Translatomics and Cardiometabolic Health: Key Insights
Cardiometabolic diseases, significant causal factors in heart attacks and strokes, are the leading cause of death worldwide. With the growing rates of cardiometabolic disorders in the Western world, a greater understanding of the mechanisms behind such disorders are increasingly required. While this has been an extensively researched area in recent times, much of what underlies the translational aspects of these conditions has been relatively understudied, largely due to the absence of suitable methodological techniques. With the advent of ribosome profiling, insights into this particular aspect of cardiometabolic disorders started to trickle into the scientific literature.
The Translational Landscape of the Human Heart
Cell, 2019; 178(1), pp.242-260.
van Heesch, S., Witte, F., Schneider-Lunitz, V., Schulz, J.F., Adami, E., Faber, A.B., Kirchner, M., Maatz, H., Blachut, S., Sandmann, C.L. and Kanda
The understanding of translational processes at the level of tissue is poorly detailed, including heart tissue. Here, the authors elucidate the translational landscape in dilated cardiomyopathy (DCM), the most common pathology leading to heart transplantation. They utilise ribosome profiling in combination with RNA-Seq to tease out the alterations between translational and transcriptional processes.
- When comparing DCM tissue to non-DCM controls, there were 327 differentially downregulated and 474 differentially upregulated genes at the translational level.
- Processes related to extracellular matrix formation (likely related to the tissue scarring seen in DCM patients) and the mechanistic target of rapamycin (mTOR) were upregulated at a translational level in DCM tissue. As a consequence of mTOR upregulation, the translation of 5’ terminal oligopyrimidine (TOP) motif-containing genes was also upregulated (these genes mostly being associated with ribosomal proteins).
- Interestingly, for most translated uORF-containing genes, there is an absence of the typical decrease in translation of the main CDS ORFs upon the translation of their uORF counterparts.
- Of the 346 potential protein truncating variants (PTVs) detected, only ~10% had an allelic imbalance of heterogenous single nucleotide-variants, and only ~17% had lower downstream occupancy of the point variant compared to upstream. This suggests these PTVs do not result in truncated proteins.
- It was found that a number of titin truncating variants (TTNtvs – being one of the main causes of DCM cases) did not result in truncated titin, and are translated by ribosomes. However, variants did induce termination of translation, and thus variation in TTNtvs do impact heart health differently.
- From 783 transcribed lncRNAs, the authors detected 169 translated short ORFs (sORFs) many of which were previously characterised as microproteins. When analysing translated lncRNAs for function, many were found to be associated with mitochondrial processes.
- Interestingly, the authors also detected circRNAs which underwent translation, producing viable proteins
The in-depth analysis carried out in this study significantly contributes to the knowledge of the translational processes taking place in the human heart, in both healthy and disease states. It demonstrated the variability in TTNtvs in terms of their translation, and subsequent implications for DCM. It also widened the scope of the translatome knowledge-base in this tissue, with a significant number of novel sORFs being discovered. It serves as an excellent foundation in the further exploration of this topic of interest.
Titin-truncating variants affect heart function in disease cohorts and the general population
Nature Genetics, 2017; 49(1), pp.46-53.
Schafer S, De Marvao A, Adami E, Fiedler LR, Ng B, Khin E, Rackham OJ, Van Heesch S, Pua CJ, Kui M, Walsh R.
Titin-truncating variants (TTNtvs) are truncated versions of the protein titin, which is responsible for passive elasticity of muscle in the heart. These variants are often associated with the pathogenicity of dilated cardiomyopathy (DCM), a condition which is the most common indication for heart transplantation. However, a significant proportion of individuals with TTNtvs do not exhibit features of DCM, and as such, the associations are unclear. Here, the authors undertook a meta-analysis of DCM and control patients, as well as generating rat models of varying TTNtvs, to gain insights into the impact of different TTNtvs on basic, as well as diseased, heart function.
- Titin, being an extremely large protein, contains 363 exons. The authors found that TTNtvs occurring in consecutive exons is significantly associated with DCM, regardless of their position within titin.
- In investigating truncations at the proximal or distal end of titin, it was found that both triggered nonsense-mediated decay (NMD) with similar efficiency. These truncations also lead to similar metabolic reprogramming, irrespective of their positioning, and resulted in accumulation of a number of molecules associated with a failing heart. While hearts displaying TTNtvs displayed somewhat higher strains and pressures in rat models at rest, these strains became increasingly significant under higher cardiac volumes and stress. This is hypothesised to be linked to an absence of mTORC1 signalling, as usually occurs in healthy hearts, due to this pathway already being elevated at basal levels of heart activity in hearts with TTNtv mutations.
- In analysing human datasets, the presence of a TTNtv in apparently healthy individuals, was found to be associated with a significantly larger left ventricular volume.
This paper gave important insights into the role of TTNtvs in the development of DCM, as well as determining the positional relevance of these TTNtvs to metabolic reprogramming. It also uncovered the physiological relevance of possessing one of these TTNtvs in individuals who do not meet the criteria for DCM. The authors outline that their demonstration of NMD associated with TTNtv alleles was made possible with the use of ribosome profiling, something not previously detected using RNA-Seq.
Translational regulation shapes the molecular landscape of complex disease phenotypes
Nature Communications, 2015; 6(1), pp.1-9.
Schafer, S., Adami, E., Heinig, M., Rodrigues, K.E.C., Kreuchwig, F., Silhavy, J., Van Heesch, S., Simaite, D., Rajewsky, N., Cuppen, E. and Pravenec, M
In this study, the authors aimed to investigate the level of influence that translation had on gene expression in rat heart and liver tissue, based on a BN-Lx reference strain and the SHR/Ola rat model of cardiovascular and metabolic health.
- It was found that, generally speaking, RNA levels largely corresponded with ribosome-protected fragments, indicating changes in transcription were largely mirrored in translation.
- Expectedly, when comparing mass spectrometry datasets (indictive of the proteome), authors found a greater correlation with Ribo-Seq datasets, as opposed to RNA-Seq, suggesting that Ribo-Seq is a better proxy for the proteome as compared to RNA-Seq.
- It was also found that genes that had a particularly low translational efficiency possessed significantly longer 3’ UTRs. Interestingly, when comparing differentially translated genes to differentially transcribed genes, there was a significant enrichment of binding sites for differentially transcribed miRNAs in the 3’ UTRs of these differentially translated genes.
- In genes that were under translational control, there was a distinct increase in the density of single-nucleotide polymorphisms in the 3’ UTRs. These alternations in 3’ UTRs resulted in significantly increased enrichment for RNA-binding proteins such as CPEB3 and SF3B4.
- In analysing KEGG pathway enrichment between rat strains, 37 and 31 pathways were enriched for the heart and liver respectively, with the majority for each only being detected using Ribo-Seq data. Such pathways included fatty acid metabolism for the heart and liver, and hypertrophic cardiomyopathy in the heart. Examples of specific genes regulated at the translational level alone include Myh6 (associated with heart rate), and Acadl (associated with metabolite levels and hepatic insulin resistance).
This paper identified certain traits of genes associated with altered translational efficiency, as well as potential involvement of miRNAs. Furthermore, they uncovered specific pathways that appear to be majorly regulated at the translational level in heart and liver tissue. As such, it highlights the importance of investigating translational processes with regard to pathological disease states in these organs, and the potential that any treatment that addresses this area may possess.
The cardiac translational landscape reveals that micropeptides are new players involved in cardiomyocyte hypertrophy
Molecular Therapy, 2021; 29(7), pp.2253-2267
Yan, Y., Tang, R., Li, B., Cheng, L., Ye, S., Yang, T., Han, Y.C., Liu, C., Dong, Y., Qu, L.H. and Lui, K.O.
The heart undergoes significant remodelling in response to physiological and/or pathological stimuli, and cardiomyocyte hypertrophy accounts for a significant proportion of this remodelling. Phenylephrine (PE) is a relevant stimulator of the pathways which induce this type of remodelling, activating the pathways of phosphatidylinositol 3-kinase (PI3K)/AKT and mitogen-activated protein kinase (MAPK), increasing protein synthesis. While transcriptional analysis of this process has been explored in great detail, translational analysis is still comparatively underdeveloped. Here, the authors utilise RNA-Seq and ribosome profiling to investigate cardiomyocyte hypertrophy induced by PE.
- In analysing KEGG pathways enriched following PE treatment of primary neonatal myocytes, it was found that genes relating to the MAPK signalling pathway and the PI3K/AKT signalling pathway, both known to regulate cardiomyocyte hypertrophy, were upregulated at both the transcriptional and translational level.
- Genes encoding ribosomal proteins were also significantly upregulated at a translational level. There was also no clear association between changes in translation, transcription, and translation efficiency for genes altered in the MAPK, PI3K/AKT and ribosome pathways. This led the authors to suggest that an increase in ribosome number, as opposed to increased translation rate, is responsible for the overall increase in translation.
- Under PE-induced cardiomyocyte hypertrophy, it appeared that uORF translation had little to no impact on the translation of the corresponding annotated CDS ORF. This is in contrast to the regularly reported inhibition of the CDS translation upon increased uORF translation.
- Through ribosome profiling, researchers were able to identify 126 novel sORF candidates. Of 15 tested, 11 were found to produce novel micropeptides. A majority of these localised to the nucleus or cytoplasm, as opposed to the mitochondria.
- Three of these novel micropeptides were found to significantly alter cardiac hypertrophy, with two of these cytoplasmic-based micropeptides promoting this process, and the remaining mitochondria-based micropeptide inhibiting this process. A number of KEGG pathways were identified which were differentially regulated depending on micropeptide expression.
The authors uncovered that an increased production of ribosomes, rather than an increased rate of translation, is responsible for the increase in protein synthesis seen during cardiomyocyte hypertrophy. Crucially, they identified three novel micropeptides that alter cell gene expression to either promote or deter cardiac hypertrophy. As such, these may potentially be targeted in alleviating conditions wherein such hypertrophy, or lack thereof, contributes to pathogenesis.
Slowed decay of mRNAs enhances platelet specific translation
Blood, The Journal of the American Society of Hematology, 2017; 129(17), pp.e38-e48.
Mills, E.W., Green, R. and Ingolia, N.T.
Platelets are nucleus-free cell fragments derived from megakaryocytes (MK). Lacking a nucleus, they do not possess the ability to create novel mRNA transcripts, and operate as such from a pool of mRNAs and ribosomes inherited from their parent MK. mRNA, as well as rRNA from ribosomes, decreases over time. However, the mechanisms of this decay are not well understood. Further, the mechanics of gene expression within platelets is not well understood generally. Researchers undertook both RNA-Seq and ribosome profiling in primary human platelets to gain insights into these processes.
- It was found that there were significantly fewer intronic reads within genes expressed in platelets, likely due to the absence of a nucleus.
- Transcripts that coded for genes related to known platelet functions, such as cytokine signalling and platelet degranulation, were enriched in both RNA-Seq and ribosome profiling libraries, suggesting platelets play an active role in synthesising the required proteins for these functions.
- Following thrombin stimulation, there is a distinct increase in ribosome occupancy, consistent with increased translational output. Certain genes involved in hemostatic plug formation were particularly upregulated.
- Comparisons between platelet RNA-Seq and publicly available MK RNA-Seq datasets essentially confirmed that platelet RNA is derived from parent MK cell RNA, with 80% of transcripts being shared between the two. The remaining 20% of transcripts belonged exclusively to MK cells. There was still significant quantitative variance between these datasets, indicating a degree of MK RNA sorting or platelet RNA decay.
- The authors determined the half-life of platelet mRNA transcripts to be roughly 5.7 hours, giving an insight into RNA decay in platelets. Histone mRNA and ribosome protein mRNA had slightly shorter and longer half-lives respectively.
- PELO, a ribosome rescue/mRNA surveillance protein, when overexpressed in a megakaryoblast cell line, significantly increased mRNA degradation. This protein is noticeably absent in platelets, and as such may help slow mRNA decay.
Here, authors confirm predictions of RNA sorting and mRNA decay models through an analysis of the platelet transcriptome. They also confirmed that the transcriptome, translatome, and proteome are largely in sync, although also demonstrated a distinct upregulation of specific genes relating to platelet function upon stimulation, suggesting certain alterations exist specifically at the level of the translatome. Significantly, they determine a rate of mRNA decay in platelets. Such information could be crucial in future platelet transfusion research.
