Translatomics
[2] Recent advancements, including single-cell ribosome profiling, have significantly improved the resolution of these studies, allowing researchers to gain insights into translation at the level of individual cells.
[5] Nearing the completion of the Human Genome Project the field of genetics was shifting its focus toward determining the functions of genes.
These collections of materials were called -omes, evoking the widespread excitement surrounding the sequencing of the human genome.
Translatomics, in combination with degradomics, aims to describe the net change to the proteome under different conditions.
In doing so, omics gain insight into different levels of regulation of gene expression and therefore genome function.
In some cases, RNA or protein abundance does not reflect function because these biomolecules may be degraded rapidly, or they may remain in a cell long after they are initially synthesized.
In polysome profiling, a sucrose gradient is used to separate molecular complexes in a cell lysate based on size.
The translation rate of mRNAs is determined based on its detection and abundance in the fractions of lower and higher molecular weight.
The full length translating mRNA (RNC-seq) involves centrifugation of lysated sample on a sucrose cushion.
[1] In Ribosome profiling, cellular mRNA including polysomes is subjected to ribonucleases, enzymes that cleave RNA.
The cell type of interest is engineered to express a ribosomal subunit fused to an epitope tag such as green fluorescent protein.
[10] After cell lysis, antibodies targeting the epitope are used to isolate mRNAs that are bound to the ribosomes containing the fusion proteins.
One method uses a variant of Stable isotope labeling by amino acids in cell culture (SILAC).
As such, pSILAC pulses have to run much longer than the translation process, making quantification of nascent peptides inaccurate.
Similar to pSILAC, AHA methods require longer pulses, thus limiting its efficacy in quantifying nascent peptides.
This, coupled with similar secondary and tertiary structures makes separating different tRNA species difficult.
Large numbers of different tRNA species cannot be fully separated by 2D-gel electrophoresis, with only 62 spots found for the 269 rat tRNAs.
[21] High performance liquid chromatography can be used to separate tRNAs based on aminoacylated tRNA isoacceptors.
[21] Mass spectrometry (MS) can be used to separate tRNAs based on unique endonuclease digestion products.
[21] Hybridization based microarrays use the 3’CCA conserved sequence in tRNAs to attach a fluorescent probe.
