Scientists pinpoint where thousands of individual proteins are made in intact tissue and individual cells | MIT News

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The following press release was issued by the Broad Institute of MIT and Harvard.

For researchers studying how proteins can cause human disease, knowing exactly where proteins are made within cells and tissues could help them learn about their role in disease and find new treatments.

Now, researchers from MIT and the Broad Institute at MIT and Harvard have developed RIBOmap, a technique that allows them to pinpoint and visualize the precise locations of thousands of proteins produced within intact tissue and even single cells. . Each colored dot in a large RIBOmap readout represents one mRNA molecule as it is used to make the corresponding protein, a process called translation.

The method, described today in Science, provides scientists with a way to learn new details about how translation is regulated within individual cell types and how it changes in disease. In the paper, the team used RIBOmap to study the translation of more than 5,000 genes in mouse brain tissue. They found fundamental differences between where mRNAs are produced and where corresponding proteins are translated in different cell types, suggesting that cells regulate translation in ways that existing transcriptomic methods, which analyze mRNA production, cannot. able to detect.

RIBOmap can reveal spatial patterns in translation at a resolution that has never been possible before, says Xiao Wang, a Central Institute Fellow and Merkin Institute Fellow at Broad and assistant professor of chemistry at MIT. When we use RIBOmap to examine individual cells within tissues, we can begin to discover how different cell types regulate translation differently.

Traslatomic instrument

Wang and his lab recognized that scientists needed new translation-focused tools. Researchers often use mRNA levels as a proxy for protein levels; in general, an increase in one mRNA molecule within a cell increases the levels of the corresponding protein. However, this is not always a strong correlation: a long-lived mRNA can be repeatedly translated into proteins, for example, leading to much higher levels of a protein than translation from a short-lived mRNA molecule. Cells also regulate translation rates of cancer cells, for example, they often turn on translation, producing more protein than healthy cells even when they have similar levels of mRNA.

To develop RIBOmap, the team started with a technique called STARmap that Wang and colleagues had previously built to visualize the spatial organization of mRNA molecules within intact tissue. RIBOmap, like STARmap, uses molecular probes that bind to specific mRNA sequences in tissues and single cells. Each probe contains a unique barcode, which allows researchers to identify each mRNA molecule. And by using a confocal microscope to analyze the fluorescent signals generated by in situ sequencing reactions in the tissue sample, the team could map the location of each mRNA. While STARmap probes can illuminate any mRNA molecule, RIBOmap probes only attach to mRNA molecules that are also bound to ribosomes, the cellular machinery that performs translation.

That means we’re only seeing RNA that’s actively being used to make proteins, says Jingyi Ren, co-author of the new paper and a graduate student in Wang’s lab. This is incredibly powerful information, because we can start drawing conclusions about what RNA is actually being translated into protein at any given time and place.

Running RIBOmap

To test the utility of RIBOmap, the team used it to map where proteins were produced from 5,413 genes expressed in the mouse brain. For some genes, they found that STARmap revealed high levels of the corresponding mRNA, but RIBOmap showed that the mRNA was not being actively translated in some brain regions. This suggests that the cells in those regions are blunting translation of the protein. In neurons, Wang and his colleagues found that RIBOmap generates high-resolution data that could even tell where within a single neuron the central cell body or long, branching neurites and associated synapses were being produced.

We think this method can be easily applied to tissues other than the brain, says postdoc Hu Zeng, co-first author of the paper along with Jiahao Huang, a graduate student, both in Wang’s lab. Since no genetic manipulation of the cells is required, it also means that we can easily apply it to isolated human tissue, not just animal models.

In the future, the researchers envision using RIBOmap to compare healthy and diseased tissue or see how drugs affect protein production within different cell types or areas of a tissue. They also plan to use the technology to study the basic mechanisms cells use to regulate translation levels.

This work was supported in part by the Searle Scholars Program, the Thomas D. and Virginia W. Cabot Professorship, the Edward Scolnick Professorship, an Ono Pharma Breakthrough Science Initiative Award, a Merkin Institute Fellowship, a Helen Hay Whitney Foundation Postdoctoral Fellowship and the National Institutes of Health.

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