Figure 1. Comparison of whole-transcriptome sequencing, polysome analysis, ribosome profiling and protein mass spectrometry methods to measure gene expression.

Transcription of nuclear localized, non-coding and coding mRNA takes place in the nucleus of eukaryotic cells (orange box). The downward arrows represent the general path of events leading from transcription to translation of mRNA. Some RNA remain in the nucleus, but coding mRNA and some non-coding RNA are exported to the cytoplasm. Once cytoplasmic, RNA can be generally categorized into three main groups; actively translated mRNA (left), RNA subjected to some level of translational control (middle) or RNA that is targeted for degradation/decay (right). As discussed in the text, actively translated and regulated mRNA (e.g. via miRNA, localization or other types of RBPs) can be found associated with the polysome fraction of the cell. Polysomes are the cellular structures where mRNA is bound by multiple ribosomes and generally lead to protein synthesis. What complicates this view is that translational control events such as miRNA-mediated regulation also seem to be associated with polysomal structures. To help dissect these types of events, methods like polysome analysis and nuclease-treated ribosome profiling approaches can be used. The arrows pointing to the right suggest the general steps involved in performing gene ‘expression’ analysis by whole transcriptome RNAseq (beige box), polysomal analysis (red box), ribosome profiling (blue box) or protein mass spectrometry (green box). Whole transcriptome sequencing involves extracting all the RNA contained within the cell, regardless of location, compartmentalization, function or translational activity. Polysome analysis involves selectively isolating the heavy fraction of mRNPs by sucrose gradient techniques or even immunoprecipitation approaches (RIP). Thus, polysome profiling is generally thought to detect the actively translating fraction of RNA in the cell. Ribosome profiling differs slightly in that nuclease is used to break apart the intact polysomal fraction into monosomes and the mRNA fragments that are protected from digestion (i.e. ribosome protect mRNA fragments) are isolated and sequenced. Protein mass spectrometry involves directly measuring the protein composition in the cell using a variety of techniques, potentially including protein labeling strategies. The results of these different approaches, despite their limitations, are all be very powerful methods to help understand and profile gene expression.