongation speed across different species. Furthermore, advanced technologies such as single-cell RNA sequencing could be employed to examine the heterogeneity of Pol II elongation speeds across different cell populations. Clinically, modulating Pol II elongation speed holds potential as a therapeutic strategy to delay or prevent age-associated diseases. Identifying biomarkers related to Pol II elongation speed could also prove useful in monitoring biological aging and the effectiveness of anti-aging interventions.
References
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