Translation regulation gets its 'omics' moment

Abstract

The fate of cellular RNA is largely determined by complex networks of protein-RNA interactions through ribonucleoprotein (RNP) complexes. Despite their relatively short half-life, transcripts associate with many different proteins that process, modify, translate, and degrade the RNA. Following biogenesis some mRNPs are immediately directed to translation and produce proteins, but many are diverted and regulated by processes including miRNA-mediated mechanisms, transport and localization, as well as turnover. Because of this complex interplay estimates of steady-state expression by methods such as RNAseq alone cannot capture critical aspects of cellular fate, environmental response, tumorigenesis, or gene expression regulation. More selective and integrative tools are needed to measure protein-RNA complexes and the regulatory processes involved. One focus area is measurements of the transcriptome associated with ribosomes and translation. These so-called polysome or ribosome profiling techniques can evaluate translation efficiency as well as the interplay between translation initiation, elongation, and termination-subject areas not well understood at a systems biology level. Ribosome profiling is a highly promising technique that provides mRNA positional information of ribosome occupancy, potentially bridging the gap between gene expression (i.e., RNAseq and microarray analysis) and protein quantification (i.e., mass spectrometry). In combination with methods such as RNA immunoprecipitation, miRNA profiling, or proteomics, we obtain a fresh view of global post-transcriptional and translational gene regulation. In addition, these techniques also provide new insight into new regulatory elements, such as alternative open reading frames, and translation regulation under different conditions.

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.

Figure 2. Example ribosome footprint

Comparison of read densities from the CWP2 gene in yeast showing a ribosome profiling sample (upper panel) and a polyA+ selected mRNAs sample (lower panel). The ribosome profiling sample results in coverage only within the open reading frame (ORF) of CWP2 and none in the 5’ or 3’ UTR regions. This reflects the primary regions of transcripts protected from nuclease digestion by bound ribosomes. In contrast, the mRNA sample results in coverage within both the ORF and the UTR regions, reflective of non-nuclease treated, intact RNA samples.

Figure 3. A model for miRNA-mediated gene regulation

To translate an mRNA, the 5’ and 3’ ends must establish communication through a network of proteins associated with the mRNP (messenger ribonucleoprotein complex)(see ref. for more details). In normal translation (left side) the ribosome subunits (green) quickly initate translation to produce nascent peptides. These transcripts will produce Ribosome Protected Fragments coincident with the density of associated ribosomes. In contrast, when a target mRNA is regulated by miRNA (right side) there is a disruption in the communication between the 5’ and 3’ ends which leads to an order of events, such as 1) Translational Repression; 2) Deadenylation and 3) eventual turnover or decay of the target mRNA. In these cases, the relative amounts of RPFs produced from miRNA regulated targets is lower than in the absence of the miRNA.