RNA Biology

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Ribosome profiling analysis of human skeletal

muscle identifies reduced translation of
mitochondrial proteins with age

Ravi Tharakan, Ceereena Ubaida-Mohien, Yulan Piao, Myriam Gorospe &

Luigi Ferrucci

To cite this article: Ravi Tharakan, Ceereena Ubaida-Mohien, Yulan Piao, Myriam Gorospe
& Luigi Ferrucci (2021) Ribosome profiling analysis of human skeletal muscle identifies
reduced translation of mitochondrial proteins with age, RNA Biology, 18:11, 1555-1559, DOI:
10.1080/15476286.2021.1875647

To link to this article: https://doi.org/10.1080/15476286.2021.1875647

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RNA BIOLOGY
2021, VOL. 18, NO. 11, 1555–1559
https://doi.org/10.1080/15476286.2021.1875647

BRIEF COMMUNICATION

Ribosome profiling analysis of human skeletal muscle identifies reduced translation

of mitochondrial proteins with age
Ravi Tharakana,b, Ceereena Ubaida-Mohiena, Yulan Piaob, Myriam Gorospeb, and Luigi Ferruccia
a
Translational Gerontology Branch, National Institutes of Health, Baltimore, MD, USA; bLaboratory of Genetics and Genomics, National Institute on
Aging Intramural Research Program, National Institutes of Health, Baltimore, MD, USA

ABSTRACT ARTICLE HISTORY

With advancing age, human muscle loses strength and function, but the molecular causes of these Received 15 August 2020
losses are unknown. Skeletal muscle shows an age-dependent decline in the levels of different proteins, Revised 8 January 2021
but whether such decline is associated with reduced translation has not been studied. To address this Accepted 9 January 2021
gap of knowledge, we used the technique of ribosome profiling to study translation in muscle from
KEYWORDS
middle-aged and old individuals. Using ribosome occupancy as a measure of translation status, several Ageing; skeletal muscle;
mRNAs showed differential translation with age. Older age was associated with lower translation of translation regulation;
myosin and titin isoforms and more broadly with the translation of proteins involved in oxidative ribosome profiling
phosphorylation encoded by the mitochondrial genome. Based on our findings, we propose that
mitochondrial proteins are less translated in old skeletal muscle.

Introduction, results, discussion transcript the ratio of translating mRNAs with overall mRNAs
present in the tissue.
Several lines of research suggest that the regulation of
Briefly, muscle biopsies were performed from vastus later­
protein translation is important for human longevity.
alis and each sample was split for both RNASeq and ribosome
Treatment with the drug rapamycin, a caloric restriction
profiling (Figure 1A). For ribosome profiling, muscle was
mimetic that prolongs life span in animal models, functions
immersed into a polysome preparation buffer containing
at least in part by reducing protein translation [1,2]. In
cycloheximide to fix ribosomes in place before digestion
C. elegans, lowering protein translation by either drug
with RNase I. Protected ribosome footprints were then
treatment or RNAi targeting ribosome components was
extracted in Trizol, purified using a Qiagen RNA kit, and
found to enhance longevity [3–6]. Similarly, in
finally isolated on a denaturing gel. In parallel, Total
D. Melanogaster, a mutation in the translation factor 4E-
RNASeq libraries were digested with proteinase K, and full-
BP decreases translation, reduces protein aggregation in
length RNA was isolated using the Qiagen Fibrous RNA kit.
muscle and increases longevity [7]. The mechanisms
Ribosome profiling (RiboSeq) libraries were single-end
whereby lowering translation promotes longevity are not
sequenced to a depth of 150 to 250 million reads, while
understood, and it is unknown whether modifications of
Total RNASeq libraries were paired-end sequenced on
translation occur during human ageing.
a NextSeq to a depth of 600 to 800 million reads. All reads
To address this gap of knowledge, we analysed the transla­
were then aligned to the known protein-coding gene tran­
tional profiles of skeletal muscle biopsies from three middle-
scriptome using Ensembl hg38.
aged and two old human subjects. The middle-aged subjects
To look systematically at the global relationship between
were 43 years old (y.o.) (Caucasian female), 46 y.o. (Caucasian
transcription and translation in our samples, we first calcu­
male), and 41 y.o. (African American female), and the old
lated the number of reads aligning to each known human
subjects were 83 y.o. (Caucasian female) and 85 y.o.
transcript (TPM, transcripts per million) for both the ribo­
(Caucasian male). Transcript-specific translation was globally
some profiling and the Total RNA libraries. In Figure 1B, each
evaluated by ribosome profiling, a technique that identifies
dot represents normalized read counts for ribosome profiling
ribosome-protected mRNA fragments by RNASeq analysis.
(y-axis, RiboSeqTPM) and their relationship with normalized
The identification of ribosome-protected mRNAs segments
read counts for Total RNA (y-axis, RNASeqTPM) for each
provides evidence that a certain mRNAs is engaged in active
transcript isoform, and shows the extent to which RNA
translation at a given point in time in a given cell or tissue.
expression and translation are related. Looking across all
Ribosome profiling and total RNA profiling were performed
transcripts and all samples, we observed a general correlation
side-by-side by using RNASeq analysis to estimate for each
between mRNA levels and levels of ribosome occupancy, but

CONTACT Luigi Ferrucci ferruccilu@grc.nia.nih.gov Translational Gerontology Branch, National Institute on Aging Intramural Research Program, NIH 251
Bayview Blvd, Baltimore, MD 21224
Supplemental data for this article can be accessed here.
© 2021 Informa UK Limited, trading as Taylor & Francis Group
1556 R. THARAKAN ET AL.

Figure 1. Workflow of ribosome profiling experiment and ribosome occupancy analysis. (A) Bergström needle biopsies from vastus lateralis muscles were obtained
from healthy adults. Samples were collected from three middle-aged and two old individuals, as shown. Ribosome profiling and Total RNA libraries (RiboSeq and
RNASeq, respectively) were prepared. The bioinformatic workflow for the ribosome profiling data filtered out contaminating non-coding RNA sequences, in particular
rRNAs, tRNAs, snRNAs, and snoRNAs, and then aligned the RiboSeq reads to the known transcriptome. In parallel, Total RNASeq data was aligned to the
transcriptome. Finally, translational efficiency (TE) was calculated for each known transcript by finding the ratio RiboSeq/RNASeq. TE was then compared between
muscle from younger and older participants. (B) Overall distributions of ribosome profiling and Total RNA sequencing data. The scatterplots show, for each known
transcript, the normalized level of reads from the ribosome profiling libraries in transcripts per million (TPM) in the y-axes (RiboSeqTPM) and the RNA sequencing in
the x-axes (RNASeqTPM). The y-axis represents the level of ribosome occupancy and hence translation level, while the x-axis is a representation of the steady-state
levels of mRNAs in the cell (the balance between mRNA transcription and mRNA degradation). Highlighted are the transcripts whose translation was significantly
different between younger and older individuals: red dots represent mitochondrial transcripts, which cluster together and were decreased with age, green dots
represent mRNAs showing decreasing translation in older muscle, and blue represent mRNAs showing increased translation in older muscle. Descriptions of
significantly changed transcripts are found in Table 1.

also some transcripts which had substantially higher or lower
ribosome binding than others. Importantly, we found that
mitochondrial transcripts, which are translated by mitochon­
drial ribosomes, were abundantly recovered by our protocol
(Figure 1B, green dots).
The ratio between ribosome profiling reads and Total RNA
reads is an estimate of the probability of ribosome binding to
a given transcript and represents the translational efficiency
(TE) of that transcript. We asked whether there were specific
transcripts whose TE was different according to age. We
divided the samples into two age groups of young (ages 43,
41 and 46) and old (ages 83 and 85). Fifteen transcripts were
significantly differentially translated between age groups
(Table 1). Consistent with previous reports of decreased over­
all skeletal muscle translation with age [8], we found that 13
of these 15 transcripts were less translated in muscle from
older participants. Interestingly, 7 of the mRNAs displaying
reduced ribosome engagement in old muscle were encoded by
the mitochondrial genome (mtDNA). The mtDNA contains
only 37 genes, of which 13 are protein-coding; all 13 proteins
are components of oxidative phosphorylation complexes [9].
Since 7 out of these 13 mRNAs appeared less translated, our
data suggest that mitochondrial transcripts are poorly trans­
lated in older human skeletal muscle. The remaining six
mitochondrial mRNAs also decreased in translation with
age, although not an extent to reach statistical significance
(Table S1). Additionally, we found that translational engage­
ment of myosin heavy chain 4 (MYH4) and two isoforms of
titin (TTN), two protein components of sarcomeres, were
lower with older age. The ribosome occupancy on mRNAs
encoding three transcription factors was also significantly
associated with age; two of them increased with age
[Poly(rC) binding protein 2 (PCBP2) and Kelch like family
member 31 (KLHL31)] and one of them decreased with age
[Zinc finger BED-type containing 9 (ZBED9)]. When exam­
ining the positions of the ribosome footprints on these tran­
scripts, we do not find patterns suggesting ribosomal pausing,
as the ribosomal profiling reads appear to be evenly distrib­
uted along the transcripts in both the young and old samples
(Supplemental Data S1). Of the two transcripts whose
 RNA BIOLOGY 1557

Table 1. Transcripts with translational efficiency significantly different between age groups. Negative fold changes indicate lower translation with older age and,
viceversa, positive fold changes indicate higher translation with older age. Significance was tested using a Wald test with Benjamini-Hochberg adjustment.
Transcript Ensembl Adjusted Gene Transcript
Identifier log2 (Fold Change) P-value Name Name
Reduced with Age
ENST00000361381.2 −1.70662 0.028 MT-ND4 Mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 4
ENST00000361899.2 −1.72477 0.032 MT-ATP6 Mitochondrially encoded ATP synthase membrane subunit 6
ENST00000361739.1 −2.07291 0.007 MT-CO2 Mitochondrially encoded cytochrome c oxidase II
ENST00000361390.2 −2.12031 0.007 MT-ND1 Mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 1
ENST00000361789.2 −2.14707 0.010 MT-CYB Mitochondrially encoded cytochrome b
ENST00000362079.2 −2.2179 0.007 MT-CO3 Mitochondrially encoded cytochrome c oxidase III
ENST00000361624.2 −2.29835 0.010 MT-CO1 Mitochondrially encoded cytochrome c oxidase I
ENST00000514057.1 −2.8333 0.014 MTATP6P1 MT-ATP6 pseudogene 1
ENST00000524745.2 −3.76914 0.040 ZBED9 Zinc finger BED-type containing 9
ENST00000615779.5 −4.2267 0.040 TTN Titin
ENST00000540040.2 −4.59802 0.001 MTRNR2L1 MT-RNR2 like 1
ENST00000255381.2 −4.69351 0.032 MYH4 Myosin heavy chain 4
ENST00000591111.5 −6.51863 0.010 TTN Titin
Increased with Age
ENST00000550733.1 5.594702 0.007 PCBP2 Poly(rC) binding protein 2
ENST00000407079.1 4.250734 0.041 KLHL31 Kelch like family member 31

ribosome occupancy increased with age, one, KLHL31 mRNA,
contains an internal ribosome entry site (IRES) [10], while the
other, PCBP2 mRNA, encodes an IRES trans-activating factor
[11], suggesting cap-independent translation might increase
overall. The transcripts showing increased translation with age
did not share other common features such as upstream open
reading frames or long untranslated regions. The mechanism
of these changes will be investigated in future work.
We and other labs have documented a decline in mito­
chondrial proteins in skeletal muscle with age [12,13]. Our
current study suggests that such a decline may involve
a decrease in the translation of mtDNA-encoded proteins.
Several studies have examined changes in the translation of
mitochondrial proteins with age using isotopic labelling of
protein translation [14–16]. In Rooyackers et al., human sub­
jects were given an infusion of labelled amino acid; analysis of
mitochondria from muscle biopsies showed reduced incor­
poration of the label and hence reduced translation [15].
Using a similar approach, Miller et al. fed mice with heavy-
isotope water, then analysed the mitochondrial fraction for
isotopic incorporation and found increased translation with
age [16]. However, these studies cannot differentiate the
translation rates of specific mRNAs, since the label is deliv­
ered to the whole animal. To our knowledge our study is the
first to examine the translation of specific mitochondrial
mRNAs in ageing skeletal muscle. In another study using rat
liver, Marcus et al. isolated mitochondria from liver samples
and then incubated inner-membrane fractions to label new
translation, finding a decrease in mitochondrial translation
with age. Our ribosome profiling data from ageing human
skeletal muscle are in line with this latter study, as we find
specific decreases in the translational engagement of mtDNA-
encoded components of the mitochondrial proteome, but no
significant differences in the translation of mitochondrial
proteins encoded by the nuclear DNA. We found no changes
in the levels of mRNAs encoding protein components of the
mitochondrial ribosome (Table S2), suggesting that the
decrease in mitochondrial translation is not due to an overall
decrease in the production of mitochondrial ribosomes.
Our results further suggest that mitochondrial biogenesis in
skeletal muscle decreases with age at least in part through
a decline in mitochondrial translation. Furthermore, it is possi­
ble that the observed age-associated changes in mitochondrial
translation occur in other tissues, since mitochondrial dysfunc­
tion is widely observed across ageing tissues. The regulation of
the mitochondrial translation machinery remains poorly under­
stood. While cytosolic translation is regulated by translation
activators and repressors, typically by binding to the 5ʹ untrans­
lated regions (5ʹUTRs) of cytosolic mRNAs, mitochondrial
mRNAs do not have 5ʹUTRs [17], and it is thus unclear how
their translation is modulated. Only one factor, translational
activator of COX I (TACO1), has so far been identified as
a regulator of mitochondrial mRNA translation, but its mole­
cular mechanism of action is unknown [18]. Progress towards
elucidating how mitochondrial translation is regulated will help
to identify the molecular factors responsible for the mitochon­
drial decline in ageing skeletal muscle.
Experimental procedures
Human subjects and biopsies
Study participants were from the Baltimore Longitudinal Study
of Ageing, a longitudinal study of ageing supported by the
National Institute on Ageing. Muscle biopsies were obtained
from healthy individuals as previously reported[19]. Briefly,
after anaesthetization with 2% Lidocaine, a 6-mm Bergström
biopsy was used to obtain 200 to 400 mg of tissue, which was
then snap frozen in liquid nitrogen and stored until further
processing. Before thawing, muscle tissue was pulverized into
a powder using a liquid nitrogen cooled mortar.
Ribosome profiling, preparation of RNASeq libraries, and
sequencing
To prepare ribosome profiling RNASeq libraries, a protocol
based on Ingolia, et al. [20], was used with some modifications.
First, pulverized muscle powder was fully homogenized by
1558 R. THARAKAN ET AL.
trituration in a polysome buffer containing 20 mM Tris-Cl, pH
7.5; 150 mM NaCl; 5 mM MgCl2; 1 mM dithiothreitol; and
100 mg/ml cycloheximide (Sigma-Aldrich). The lysate was then
cleared by centrifugation at 20,000 x g for 10 minutes at 4°C,
then treated with 1% NP-40 (Sigma-Aldrich) and Turbo DNase
(ThermoFisher) at 25 U/ml for 10 minutes on ice, and cleared
again by centrifugation at 20,000 x g for 10 minutes at 4°C. The
RNA concentration was then measured using Qubit High
Sensitivity RNA kit according to the manufacturer’s protocol,
and RNase I (ThermoFisher) was added to a concentration of
1 µg RNA:10 units RNase I. Digestion was performed at room
temperature with agitation for 45 minutes. The digestion was
stopped with 200 U of RNase inhibitor (SuperaseIn,
ThermoFisher) and the digested sample was layered over 1 ml
of 1 M sucrose cushion containing RNase inhibitor in a 13-mm
x 51-mm ultracentrifugation tube (Beckman-Coulter), and
ribosomes were then pelleted by ultracentrifugation in a TLA
100.3 rotor for 2 hours at 70,000 rpm at 4°C. The resulting
pellet was then resuspended in Qiazol (Qiagen), purified, and
separated on a 15% TBE-urea gel. Footprint size selection,
dephosphorylation, and linker ligation were performed accord­
ing to the Ingolia procedure with the following modifications:
footprint sizes were selected between 28 and 34 nucleotides, the
Universal miRNA Cloning Linker (NEB) was used, and T4
RNA Ligase 2, truncated K227Q (NEB) was used. For rRNA
depletion, the RiboZero kit (Illumina) was used after linker
ligation, using the manufacturer’s protocol, with 8 µl of rRNA
removal solution, 18 µl of RNase-free water, and 4 µl RiboZero
buffer, for a total volume per sample of 40 µl. The reaction was
then purified using the RNA clean and concentrator-5 (Zymo),
eluting into 10 µl water. Reverse transcription, circularization,
and PCR amplification were performed according to Ingolia
et al., except that CircLigase 2 was substituted and ATP omitted
from the circularization reaction. Ribosome profiling libraries
were sequenced using an Illumina HiSeq 2500, single-end, 75-
bp reads, with a maximum depth per sample of between
150 million and 250 million reads.
To prepare Total RNASeq libraries, pulverized tissue was
homogenized in a buffer containing proteinase K. Digestion
was performed at 55°C for 10 minutes. Samples were then
extracted using the Qiagen Fibrous Tissue kit. Total RNAs
were then extracted using the Qiagen Fibrous Tissue kit. After
quality checking on the Agilent Bioanalyzer, the rRNA was
depleted using the RiboMinus kit according to the manufac­
turer’s protocols. cDNA was prepared using a NuGen Ovation
RNA-Seq system V2 and fragmented by sonication using
a Biorupter for library preparation. Libraries were prepared
using the Illumina TruSeq ChIP library preparation kit. RNA-
seq libraries were sequenced paired-end on an Illumina
NextSeq, with a read depth between 600 million and
800 million reads.
Bioinformatics
Ribosome profiling libraries were processed by clipping the
cloning linker sequence CTGTAGGCACCATCAAT.
Ribosome profiling libraries are well known to be contaminated
with rRNAs, which are part of the ribosome itself, not translated
sequences. Furthermore, other noncoding RNAs, specifically
tRNAs, snRNAs, snoRNAs, are also bound to the ribosome
itself and should not be considered translated sequences [21].
Therefore, the ribosome profiling reads were first aligned to
a database of known human rRNAs, tRNAs, snRNAs and
snoRNAs and any aligning reads were filtered out. The average
rate of contamination across all libraries is shown in Figure 1.
The filtered reads from the ribosome profiling libraries
(RiboSeq) as well as the Total RNA (RNASeq) reads were
aligned to the Ensembl human transcriptome (Homo_sapiens.
GRCh38.cdna) using Salmon [22]. Finally, the translational
efficiency of each transcript was determined by taking the
ratio of the ribosome profiling reads to the Total RNA reads
for each transcript, and the two age groups were compared
using RiboRex, with transcripts [23]. Transcripts were included
in the analysis only if they were detected in all five samples and
above a minimum TPM of 0.001 in RiboSeq. Upstream open
reading frames and untranslated region lengths were deter­
mined using Ensembl Human (GRCh38.p13) genome annota­
tions. For mitoribosome expression analysis, results of Salmon
alignment were analysed for differential expression with
DESeq2 [24,25].
Acknowledgments
This work was supported in its entirety by the National Institute on
Aging Intramural Research Program, NIH. We would like to acknowl­
edge the Baltimore Longitudinal Study of Aging subjects and team for
the muscle biopsy samples. We would also like to acknowledge Nicholas
Ingolia and William Wood for technical advice and assistance.
Funding
This research was supported by the Intramural Program of the National
Institutes of Aging.
Author contributions
R. Tharakan, M. Gorospe, and L. Ferrucci conceived and designed the
project. R. Tharakan and Y. Piao performed ribosome profiling experi­
ments. R. Tharakan, C. Ubaida-Mohien, and L. Ferrucci analyzed data.
All authors contributed to writing of the manuscript.
Data availability statement
Raw RNASeq datasets from all samples, both RiboSeq and TotalRNASeq,
are openly available in NCBI GEO at www.ncbi.nlm.nih.gov/geo with
accession number GSE152558.25
Disclosure of potential conflict of interest
No potential conflict of interest was reported by the authors.
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