Complete Differential Gene Expression via Translatomics

Complete Differential Gene Expression

Identifying what proteins are being expressed at a cellular level in response to a drug or as a consequence (or cause) of a pathological state is fundamental to our understanding of disease phenotypes and a logical starting point for the design of therapeutics for targeting them. However, researchers have often limited their analysis to the use of RNA-seq, which on its own provides no information on whether mRNAs are subsequently being translated into proteins. In contrast, an integrated analysis of RNA-seq and Ribo-seq data allows for a multi-layered categorisation of expression changes. There is a growing appreciation for the added value Ribo-seq or ribosome profiling can bring to these types of studies, particularly in relation to biological stresses and conditions where a large translational response is known to occur.

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, M.

In recent times, gene expression in human tissue has been primarily studied on the transcriptional level. In a large collaborative event, the authors wished to investigate translational regulation of gene expression in human heart tissue. Using ribosome profiling and mRNA sequencing in conjunction with de novo transcriptome assemblies, the authors investigated the translational landscape of 80 human hearts, using both DCM (dilated cardiomyopathy) patients and non-DCM controls. DCM is a disease of the heart muscle tissue that causes the left ventricle to stretch and thin over time, becoming weaker as time progresses.

Key Findings

The authors showed extensive translational regulation of gene expression in DCM patients, with 2,648 genes having significant expression differences in ribosome occupancy, only of which 964 could be attributed to commensurate changes in mRNA levels.
A cluster of genes involved in extracellular matrix (ECM) production, a hallmark of the fibrotic response to cardiac damage, were shown to be translationally upregulated, while genes related to mitochondrial function were both transcriptionally and translationally downregulated – a reflection of the energy-deficient state of the failing heart.
5′ terminal oligopyrimidine (TOP) motif-containing mechanistic target of rapamycin (mTOR) target genes encoding ribosomal proteins were found to be translationally upregulated in response to decreased mRNA expression in diseased hearts, highlighting the key role of the mTOR pathway in regulating translation during end stage cardiac dilation.

Implications

The first paper to extensively characterize cardiac translational processes using a large number of clinical tissue samples, this work highlights just how big a role translational regulation of gene expression plays in stressed or diseased heart tissue. In addition to its ability to provide a more complete profile of gene expression than RNA-seq alone can provide, the authors also show case how ribo-seq can be used as a critical component of a platform to identify novel proteins and validate their function.

Quantifying absolute protein synthesis rates reveals principles underlying allocation of cellular resources.

Cell, 2014; 157(3), pp.624-635
Li, G.W., Burkhardt, D., Gross, C. and Weissman, J.S.

The authors present a genome-wide approach for measuring absolute protein synthesis rates by taking relative synthesis rates based on average ribosome density from ribosome profiling data and normalising them for each protein by the total amount of proteins synthesized during a single cell cycle. Using data generated from E. coli, the authors look at the extent of which protein synthesis is controlled to optimize function and efficiency. As protein biosynthesis is the largest consumer of energy during cellular proliferation (accounting for ~50% of the energy consumption in bacteria, and ~30% of the energy consumption in mammalian cells), the authors wished to investigate how a cell allocates its synthesis capacity for each protein as this is a key step for how a cell regulates its diverse cellular functions.

Key Findings

Bacterial operons, which encode multiple subunits of the same protein complex on a single mRNA transcript, have their individual genes translated in proportion to their stoichiometry. The principle of proportional synthesis was also found to hold for multi-subunit complexes in yeast, despite individual subunits being encoded on different mRNAs in eukaryotes.
Differential translation rate of individual proteins within functional modules, such as toxin and anti-toxin pairs or two-component signalling systems, were adjusted to match the functional requirements, with the limiting component often found to be synthesized in excess.
The authors identified MetE to be the rate limiting enzyme involved in L-methionine biosynthesis, which in turn limits overall rates of protein synthesis. Synthesis of MetE was shown to be finely tuned, with the fastest growth rates being achieved at an optimal MetE expression equating to 7% of protein synthesis capacity. Lower expression levels reduces the growth rate due to insufficient methionine production, whereas higher levels also negatively affected the growth rate by diverting cellular protein synthesis capacity from the translation of other essential genes.

Implications

The authors illustrate here the capacity to measure absolute synthesis rates for cellular proteins and its utility for deciphering the logic behind the design principles of biological networks. They identify the rules underlying the observed synthesis rates for many distinct classes of proteins. By providing an approach for identifying the control points of a broad range of cellular and engineered metabolic pathways, the authors demonstrate the applicability of ribosome profiling to not only better understand cellular biochemistry but also to optimise the manufacture of biological products and therapeutics.

The translational landscape of the mammalian cell cycle

Molecular Cell, 2013; 52(4), pp.574-582
Stumpf, C.R., Moreno, M.V., Olshen, A.B., Taylor, B.S. and Ruggero, D.

During cell division, there is a fine tuned, time-sensitive control of the expression of various proteins throughout the different phases of the cell cycle. These periods act as checkpoints, ensuring accurate completion of cell division. The authors investigated gene expression that occurs during cell-cycle progression, which remains relatively unknown from a translational point of view. Using ribosome profiling;/they identified widespread translational regulation of critical cell cycle genes.

Key Findings

Widespread translational regulation was observed with 12% of all expressed transcripts exhibiting significant changes in ribosome occupancy in any of the 3 cell cycle phases investigated (G1, S and M).
Translational regulation of RICTOR (RPTOR Independent Companion Of MTOR Complex 2) – whose translational efficiency is up-regulation 3-fold between G1 and S phases of the cell cycle and down-regulated 13-fold between the S and M phases – is implicated in modulating mTOR’s control of protein synthesis and is critical for S-phase to mitosis progression.
Groups of translationally co-regulated mRNAs were identified relating to the control of metabolism, nuclear transport, and DNA repair.
5′ UTR-mediated regulation of components of the cohesin and condensin complexes exhibited high levels of ribosome occupancy during G1 and S-phase that decreased in mitosis. Both complexes are loaded into DNA during S phase or G2 in order to prepare chromosomes for segregation during mitosis.

Implications

This piece sheds light on the level to which crucial genes involved in cell cycle progression are translationally regulated. Future understanding of the mechanisms underlying the translational regulation of these genes could lead to discovery of a new class of potential drug targets for the treatment of cancers and other diseases associated with cell cycle dysregulation.

Circadian and feeding rhythms differentially affect rhythmic mRNA transcription and translation in mouse liver

Proceedings of the National Academy of Sciences, 2015; 112(47), pp.E6579-E6588.
Atger, F., Gobet, C., Marquis, J., Martin, E., Wang, J., Weger, B., Lefebvre, G., Descombes, P., Naef, F. and Gachon, F

Diurnal oscillations of gene expression are known to be a part of the rhythmic physiology in most organisms. These shifts are controlled by the interplay between the circadian clock and feeding patterns. Significant efforts have been made to understand this interaction but a complete picture of the resulting diurnal transcription–translation processes is still missing. Through the use of Ribo-seq and RNA-seq the authors investigated the regulatory landscape of mice with intact or deficient circadian clocks by subjecting them to different feeding regimens.

Key Findings

Circadian and feeding cycles regulate changes in transcription which in turn drive changes in rhythmic mRNA accumulation and translation for most genes.
Two distinct subsets of genes exhibited rhythmic changes in translation despite displaying constant mRNA levels.
By day, genes containing Translation Initiator of Short 5′-UTR (TISU) motifs translated nuclear encoded genes related to mitochondrial function.
By night, genes containing 5′-Terminal Oligo Pyrimidine Tract (TOP) elements translated genes related to the translational machinery itself.
Restricting mice feeding to night-time led to narrower phases and higher amplitudes in the rhythmic translation of both TOP and TISU genes.
Knock-out of Bmal1, a transcription factor which plays a key role in the regulation of circadian rhythms, was only found to effect rhythmic translation of a small number of genes with no clear mechanism behind the regulation being apparent.

Implications

The authors found that feeding-fasting cycles appear to be the main timing cues for the liver “clock”, going as far as bypassing the master clock contained within the brain. This brings a hypothesis that the desynchrony of master and peripheral clocks under altered feeding conditions can disrupt the homoeostasis of an organism, which can increase the risk of chronic diseases. Therefore, the translational control of ribosome biogenesis and mitochondrial function by the circadian clock and feeding rhythms could provide key factors in pathologies associated with circadian disruption.

Oxygen and glucose deprivation induces widespread alterations in mRNA translation within 20 minutes

Genome Biology, 2015; 16(1), pp.1-14.
Andreev, D.E., O’Connor, P.B., Zhdanov, A.V., Dmitriev, R.I., Shatsky, I.N., Papkovsky, D.B. and Baranov, P.V.

Oxygen and glucose metabolism play pivotal roles in many physiological conditions. The World Health Organisation denote that ischemic heart disease and stroke are the leading causes of death in humans. Both of these diseases are characterized by the interruption of blood flow (which disrupt oxygen and glucose supply) to various tissues, causing severe damage. The authors used ribosome profiling to assess gene expression in the neuronal cell line PC12 over varying durations of oxygen and glucose deprivation (OGD), with the intent to further understand the mechanisms involved.

Key Findings

While only a little over a hundred genes had RNA levels affected by OGD, widespread translational regulation was observed in around 3,000 genes in the same period.
Translational responses to OGD occurred rapidly with most changes occurring in as little as 20 minutes and amplifying with increasing duration of exposure.
Genes associated with the oxidative phosphorylation pathway and genes associated with neurological disorders whereby this pathway plays a major role (Parkinson’s, Huntington’s and Alzheimer’s) were found to be overrepresented among genes with differentially expressed ribosome occupancy.
The mechanisms underlying these changes are likely multifactorial, encompassing increased translation of 5’leaders, OGD-induced ribosome pausing, reduced stringency of start codon selection and improved accuracy of translation termination.

Implications

The authors were able to uncover novel regulatory mechanisms of the translational response to OGD in mammalian cells, that were different from more well-known pathways like HIF (hypoxia inducible factors) signalling. The large rapid response in gene expression identified by the authors would have been beyond the power of RNA-seq to reveal.