The central dogma of biology states that DNA codes for RNA, which codes for protein. Of course, there are lots of steps in between: factors like regulatory mechanisms, editing, and post-translational modifications can impact polymer stability or the efficiency of the processes involved. It is important to note that proteins are the real workhorses inside of the cells, not mRNAs.
Next-generation RNA sequencing was first employed ~20 years ago. Before we could really investigate RNA, we made one key assumption: the level of RNA transcripts would match the level of the corresponding protein. As we will see momentarily, that assumption is deeply flawed.
RNA vs. Protein: Why the Disconnect?
There are quite a few variables involved in the relationship between the levels of RNA and protein. We need to take into account the cell state, methods of measurement, rate of translation, post translational modifications to the protein, and the stability of the mRNA vs. the stability of the protein in question. Messenger RNA (mRNA) is the fully matured form of RNA that moves from the nucleus to the cytoplasm to be translated to a protein. In general, mRNAs have rapid turnover in the cytoplasm, whereas proteins can be either short-lived or stabilized to remain active for extended periods of time.
With the ability to measure transcript and protein levels in individual cells, it is now possible to accurately compare them to each other. Single-cell RNAseq and mass spectrometry have provided data demonstrating that at steady state, mRNA and protein levels are generally similar for the majority of targets.
That said, cellular stress or differentiation can lead to a lag between transcription and translation so that levels no longer correlate well. Another factor controlling translation is the location of the mRNA relative to the ribosomes. Ribosomes are not homogeneously distributed throughout the cytoplasm, so there could be competition to get a place on one.
Resource limitation can also delay protein translation as it’s a very expensive process for cells. Finally, the rate of translation will also be impacted by the sequence of the mRNA itself, specifically the distribution of codons and their availability.
Traditional Detection Limitations
Historically, RNA and protein technologies have had limitations in the depth and accuracy of data they could provide. RNA sequencing and bulk transcriptomics give researchers the average expression across cells/tissues, rather than the actual expression of any individual cell or cell types. Proteomics has often relied on immunoassays, but these are somewhat limited in plex when they offer absolute quantification.
The use of single-cell RNA-seq to generate large atlases of whole transcriptomes for a staggering variety of cell types has been a boon to researchers. Groups such as the Human Protein Atlas doing the same for protein finally enables researchers to take a close look at the differences between transcripts and proteins.
The Spatial Dimension of Biology
The cell’s environment has an impact on transcription and translation that we can study using spatial biology. Spatial biology is the study of cells in their native tissue context.
New technologies like 10x Genomics Xenium now allow scientists to analyze transcript and protein levels in the same cell concurrently. This opens the door to understanding how the cell’s neighbors are behaving relative to the cell itself, and to measuring their impact on their local microenvironment in the tissue. For example, researchers can now ask, “Are immune cells near the tumor nest expressing different transcripts and proteins than those near the invasive margin?”
An example of the difference between Xenium RNA (left) and Xenium protein (right). The purple section is expressing a unique set of proteins when compared to the blue sections. When looking at only RNA, the blue and purple sections appear similar.
Case Studies of RNA/Protein Mismatch in Context
With new technologies generating novel insights, several high-profile studies have already identified mismatches in protein and RNA expression. These discoveries have implications on our understanding of human health and disease.
In solid tumors, despite attempts to vascularize themselves, the tumor microenvironment frequently suffers from hypoxia due to limited access to oxygen from blood vessels. In cases of hypoxia, transcription and translation processes are significantly altered. The endoplasmic reticulum (ER) monitors the cell’s homeostasis and preferentially translates proteins involved in stress response. Additionally, states of hypoxia also suppress the rate of translation by blocking the activity of a key initiation factor.
RNA and protein technologies used in neuroscientific studies have uncovered important mechanisms of neuronal activity. We now know that mRNAs are transported to the synapse and undergo translation upon stimulation at the synapse. Data have shown there are hundreds of mRNAs localized in dendrites but even more interestingly, these seem to be dynamically regulated. For example, in response to neuronal activity, transcripts of BDNF and trkB increase and travel rapidly to the synapse. Dopamine receptor activation leads to glutamate receptor 1 and 2 being transported to the synapse.
Many available protein technologies and panels focus on inflammation response. In this study, researchers found that inflammation is triggered by the innate immune system releasing cytokines that activate downstream pathways to the site of injury. This system has to be tightly regulated or the host will suffer severe consequences such as cytokine storm, toxic shock, cardiovascular disease, and inflammatory diseases.
Since these activities require a rapid and strong response, the mRNAs are transcribed, but subjected to translational control. The mRNAs are bound by RNA-binding proteins that block translation via steric hindrance. Upon injury, the expression of another protein derepresses the translational block and allows the inflammatory cytokine to be translated to a protein.
How Spatial Biology Bridges the Gap
With the recent release by 10x Genomics of protein panels that can co-detect on the same tissue section as RNA panels, a new level of understanding for cancer, neuroscience, and immunology is on the horizon. As mentioned earlier, RNA and protein levels don’t always correlate. It’s critical to know the protein levels, since they are doing the majority of the work in the cell. This opens the door to identify new biomarkers and drug targets, and to deepen our understanding of disease states in a totally unprecedented manner.
Single-cell RNA sequencing is an invaluable tool for researchers today. But we must remember that mRNA levels do not always correlate with protein levels. By using the newest spatial biology techniques, scientists can unlock deeper insights into health and disease with the promise of more targeted treatments.
