Translating the Future of RNA Genomics
Translation – the process by which RNA is converted into protein – is a key element in the central dogma of molecular biology.
How Translation Works
Of course, it’s not quite as simple as depicted above. Transcription and translation are both extremely complex processes. Transcription is the process by which transcriptase reads DNA and generates RNAs.
Translation creates proteins when ribosomes occupy and translate the mRNA, and the resulting set of expressed proteins (the proteome) is the foundation for cellular function. Determining the composition of the proteome and relative abundance of proteins in biological and clinical samples is a pillar of biomedical research. To explore this, we must first understand translation itself.
Translation is a four-step process, consisting of initiation, elongation, termination, and ribosome recycling. Many steps must occur correctly and concurrently for a successful round of translation to take place. This process is richly regulated and there are many factors affecting the steps outlined above.
Environmental conditions such as temperature and nutrient availability can determine which proteins are translated by a cell. For example, in healthy, pro-growth conditions a cell will synthesize growth proteins. However, in stressful conditions like heat shock where the temperature is higher than prefers, heat shock associated proteins will be synthesized instead. As the conditions a cell experiences change, so do the proteins it translates.
The structure of the mRNA itself will also affect its translational activity. An mRNA that is rich with secondary structures may not be easily navigated by ribosomes and may require helicases to aid in translation. A more simply structured mRNA on the other hand can be more easily bound by ribosomes which can result in a more abundantly expressed protein. Other structural features including the 5’ cap and poly-A tail influence translation as well.
While all the aforementioned factors affect translation, translation itself is ultimately performed by the ribosome. Thus, to assess the complexity of the proteome, one must be able to quantitate transcripts associated with ribosomes that are actively being translated. The ability to do this with high accuracy will yield an accurate and unbiased representation of protein levels. This type of data is referred to as Ribosome Occupancy, or “RO” for short.
Historically, the scientific community has had to make a choice when researching ribosome occupancy. On one hand, transcriptional methods are used to investigate RNA levels, whereas translational methods interrogate protein levels. Traditional polysome profiling, a way to look at ribosome-bound RNA levels, is well substantiated in literature but can be both time consuming and laborious. To look at protein levels researchers often turn to mass spectrometry, a less accessible technology compared to NGS that only elicits steady state levels of protein without insight into protein production.
While commercial next-gen sequencing methods like RNA-Seq continue to become more accessible in both price and ease of use, they have so far been unable to capture ribosome associated transcripts. As a result, the research community is left with limited options to meaningfully answer scientific questions about ribosome occupancy and changes in translation using NGS alone. Therefore, bridging the gap between mass spec and RNA-Seq with a single assay would provide a significant benefit to researchers by enabling translational interrogation using readily available NGS methods.
Capture Ribosome Occupancy with eRibo
Eclipsebio has developed a suite of products called eRibo that do exactly that: delivering meaningful translational data on multiple levels, utilizing the speed and efficiency offered by NGS. All eRibo methods provide a comprehensive dual-output of both RO and RNA-Seq datasets from one single streamlined workflow. Each of the three methods utilizes a hybridized approach to explore active translation, giving the researcher the ability to choose exactly which aspects of RNA translation they want to interrogate.
Eclipsebio's eRibo Product Family
- eRibo Total provides ribosome occupancy data and RNA-Seq with full transcript length coverage, which enables the identification of splice variants and changes in translation.
- eRibo 3’ is a rapid, easy, and efficient method of quantifying ribosome occupancy. With a sequencing library focused at the 3’ end of transcripts, gene-level expression data and gene counting can occur with higher multiplexing capabilities
A myriad of research questions can be addressed with accurate ribosome profiling data. In academic research, RO data can be used to make mechanistic and interaction discoveries. The biopharmaceutical world can utilize RO and ribosome profiling to understand a cell’s response to drug treatments, e.g. whether their protein of interest is being up- or down-regulated as intended.
Eclipsebio’s eRibo product family addresses these applications and many more. Stay tuned – in a future blog post, we will do a deep dive into each of the eRibo methods so that you can better understand what each of them offer to help you accelerate your research.
Figures created with biorender.com