Translational Medicine of Aging 5 (2021) 31e38

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Translational Medicine of Aging

journal homepage: www.keaipublishing.com/TMA

Transcription errors in aging and disease

M.E. Anagnostou a, 1, C. Chung a, 1, E. McGann a, 1, B.M. Verheijen a, 1, Y. Kou b, L. Chen b, M. Vermulst a, *
a
University of Southern California, School of Gerontology, Los Angeles, CA, 90089, USA
b
University of Southern California, Department of Cellular and Molecular Biology, Los Angeles, CA, 90089, USA

a r t i c l e i n f o a b s t r a c t

Article history: Accurate transcription is required for the faithful expression of genetic information. However, little is
Received 28 June 2020 known about the fidelity of RNA synthesis or the biological consequences of transcription errors in living
Received in revised form cells. Here, we review recent developments in massively parallel sequencing technology that allow the
31 March 2021
fidelity of transcription to be measured with unprecedented accuracy and summarize new observations
Accepted 26 May 2021
Available online 10 June 2021
that provide insight into the role of transcription errors in aging and disease. Taken together, these
developments have the potential to change our understanding of mutagenesis itself, and significantly
enhance our understanding of the origins of human disease.
Keywords:
Aging
© 2021 The Authors. Publishing services by Elsevier B.V. on behalf of KeAi Communications Co. Ltd. This
Transcription is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/
Mutagenesis 4.0/).
Protein aggregation
Polymerases

1. Introduction
Biological reactions are remarkably precise. Even though pro-
teins encounter millions of potential substrates, binding partners
and co-factors every day, they consistently select the correct mol-
ecules to interact with. As a result, the right reactions take place at
the right time and the right place. This precision is enabled by
optimized topology and charges that perfectly fit the molecules a
protein needs to interact with, while excluding those that merely
resemble them [1]. Some molecules are nearly identical though,
which means that even with the most sophisticated safety mea-
sures in place, sporadic errors are unavoidable. For example, RNA
and DNA differ by just a few atoms, which makes it difficult for
proteins to distinguish between them. As a result, DNA polymerases
sporadically insert RNA bases into DNA strands, despite the pres-
ence of multiple safety mechanisms specifically designed to pre-
vent this problem [2]. Because of the complexity of living
organisms, similar errors can be made in any other biological
reaction.
For the vast majority of these reactions though, it is unknown
how frequently mistakes are made or what their biological conse-
quences are. After all, most scientific experiments are designed to
* Corresponding author.
E-mail address: vermulst@usc.edu (M. Vermulst).
1
these authors contributed equally to this manuscript.
understand how a reaction works, not how often it doesn't work.
And in many cases, these experiments are performed in vitro, in the
presence of purified molecules that are manipulated under highly
controlled conditions. The reality though, is that most reactions
take place at incredible speeds in complex environments filled with
countless closely related molecules. It is very difficult to achieve
perfect accuracy under those conditions. The proteins that catalyze
these reactions are also subject to various forms of evolutionary
pressure, some of which refine other features at the expense of
accuracy.
A useful example to illustrate this concept of evolutionary trade-
offs is DNA replication. More than any other process, the fidelity of
DNA replication is essential for human health [3]. By limiting the
accumulation of mutations, accurate DNA synthesis prevents
carcinogenesis and lowers the risk of inborn diseases [4,5]. And
because a single mutation is sufficient to cause disease, DNA
polymerases have evolved to commit ~1 mistake every million
bases [6]. Although that sounds impressive, it is important to put
this number into context. Our genome consists of 3 billion bases,
which means that with an error rate of 1
106, 3000 new mu-
tations are introduced every cell division. That is approximately the
same mutation rate as human cancers [4,7]. Surprisingly then, the
fidelity of DNA polymerases is grossly insufficient to prevent dis-
ease. If it wasn't for mismatch repair, a back-up system that
removes >95% of the errors made during DNA replication [8], the
error rate of DNA polymerases would endanger our long-term
health, and perhaps the survival of our species.

https://doi.org/10.1016/j.tma.2021.05.002
2468-5011/© 2021 The Authors. Publishing services by Elsevier B.V. on behalf of KeAi Communications Co. Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
M.E. Anagnostou, C. Chung, E. McGann et al.
How is that possible? How can these enzymes, which are
responsible for propagating life itself, not be up to the task? The
answer to that question lies in a complicated set of trade-offs that is
key to DNA synthesis. To safely replicate billions of bases, DNA
polymerases must not only be accurate, they must also be fast. And
the faster a polymerase is, the less accurate it becomes [9]. Thus,
every polymerase is the result of a careful trade-off between ac-
curacy and speed, where some accuracy is sacrificed to accelerate
elongation.
This trade-off is not unique to DNA polymerases. Over the course
of evolution, the accuracy of almost every protein has been
compromised for similar reasons. Sometimes that meant sacrificing
fidelity for speed, and in other cases for flexibility, efficiency, or
other aspects of protein function. And importantly, our health de-
pends on the accuracy of all of these proteins. A deeper under-
standing of biological fidelity is therefore key to understand the
origins of human disease. We anticipate that these insights will be
especially important for the aging field. One of the guiding prin-
ciples of aging research is that the power of natural selection wanes
with age [10,11]. For example, the balance between protein syn-
thesis, folding, and destruction (proteostasis) is optimal when
we're young, which prevents the occurrence of proteotoxic diseases
[12e14]. Because there is no evolutionary pressure to keep pro-
teostasis functioning as efficiently when we're old it declines with
age, which coincides with the onset of age-associated protein
misfolding diseases. The same argument can be made for the ac-
curacy of biological reactions. Proteins only need to work as well, or
be as accurate, as it takes to reach a certain age. In the case of DNA
polymerases that means that the fidelity of DNA synthesis was only
allowed to evolve to such a degree that it prevents cancer prior to
middle age, but not beyond. However, the same principle applies to
the accuracy of other processes in our cells, suggesting that the
fidelity of other process is equally important for human health.
A strong candidate for this idea is transcription. For many years,
it has been suspected that transcription errors play an important
role in age-related diseases, including Alzheimer's disease and
cancer [15,16]. In addition, we recently found that transcription
errors can perturb several basic biological processes that are key to
human aging, including proteostasis, metabolic pathways and NAD
metabolism [17,18]. These experiments also demonstrated that
transcription errors increase with age in yeast [19], suggesting that
they may contribute to the steady decline of these processes with
age. In this review, we will investigate how transcription errors
could affect human aging, explain how they can be detected in a
massively parallel fashion, and speculate how these techniques
may need to evolve in the future to answer even more sophisticated
research questions.
2. Transcription errors can mimic mutations
DNA replication errors are a powerful source of human disease.
The mutations that are generated by these errors give rise to
mutant proteins that drive the pathology of countless genetic dis-
eases. Mutant proteins can also be generated by errors that occur
during other processes though, including transcription and trans-
lation. It is commonly assumed though, that these errors are
random, temporary events that do not have a lasting impact on
cellular function, let alone human physiology. Several lines of evi-
dence now demonstrate though, that transcription errors are not
always random or temporary, and that they can have lasting con-
sequences for human health. For example, in patients with non-
familial cases of Alzheimer's disease transcription errors in the
UBB and APP genes give rise to toxic versions of ubiquitin-B and
32
Translational Medicine of Aging 5 (2021) 31e38
Fig. 1. Transcription errors and protein aggregation. A. The first example of “mo-
lecular misreading”. A GA dinucleotide repeat provokes transcription errors in the b-
APP gene, leading to a small protein fragment. B. Yeast cells that express an error prone
version of the major catalytic subunit of RNA polymerase II make more transcription
errors than WT cells. As a result, protein quality control is overwhelmed and TDP-43
and Ab1-42 are able to evade this machinery and seed aggregates. C. Graphical
depiction of B.
amyloid precursor protein that promote disease progression [15,20]
(Fig. 1). Similarly, errors in the RAS gene can activate oncogenic
pathways in human cell lines [21]. These observations were espe-
cially informative because the transcription errors responsible for
these phenotypes occurred repeatedly at the same position. As a
result, they produced a steady stream of a single, mutated protein,
thereby effectively mimicking a mutation. There are at least two
templates that allow errors to occur repeatedly at the same posi-
tion: templates that are naturally error prone, and templates that
are artificially error prone due to DNA damage. For example, the
dinucleotide tracts that promote transcriptional mutagenesis in the
UBB and APP gene are examples of naturally error prone templates.
Measurements in S. cerevisiae indicate that these tracts can increase
the error rate of transcription >100-fold [22]. Conversely, DNA
damage engineered into the RAS gene [23,24] is an example of a
damaged template that promotes transcriptional mutagenesis
(Fig. 2). DNA lesions can raise the error rate of transcription
100,000-fold, from one mistake every 200,000 bases to one
mistake every 2 bases [16,21,25,26]. Remarkably then, these ex-
periments demonstrate that DNA damage does not need to be
converted into a mutation to produce mutant proteins, the damage
itself is sufficient.
Although damaged bases and natural hotspots both result in
increased error rates, they have different consequences for a cell.
Natural hotspots are only embedded in a subset of genes and will
thus affect those genes exclusively. In contrast, DNA damage occurs
stochastically, which means that it can affect every gene inside a
cell. As a result, natural hotspots are likely to cause the same
phenotype in every cell, while DNA damage can produce a unique
phenotype in every cell, depending on the genes that are damaged.
How severe this phenotype is and how long it will last will depend
on the amount of DNA damage a cell incurs and how quickly it is
repaired. For example, neurons produce ample amounts of reactive
oxygen species (a source of DNA damage that causes transcription
errors [27,28]) and live for extended periods of time, so that there is
ample time for damage to accumulate on the genome. In addition,
post-mitotic cells are known to have limited DNA repair capacity
[29,30], which means that this damage is likely to persist.
Accordingly, neurons might be more sensitive to damage induced
transcription errors than other cell types. It is interesting to note
M.E. Anagnostou, C. Chung, E. McGann et al. Translational Medicine of Aging 5 (2021) 31e38

Fig. 2. Mechanisms by which transcription errors can impact on cellular health. A. Unless repaired, DNA damage can provoke transcription errors (red dots) during consecutive
rounds of transcription. As a result, a single lesion (O [6]-methyl guanine, O [6]-me) can have the same effect on cellular function as a heterozygous mutation (red band on
chromosome). RNAPII is depicted as light blue proteins, DNA is in orange and RNA in black. B. Numerous hotspots exist in the genome that can increase the error rate of tran-
scription up to 100-fold. These hotspots provide a constant stream of aberrant proteins throughout the lifespan of a cell. C. Random transcription errors can affect any gene in the
cell (4 genes are depicted with 4 unique colors). These errors tend to induce protein misfolding, which applies pressure to various protein quality control systems in the cell,
eventually overwhelming them. D. Even a single, random transcription error can cause cellular dysfunction if it has a highly specific biological consequence, or if the consequences
of the error are amplified through a self-sustaining mechanism. For example, a protein that now has prion-like properties will force other proteins to assume its conformation,
thereby amplifying itself into proteinaceous fibers. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

therefore, that neurons also suffer from protein aggregation, one of
the primary consequences of transcription errors.
3. Transient transcription errors can overwhelm the protein
quality control machinery
Transcription errors do not need to mimic mutations to affect
cellular function though. Even transient errors, which are errors
that occur only once at each position, can compromise cellular
health. To understand how these errors affect cellular health, we
studied yeast cells that express an error prone RNA polymerase II,
which commits random errors across the entire transcriptome.
Using these cells, we discovered that random errors tend to affect
the structural integrity of proteins, which induces protein mis-
folding [17]. Most amino acids are part of structural motifs, so
replacing a hydrophobic amino acid with a hydrophilic one (or vice
versa) can result in protein misfolding [31]. Similar problems arise
when other substitutions are made. Individually, these misfolded
proteins have little effect on cellular health, but together they can
overload the protein quality control machinery (Fig. 2) and prevent
the degradation of toxic proteins that are normally targets for this
machinery, including TDP-43 (amyotrophic lateral sclerosis, ALS,
Fig. 1), Ab1-42 (Alzheimer's disease, Fig. 1), HTT103Q (Huntington's
disease), and RNQ1 (a prion) [17]. Surprisingly then, transcription
errors have very specific effects on cellular function, even if they
occur randomly throughout the transcriptome. Random errors also
deregulate bioenergetics, nucleotide synthesis, nitrogen availability
and NAD metabolism, most likely due to similar reasons: their
cumulative burden puts constant pressure on these pathways until
they collapse under their weight [22]. If transcription errors have
the same effect on human cells, they could affect the age of onset,
progression and severity of multiple diseases caused by protein
aggregation and may contribute to various metabolic syndromes by
depleting molecules that are essential for human health.
4. Even single transcription errors can cause dysfunction
However, transcription errors do not need to mimic mutations
or occur en masse to affect cellular health. Even a single error can
change the fate of a cell. For example, a recent study demonstrated
that errors that open up a membrane channel in bacteria can allow
molecules to enter the cell that stimulate additional channels to
open up [32]. In this study, the authors made clever use of the lac
operon in bacteria, a model system for gene regulation with
bistable behavior (i.e., it exists in an ON or OFF state). Transient
errors in the lac repressor lacI led to derepression of the lac operon
(switching it from an OFF to ON state), resulting in the appearance
of lacY permease. This permease is a channel protein that allows
cellular influx of a lac inducer that activates permease synthesis.
Thus, through this feed-forward loop, the consequences of the
initial mistake were amplified and allowed for a heritable change in
the physiology of the cell and all of its progeny. This observation is
especially instructive because it is easy to imagine how errors in
other genes could result in a similar cascade of events.
We suspect that one of these cascades involves prion-like
behavior. It is now becoming clear that many age-related neuro-
degenerative diseases are caused by proteins that, for unknown
reasons, have acquired prion-like behavior [33,34]. Many proteins
that have the potential to display this behavior are metastable.
Because of this instability, a single, well placed mutation can be
sufficient to change a properly folded protein into a protein with
prion-like properties, initiating an auto-catalytic cascade of self-
assembly into toxic amyloid structures [34,35]. Because the error
rate of transcription error is >100-fold higher than the mutation
rate, we anticipate that erroneous transcripts are constantly
generated that promote prion-like behavior. Once generated, these
prions will continue to self-assemble long after the transcript that
generated them has been degraded, potentially amplifying the
biological consequences of the initial error in perpetuity. Given the
rate of RNA synthesis, as well as the longevity and error rate of
transcription of mammalian cells, it is likely that every error that
could conceivably result in a prion-like protein will be produced
countless times over the lifespan of a human neuron. Because our
brains house >80 billion neurons, it is possible that this mechanism
could result in ample mutant proteins to initiate disease.
Taken together, these observations could have a lasting impact
on our understanding of human aging and disease. Every day, pa-
tients enter the clinic with complex diseases for which there is no
clear cause. Scientists have traditionally searched for the origins of
these diseases by sequencing the genome of human patients. The
ideas described above, that lay out the impact of re-occurring er-
rors, transient errors and single errors on cellular function, suggests

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M.E. Anagnostou, C. Chung, E. McGann et al. Translational Medicine of Aging 5 (2021) 31e38

that in some cases, the mutations responsible for the disease may sequencing; however, a crucial drawback of this strategy is that the
not be present in the genome, but rather in the transcriptome. If so, reverse transcriptases (RTs) used to generate cDNA libraries are
a deeper understanding of transcriptional mutagenesis could ulti- notoriously error-prone and tend to make 1 error every ~20,000
mately transform our understanding of the origins of human bases [46,47], whereas RNA polymerases make 1 error every
disease. ~200,000 bases [18,48]. As a result, standard cDNA libraries are
littered with RT errors that are indistinguishable from true tran-
5. Transcription errors in aging cells scription errors (Fig. 3). One solution to this problem is to reverse
transcribe the same mRNA molecule multiple times. If multiple
Transcription errors are especially interesting from an aging cDNA copies are made of a single mRNA molecule, a true
perspective. As described above, they exacerbate the aggregation of
proteins associated with neurodegenerative disorders by at least
two mechanisms: i) by overwhelming the protein quality control
machinery and ii) by generating highly specific disease-related
peptides [15,20]. These observations suggest that transcription er-
rors do not only generate the peptides that aggregate during dis-
ease progression [15,20], but that they can also provide the very
conditions that allow these proteins to survive inside cells and seed
aggregates [17]. The combination of these two events could be a
powerful driver for disease progression.
There are other links between aging and transcription errors as
well though. For example, the error rate of transcription seems to
increase with age in yeast [17]. If the same holds true for humans,
transcription errors could change the age of onset, severity and
progression of various diseases caused by protein aggregation. One
potential mechanism by which aging could accelerate transcrip-
tional mutagenesis is DNA damage [23,24]. It has long been known
that cells accumulate DNA damage with age [36,37]. A significant
amount of this damage results from defective respiration, which
allows reactive oxygen species to leak out of mitochondria [38,39].
For example, an age-dependent increase in oxidized DNA lesions
and oxidized proteins has been reported in the brain [40] and
oxidative damage is a powerful source of transcription errors
[21,25,26]. Salient examples of oxidative DNA lesions that are prone
to transcriptional mutagenesis include the uracil intermediate
dihydrouracil [41,42], and the modified guanine 8-oxoguanine
[21,43,44]. When RNAPII encounters these lesions it tends to mis-
incorporate an adenine opposed the damaged base that can escape
intrinsic proofreading [45]. Thus, highly metabolic cells like neu-
rons that accumulate oxidative damage as they age [37] are at
increased risk for an age-related increase in transcriptional muta-
genesis, which is further compounded by the relatively inefficient
DNA repair capabilities of non-dividing cells [30] and their
remarkable longevity, which allows lesions to accumulate over long
periods of time. Mitochondrial dysfunction, reactive oxygen species
and DNA damage have long been implicated in age-related dis-
eases, although it remains unclear what the mechanistic basis for
this relationship is. The considerations described above suggest
that one way by which oxidative damage can promote neurode-
generative diseases is by increasing transcriptional mutagenesis.
How hazardous transcription errors are to aging organisms
depends on the frequency with which they occur. Therefore, it will
be important to measure the error rate of transcription in human
cells. These measurements could help us determine how many
misfolded proteins are generated by transcription errors, identify
hotspots in the human genome that result in toxic proteins, and
determine which genetic and environmental factors control the
error rate. Technical limitations have thus far prevented a system-
Fig. 3. Core concept of the circle-sequencing assay. Data from traditional cDNA li-
atic study though, and to solve this problem we recently modified
braries are littered with reverse transcription ( ) and sequencing errors ( ) that are
an assay from the virology field to detect transcription errors in a indistinguishable from true transcription errors ( ). To remove these artifacts from our
massively parallel fashion [18]. data, we circularize RNA prior to reverse transcription. These circularized molecules
are then reverse transcribed in a rolling circle fashion to generate linear cDNA mole-
6. Detecting transcription errors with massively parallel cules made up of tandem repeats of the original template (blue strands). If a tran-
scription error was present in the original template, this error will be present in all
sequencing technology copies of the cDNA molecule, while artifacts occur in only one. (For interpretation of
the references to color in this figure legend, the reader is referred to the Web version of
The most intuitive way to detect transcription errors is cDNA this article.)

34
M.E. Anagnostou, C. Chung, E. McGann et al. Translational Medicine of Aging 5 (2021) 31e38

transcription error will be present in every copy, while an RT error
would appear only once (Fig. 3). This is the core idea behind the
circ-seq assay, an assay that was originally designed for high-
fidelity sequencing of RNA viruses [49,50]. The circ-seq assay
carries this name because a key step in its protocol is RNA circu-
larization. These circular RNA molecules are reverse transcribed in a
rolling-circle fashion, so that each cDNA molecule consists of a
tandem repeat of the original template. These concatemers are then
sequenced and analyzed using advanced bio-informatics to
distinguish transcription errors from technical artifacts [51]. This
assay is highly reproducible, extremely precise, and provides the
first genome-wide view of transcriptional mutagenesis at single
base resolution [18,51] (Fig. 4). As a result, it has opened up the field
of transcriptional mutagenesis to widespread experimentation.
Because of its novelty, the circ-seq assay is a powerful tool for
discovery. For example, by applying it to the budding yeast
S. cerevisiae [18], we recently provided the first reasonable estimate
of the error rate of transcription in eukaryotic cells. In that study,
we identified errors in every class of RNA molecules, including
rRNA, mRNA, tRNA, snRNA, snoRNA, lncRNA and mitochondrial
RNA, demonstrating that transcription errors can occur anywhere
in the transcriptome (Fig. 4). Because different RNA species are
synthesized by different RNA polymerases, we were also able to
calculate the individual error rates of RNA polymerase I, II, III and
the mitochondrial RNA polymerase. We found that each of these
RNA polymerases makes the same type of errors as DNA poly-
merases, including single base substitutions, insertions and de-
letions. Remarkably though, RNA polymerases made these errors
>100-fold more frequently than DNA polymerases, suggesting that
due to its relatively high error rate, transcription errors are a con-
stant source of mutated and misfolded proteins.
Another advantage of the circ-seq assay is that it generates large
amounts of data. For example, in just one study we were able to
interrogate >40 billion bases of the yeast transcriptome and iden-
tified >200,000 transcription errors [18]. That resolution allowed
us to determine how various static and dynamic features of the
genome affect the fidelity of transcription, including specific DNA
sequences, histone modifications, and nucleosome coverage

Fig. 4. Genome wide detection of transcription errors in yeast. A. Yeast chromosomes were laid out in an end-to-end fashion. A checkmark was placed where errors were
detected (200K total), demonstrating that the circ-seq assay detects transcription errors in a genome-wide fashion. B. By zooming into one chromosome, the error distribution over
different genes can be examined. Here, the total number of errors detected in each gene is depicted. Highly transcribed genes have the highest peaks because they were sequenced
the most. C. By zooming into one transcript, the distribution of errors across that transcript can be examined. At this level, the effect of each error on RNA and protein structure and
function can be determined. Here, the number of errors at each position is depicted as a balloon. Green balloons indicate errors that change the start codon. Purple balloons change
the stop codon. Red balloons generate premature stop codons. Blue balloons indicate non-synonymous changes and yellow balloons indicate synonymous changes. Approximately
10% of all errors detected in the Adh1 transcript are depicted. D. Further magnification allows the errors to be examined at the single base pair level. Yellow bases indicate syn-
onymous errors. Blue bases indicate non-synonymous errors and red errors indicate a premature stop codon. D indicates the deletion of one or two bases, þ indicates insertion of a
base. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

35
M.E. Anagnostou, C. Chung, E. McGann et al.
[18,24]. For example, previous studies identified hotspots for
transcription errors in the human UBB and APP genes [20], and in
the gene encoding rat and human vasopressin [15,52]. Consistent
with this idea, we found that similar dinucleotide repeats are also
hotspots for transcription errors in yeast and can increase the error
rate of transcription >100-fold [18]. This observation provides
important insight into the steady stream of mutant UBB and APP
proteins that arise as a result of these hotspots. Another factor that
alters the fidelity of transcription is expression rate. Interestingly,
rarely transcribed genes display higher error rates than other genes
[24]. This is a biologically relevant observation because the fewer
transcripts are made, the greater the impact of a mutant copy is.
Finally, a number of elements, such as specific histone species and
transcription factors seem to be associated with a 10-fold increase
in certain errors, suggesting a highly specific mutational mecha-
nism [24]. Future work will need to determine what that this
mysterious mechanism is. Regardless though, these observations
suggest that different cells may display different error rates in the
same genes, depending on their chromatin landscape [24].
A final advantage of the circ-seq assay is its adaptability. Because
it uses RNA molecules as its substrate [51], it can be applied to any
organism of choice, including human cells. As a result, a large
amount of data is likely to emerge in the coming years that de-
scribes the landscape of transcription errors in numerous model
organisms, including human cells. That data will be an important
step forward for the field. First, this data will allow us to identify
every hotspot for transcription errors across the human genome,
and thus every gene that is at risk for excessive transcriptional
mutagenesis. Accordingly, these measurements will help us predict
the types of diseases these errors might contribute to, similar to the
UBB and APP genes in Alzheimer's disease [20]. By identifying these
hotspots, similar disease-related RNA species may be detected, and
based on the precise molecular sequence of the resulting tran-
scripts, it may be feasible to specifically target these mutant RNA
molecules, e.g., using antisense oligonucleotides [53]. Second, by
cross-referencing the locations of transcription errors with various
static and dynamic features of the genome, many risk factors for
transcription errors may be identified. These results could lead to
the surprising conclusion that identical genes and DNA sequences
will display vastly different error rates depending on the epi-
genomic state of these genes. Third, this data would allow us to
identify genes and factors that directly control the error rate of
transcription in human cells. Once identified, these genes could be
knocked out to dissect how transcription errors affect the biology of
human cells in unprecedented detail.
Finally, we could also use the tools described above to help us
identify factors or molecules that increase the fidelity of tran-
scription. For example, it would be interesting to explore the effects
of different genetic contributors, cellular stressors and specific in-
terventions like exercise and dietary restriction on enhancing
transcriptional fidelity. After all, if genotoxic stress and DNA repair
are powerful modifiers of transcriptional mutagenesis [24], it is
plausible that transcriptional fidelity can be modulated by these
same variables. If so, the tools described above could be a powerful
ally in our efforts to combat diseases caused by transcriptional
mutagenesis. It may also be possible to improve the fidelity of
transcription under normal conditions. For example, several fidelity
factors have now been identified in yeast for RNA polymerase I and
II, and clear homologs of these genes exist in humans [22]. It is
possible that overexpression of these genes will lead to increased
fidelity of transcription in both yeast and human cell lines. In
addition to an overall decline in transcriptional fidelity, there might
be a role for specific RNA mutants contributing to pathology, e.g.,
36
Translational Medicine of Aging 5 (2021) 31e38
the APP and UBB frameshift mutants found in Alzheimer's disease
patient brains [20]. It may be feasible to specifically target these
mutant RNA molecules, e.g., using antisense oligonucleotides [53].
7. Potential diseases caused by an increased error rate of
transcription
Using the circ-seq assay, as well as genetic assays, we and others
previously identified mutations in subunits of RNA polymerase I
and II that raised the error rate of transcription >5-fold
[18,24,32,54e59]. By identifying these genes, two exciting oppor-
tunities have arisen. First, because these proteins are conserved
from yeast to humans, these observations make it possible to
mutate these genes in higher organisms, which would allow us to
understand the impact of transcription errors on mammals. Sec-
ond, it provides an opportunity to identify human subjects with
mutations in these genes. If so, these patients may display error
prone transcription and could reveal the biological consequences of
transcription errors on human biology. Intriguingly, two cohorts of
pediatric patients were recently identified that carry mutations in
POLR2A, the major catalytic subunit of RNA polymerase II [60,61].
These patients suffer from various metabolic and neurological
symptoms that vary from mild hypotonia and delayed development
to severely decreased muscle tone, loss of hearing and vision,
enlarged ventricles, white matter abnormalities and cerebellar
problems. Ultimately, these problems cause craniofacial distur-
bances, behavioral problems and learning disabilities in the pa-
tients. A structural analysis of the mutations that cause the disease
demonstrated that they cluster in regions that are essential for
transcriptional fidelity. For example, several mutations affect the
trigger loop. Some mutations that affect the trigger loop allow
incorrect nucleotides to remain in the active site longer than usual,
increasing their chance of misincorporation [59]. The best example
of this phenomenon is the yeast Rpb1E1103G allele, the most widely
studied allele of error prone transcription today [54,56,57]. Rpb1 is
the yeast homolog of POLR2A and mutation of aa1103 results in a 5-
fold increase in transcription errors [18], increased protein aggre-
gation and profound changes in bio-energetics, NAD metabolism,
nitrogen usage and purine synthesis. Remarkably, two patients
carry mutations in POLR2A that correspond to aa1102 and 1101 of
Rpb1, just upstream of aa1103, while other mutations affect both
the trigger loop and the quay region, which interacts with the
trigger loop. Further mutations affect the active site (which is
essential for the selection of correct nucleotides), and a region that
mediates the interactions between POLR2A and TCEA2, a fidelity
factor homologous to the yeast TFIIS protein. These observations
strongly suggest that these patients may display error prone tran-
scription. If so, these patients could unambiguously demonstrate
how transcription errors affect human health.
8. Conclusion
The fidelity of biological reactions is an essential component of
life itself. For life to thrive, reactions need to occur at the right place
and the right time, and with the right molecules. This precision
limits the resources that are lost by inaccuracies, allows for a more
robust and targeted response to stimuli and prevents the genera-
tion of toxic side products. Evolutionary considerations dictate
though, that reactions only need to be precise enough for an or-
ganism to reach its expected lifespan in the wild [10,11]. For
example, the error rate of DNA replication is sufficient to prevent
carcinogenesis early in life, but the accumulation of these errors is
estimated to be responsible for the majority of cancers later in life.
M.E. Anagnostou, C. Chung, E. McGann et al.
The same concept may apply to other processes. Here, we argue
that one of these processes is the fidelity of transcription. In recent
years it has become clear that transcription errors arise 100-fold
more frequently than genetic mutations. And unlike mutations,
they are generated continuously, which means that countless
mutated proteins are generated over the lifespan of a cell. These
proteins will undoubtedly have many expected and unexpected
consequences, some of which are bound to be detrimental to hu-
man health. By revealing the sources and consequences of these
errors, we expect that future studies will ultimately transform our
understanding of mutagenesis itself. And in doing so, provide key
insight into the biological basis of aging and age-related diseases.
Funding sources
This work was made by possible by National Institute on Aging
award R01AG054641 (M.V.), and an AFAR young investigator award
in Alzheimer's disease (M.V.)
Declaration of competing interest
The authors declare no competing interests.
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