Tuesday, August 11, 2015

Penn Science Spotlight: Learning how T cells manage the custom RNA business

Chris Yarosh

This Science Spotlight focuses on the research I do here at Penn, the results of which are now in press at Nucleic Acids Research1. You can read the actual manuscript right now, if you would like, because NAR is “open access,” meaning all articles published there are available to anyone for free. We’ve talked about open access on this blog before, if you’re curious about how that works. 

First, a note about this type of science. The experiments done for this paper fall into the category of “basic research,” which means they were not designed to achieve an immediate practical end. That type of work is known as “applied” research. Basic research, on the other hand, is curiosity-driven science that aims to increase our understanding of something. That something could be cells, supernovas, factors influencing subjective well-being in adolescence, or anything else, really. This isn’t to say that basic research doesn’t lead to advances that impact people’s lives; quite the opposite is true. In fact, no applied work is possible without foundational basic work being done first. Rather, the real difference between the two categories is timeline and focus: applied research looks to achieve a defined practical goal (such as creating a new Ebola vaccine) as soon as possible, while basic research seeks to add to human knowledge over time. If you’re an American, your tax dollars support basic research (thanks!), often through grants from the National Institutes of Health (NIH) or the National Science Foundation (NSF). This work, for example, was funded in part by two grants from the NIH: one to my PhD mentor, Dr. Kristen Lynch (R01 GM067719), and the second to me (F31 AG047022). More info on science funding can be found here.

Now that you've gotten your basic research primer, let's talk science. This paper is primarily focused on how T cells (immune system cells) control a process called alternative splicing to make custom-ordered proteins. While most people have heard of DNA, the molecule that contains your genes, not everyone is as familiar with the RNA or proteins. I like to think of it this way: DNA is similar to the master blueprint for a building, specifying all of the necessary components needed for construction. This blueprint ultimately codes for proteins, the molecules in a cell that actually perform life’s work. RNA, which is “transcribed” from DNA and “translated” into protein, is a version of the master blueprint that can be edited as needed for different situations. Certain parts of RNA can be mixed and matched to generate custom orders of the same protein, just as you might change a building’s design based on location, regulations, etc. This mixing and matching process is called alternative splicing (AS), and though it sounds somewhat science-fictiony, AS naturally occurs across the range of human cell types.



While we know AS happens, scientists haven’t yet unraveled the different strategies cells use to control it. Part of the reason for this is the sheer number of proteins involved in AS (hundreds), and part of it is a lack of understanding of the nuts and bolts of the proteins that do the managing. This paper focuses on the nuts and bolts stuff. Previous work2 done in our lab has shown that a protein known as PSF manipulates AS to produce an alternate version of a different protein, CD45, critical for T cell response to antigens (bits of bacteria or viruses). PSF doesn’t do this, however, when a third protein, TRAP150, binds it, although we previously didn’t know why. This prompted us to ask two major questions: How do PSF and TRAP150 link up with one another, and how does TRAP150 change PSF’s function?

My research, as detailed in this NAR paper, answers these questions using the tools of biochemistry and molecular biology. In short, we found that TRAP150 actually prevents PSF from doing its job by binding in the same place RNA does. This makes intuitive sense: PSF can’t influence splicing of targets it can’t actually make contact with, and it can't contact them if TRAP150 is gumming up the works. To make this conclusion, we diced PSF and TRAP150 up into smaller pieces to see which parts fit together, and we also looked for which part of PSF binds RNA. These experiments helped us pinpoint all of the action in one region of PSF known as the RNA recognition motifs (RRMs), specifically RRM2. Finally, we wanted to know if PSF and TRAP150 regulate other RNA molecules in T cells, so we did a screen (the specific technique is called “RASL-Seq,” but that’s not critical to understanding the outcome) and found almost 40 other RNA molecules that appear to be controlled by this duo. In summary, we now know how TRAP150 acts to change PSF’s activity, and we have shown this interaction to be critical for regulating a bunch of RNAs in T cells.

So what are the implications of this research? For one, we now know that PSF and TRAP150 regulate the splicing of a range of RNAs in T cells, something noteworthy for researchers interested in AS or how T cells work. Second, we describe a mechanism for regulating proteins that might be applicable to some of those other hundreds of proteins responsible for regulating AS, too. Finally, PSF does a lot more than just mange AS in the cell. It actually seems to have a role in almost every step of the DNA-RNA-protein pathway. By isolating the part of PSF targeted by TRAP150, we can hypothesize about what PSF might do when TRAP150 binds it based on what other sections of the protein remain “uncovered.” It will take more experiments to figure it all out, but our data provide good clues for researchers who want to know more about all the things PSF does.

A map of the PSF protein. Figure adapted from Yarosh et al.WIREs RNA 2015, 6: 351-367. doi: 10.1002/wrna.1280
Papers cited:
1.) Christopher A. Yarosh; Iulia Tapescu; Matthew G. Thompson; Jinsong Qiu; Michael J. Mallory; Xiang-Dong Fu; Kristen W. Lynch. TRAP150 interacts with the RNA-binding domain of PSF and antagonizes splicing of numerous PSF-target genes in T cells. Nucleic Acids Research 2015;
doi: 10.1093/nar/gkv816

2.) Heyd F, Lynch KW. Phosphorylation-dependent regulation of PSF by GSK3 controls CD45 alternative splicing. Mol Cell 2010,40:126–137.

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