Translational control in endothelial cells

Abstract

Cellular phenotype and function is ultimately determined by the synthesis of proteins derived from a genetic blueprint. Control of gene expression occurs at multiple checkpoints, including the transcription of DNA into RNA and the translation of RNA into protein. Translational control mechanisms are important regulators of cellular phenotype, controlling up to 10% of overall cellular gene expression, yet they remain relatively understudied when compared with transcriptional control mechanisms. Specific regulation of protein synthesis from messenger RNA transcripts allows cells to temporally unlink translation from transcription and provides a mechanism for a more rapid response to environmental signals than if transcription were required. We discuss some of the fundamental concepts of translational control, tools for studying it and its relevance to vascular cells, in particular the endothelium.

Figure 1

Control of gene expression – Classical view of gene regulation in which an extracellular signal triggers a sequential series of temporally related events leading from DNA to mRNA to protein.

Figure 2

Initiation of translation in eukaryotic cells - The traditional scanning model illustrates “cap-dependent” initiation of translation in which eukaryotic initiation factors (eIFs) facilitate ribosome binding to the capped 5’ end of an mRNA transcript. “Cap-independent” translation is also depicted in which an internal ribosome entry site (IRES) with a complex secondary structure facilitates ribosome binding in the 5’ untranslated region (UTR).

Figure 3

Ribosome profiling – A) Efficiently translated mRNA transcripts (polysomes) are separated from inefficiently translated mRNA transcripts (monosomes) using sucrose density gradient centrifugation. Because individual ribosomes are so heavy, discrete bands are produced that correspond to an integer number of ribosomes in a polysome. Following centrifugation, the gradients are passed through a spectrophotometer which generates tracings based on the presence of genetic material to facilitate separation of the monosome fraction from the polysome fraction. B) In general, conditions characterized by high translational activity (such as cell growth or proliferation) will have mRNA predominantly associated with polysomes (dashed line) while conditions characterized by low translational activity (such as starvation) will have mRNA predominantly associated with monosomes (solid line).

Figure 4

Translation state array analysis (TSAA) – This high-throughput method for assessing the translational state (TS) of a large number of mRNAs combines ribosomal profiling and microarray technology. Cellular mRNAs are separated into inefficiently translated fractions (monosomes) or efficiently translated fractions (polysomes). The mRNAs are then converted into cDNA labeled with a fluorescent marker (Cy3 is green and Cy5 is red) and hybridized to an array chip containing thousands of genes. If there is a larger amount of mRNA for a certain gene sequence in the monosome fraction, that spot on the chip (representing that gene) will fluoresce green and will have a corresponding TS <1 (where TS represents the mRNA in the polysome fraction divided by the mRNA in the monosome fraction). If there is a larger amount of mRNA for a certain gene sequence in the polysome fraction, that spot on the chip will fluoresce red and will have a corresponding TS >1. If there are equivalent amounts of mRNA for both the monosome and polysome fractions, then that spot will fluoresce yellow and have a corresponding TS≈1. If there is no hybridization of the probes to the gene in question, the spot will not fluoresce. Once the translational states are determined for each gene on the control array, they can be compared with the translational states for each gene on the experimental array. A ratio of the TS for each gene under experimental conditions divided by the TS for control conditions will yield a translational index (TLI). This value represents the redistribution of mRNA between monosome and polysome fractions for the given experimental condition.

Figure 5

Parallel signal inputs can regulate transcription and translation independently - Contemporary view of factors controlling gene expression in which there may be multiple signaling inputs occurring simultaneously to modulate changes in a cell’s gene expression profile.