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Translation elongation rate varies among organs and decreases with age

Gerashchenko, Maxim V, Peterfi, Zalan, Yim, Sun Hee, Gladyshev, Vadim N·2021·Nucleic Acids Research
DOI 10.1093/nar/gkaa1103PMC PMC7826258
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Abstract

There has been a surge of interest towards targeting protein synthesis to treat diseases and extend lifespan. Despite the progress, few options are available to assess translation in live animals, as their complexity limits the repertoire of experimental tools to monitor and manipulate processes within organs and individual cells. It this study, we developed a labeling-free method for measuring organ- and cell-type-specific translation elongation rates in vivo. It is based on time-resolved delivery of translation initiation and elongation inhibitors in live animals followed by ribosome profiling. It also reports translation initiation sites in an organ-specific manner. Using this method, we found that the elongation rates differ more than 50% among mouse organs and determined them to be 6.8, 5.0 and 4.3 amino acids per second for liver, kidney, and skeletal muscle, respectively. We further found that the elongation rate is reduced by 20% between young adulthood and mid-life. Thus, translation, a major metabolic process in cells, is tightly regulated at the level of elongation of nascent polypeptide chains.

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Figure 1. Evaluating translation inhibitors in vivo. (A) Single injection done to retroorbital cavity around the eye. (B) Both translation inhibitors show similar efficiencies and uptake times following injection into the bloodstream. Using sucrose gradient profiles of the mouse liver as an example, we demonstrate that harringtonine depletes polysomes while co-injection with cycloheximide preserves polysomes. (C) Harringtonine performance in 7 organs. It effectively stalls ribosomes at the vicinity of start codons. Post-injection incubation time was 5 min except testes, where up to 30 min were required to pass the blood-tissue barriers. Zero corresponds to the start of the open reading frame. 100 nucleotides from the 5′-UTR were added to allow mapping footprints over the start codon.
Figure 1. Evaluating translation inhibitors in vivo. (A) Single injection done to retroorbital cavity around the eye. (B) Both translation inhibitors show similar efficiencies and uptake times following injection into the bloodstream. Using sucrose gradient profiles of the mouse liver as an example, we demonstrate that harringtonine depletes polysomes while co-injection with cycloheximide preserves polysomes. (C) Harringtonine performance in 7 organs. It effectively stalls ribosomes at the vicinity of start codons. Post-injection incubation time was 5 min except testes, where up to 30 min were required to pass the blood-tissue barriers. Zero corresponds to the start of the open reading frame. 100 nucleotides from the 5′-UTR were added to allow mapping footprints over the start codon.CC BY-NC · PubMed Central
Figure 2. Experimental setup for double injections. (A) Representative electrocardiograms (ECG) from injected mice. The upper track shows the slowdown of the beat rate in response to 5 mg/ml harringtonine. The lower track shows no difference after the injection of 2.5 mg/ml harringtonine. The effect of harringtonine appears roughly 20 s after the injection. The red line marks the injection event. (B) High doses of harringtonine severely drop heartbeat rate. We established that 2.5 mg/ml had no effect on the beat rate, whereas 5 mg/ml decreased it twice. 10 mg/ml caused cardiac arrest in most cases. All injections were done via the tail vein − injection volume was 200 ul and the weight of mice was 20–25 g. (C) The layout of mouse organs susceptible to translation inhibitors. Organs marked in green are permeable for both harringtonine and cycloheximide within 15 s post-injection. Translation elongation rate can be directly measured in these organs. Testes (shown in yellow) are partially permeable, and brain is mostly unaffected by inhibitors.
Figure 2. Experimental setup for double injections. (A) Representative electrocardiograms (ECG) from injected mice. The upper track shows the slowdown of the beat rate in response to 5 mg/ml harringtonine. The lower track shows no difference after the injection of 2.5 mg/ml harringtonine. The effect of harringtonine appears roughly 20 s after the injection. The red line marks the injection event. (B) High doses of harringtonine severely drop heartbeat rate. We established that 2.5 mg/ml had no effect on the beat rate, whereas 5 mg/ml decreased it twice. 10 mg/ml caused cardiac arrest in most cases. All injections were done via the tail vein − injection volume was 200 ul and the weight of mice was 20–25 g. (C) The layout of mouse organs susceptible to translation inhibitors. Organs marked in green are permeable for both harringtonine and cycloheximide within 15 s post-injection. Translation elongation rate can be directly measured in these organs. Testes (shown in yellow) are partially permeable, and brain is mostly unaffected by inhibitors.CC BY-NC · PubMed Central
Figure 3. Measuring organ-specific translation elongation rate in vivo. (A) ECG monitoring of heart function before and after drug injection to ensure the heartbeat rate is unaffected at the chosen dosage. (B) Experimental design for a time-resolved study. (C) Linear regression analysis of injection timepoints to infer the elongation rate. (D) Comparison of mean translation elongation rates in three organs. Statistical significance was accessed with a linear model distance ∼ time, error bars represent the standard error and the number of samples is identical to the panel E (excluding controls). (E) Metagene analysis of ribosome profiling sequences. Genes longer than 2000 nucleotides are presented (refer to Supplementary Figure S4 for gene length selection). The first 1500 nucleotides are shown starting from the first codon of the reading frame. Solid lines represent mean signal from biological replicates, and light shading corresponds to a standard deviation. 0 s timepoint refers to control animals injected with PBS.
Figure 3. Measuring organ-specific translation elongation rate in vivo. (A) ECG monitoring of heart function before and after drug injection to ensure the heartbeat rate is unaffected at the chosen dosage. (B) Experimental design for a time-resolved study. (C) Linear regression analysis of injection timepoints to infer the elongation rate. (D) Comparison of mean translation elongation rates in three organs. Statistical significance was accessed with a linear model distance ∼ time, error bars represent the standard error and the number of samples is identical to the panel E (excluding controls). (E) Metagene analysis of ribosome profiling sequences. Genes longer than 2000 nucleotides are presented (refer to Supplementary Figure S4 for gene length selection). The first 1500 nucleotides are shown starting from the first codon of the reading frame. Solid lines represent mean signal from biological replicates, and light shading corresponds to a standard deviation. 0 s timepoint refers to control animals injected with PBS.CC BY-NC · PubMed Central
Figure 4. Ribosome footprint profiles of representative transcripts. Transcripts were selected among those highly expressed in three organs: liver, kidney and skeletal muscle. For each transcript, the entire ORF was selected with extra 100 nucleotides from 5′- and 3′-UTRs. The ribosomal run-off is a consequence of the harringtonine injection. The entire ORF with 100 nucleotides from the 5′ and 3′ UTRs is shown. The sharp peak on the left side of each plot corresponds to ribosomes stalled at the translation start site by harringtonine. Annotated ORF is marked with the grey rectangle.
Figure 4. Ribosome footprint profiles of representative transcripts. Transcripts were selected among those highly expressed in three organs: liver, kidney and skeletal muscle. For each transcript, the entire ORF was selected with extra 100 nucleotides from 5′- and 3′-UTRs. The ribosomal run-off is a consequence of the harringtonine injection. The entire ORF with 100 nucleotides from the 5′ and 3′ UTRs is shown. The sharp peak on the left side of each plot corresponds to ribosomes stalled at the translation start site by harringtonine. Annotated ORF is marked with the grey rectangle.CC BY-NC · PubMed Central
Figure 5. Time- and age-dependent changes in translation. (A) Ribosome footprint coverage of ATF4 transcript. This gene is a classic example of post-transcriptional regulation mediated by upstream reading frames (uORFs). It has 2 uORFs, shown in orange. Shortly after the injection of harringtonine, ribosomes are stalled at the start codons of the corresponding uORFs. Interestingly, there are two peaks in skeletal muscle samples: one at the first uORF and another at the second. However, there is only a single peak in liver and kidney. (B) The elongation rate declines with age. Left: estimation of translation elongation in liver was done as in Figure 3E, but 18-month-old mice were used. Right: Liver translation between two groups of mice (3- and 18-month-old) was compared. Mean elongation rates (aa/sec) were measured as 6.8 at the young age and 5.5 at the older age.
Figure 5. Time- and age-dependent changes in translation. (A) Ribosome footprint coverage of ATF4 transcript. This gene is a classic example of post-transcriptional regulation mediated by upstream reading frames (uORFs). It has 2 uORFs, shown in orange. Shortly after the injection of harringtonine, ribosomes are stalled at the start codons of the corresponding uORFs. Interestingly, there are two peaks in skeletal muscle samples: one at the first uORF and another at the second. However, there is only a single peak in liver and kidney. (B) The elongation rate declines with age. Left: estimation of translation elongation in liver was done as in Figure 3E, but 18-month-old mice were used. Right: Liver translation between two groups of mice (3- and 18-month-old) was compared. Mean elongation rates (aa/sec) were measured as 6.8 at the young age and 5.5 at the older age.CC BY-NC · PubMed Central

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Citations

Cites

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Metadata

Authors
Gerashchenko, M. V., Peterfi, Z., Yim, S. H., & Gladyshev, V. N.
Affiliations
Division of Genetics, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA
ORCID
Venue
Nucleic Acids Research
Publisher
Oxford University Press (OUP)
Published
2020-12-02
Volume
49
Issue
2
Pages
e9-e9
ISSN
  • 0305-1048
  • 1362-4962
DOI
10.1093/nar/gkaa1103
PMC
PMC7826258
Cited by
98
References
7
Language
en
Licence
creativecommons.org/licenses/by-nc/4.0/ (vor)
Funders
NIH (DK117149, AG065403, AG047200)

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Gerashchenko, M. V., Peterfi, Z., Yim, S. H., & Gladyshev, V. N. (2021). Translation elongation rate varies among organs and decreases with age. Nucleic Acids Research. https://doi.org/10.1093/nar/gkaa1103