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.

Tuesday, June 30, 2015

Training the biomedical workforce - a discussion of postdoc inflation


By Ian McLaughlin


Earlier this month, postdocs and graduate students from several fields met to candidly discuss the challenges postdocs are encountering while pursuing careers in academic research.  The meeting began with an enumeration of these challenges, discussing the different elements contributing to the mounting obstacles preventing postdocs from attaining faculty positions – such as the scarcity of faculty positions and ballooning number of rising postdocs, funding mechanisms and cuts, the sub-optimal relationship between publications and the quality of science, and the inaccurate conception of what exactly a postdoctoral position should entail.


From [15]

At a fundamental level, there’s a surplus of rising doctoral students whose progression outpaces the availability of faculty positions at institutions capable of hosting the research they intended to perform [10,15].  While 65% of PhDs attain postdocs, only 15-20% of postdocs attain tenure-track faculty positions [1].  This translates to significant extensions of postdoctoral positions, with the intentions of bolstering credentials and generating more publications to increase their appeal to hiring institutions.  Despite this increased time, postdocs often do not benefit from continued teaching experiences, and are also unable to attend classes to cultivate professional development.


From [10]
Additionally, there may never be an adequate position available. Instead of providing the training and mentorship necessary to generate exceptional scientists, postdoctoral positions have become “holding tanks” for many PhD holders unable to transition into permanent positions [5,11], resulting in considerably lower compensation relative to alternative careers 5 years after attaining a PhD.

From [13]

Perhaps this wouldn’t be quite so problematic if the compensation of the primary workhorse of basic biomedical research in the US was better.  In 2014, the US National Academies called for an increase of the starting postdoc salary of $42,840 to $50,000 – as well as a 5-year limit on the length of postdocs [1].  While the salary increase would certainly help, institutions like NYU, the University of California system, and UNC Chapel Hill have explored term limits.  Unfortunately, a frequent outcome of term limits was the promotion of postdocs to superficial positions that simply confer a new title, but are effectively extended postdocs. 

Given the time commitment required to attain a PhD, and the expanding durations of postdocs, several of the meeting’s attendees identified a particularly painful interference with their ability to start a family.  Despite excelling in challenging academic fields at top institutions, and dedicating professionally productive years to their work, several postdocs stated that they don’t foresee the financial capacity to start a family before fertility challenges render the effort prohibitively difficult.

However, administrators of the NIH have suggested this apparent disparity between the number of rising postdocs and available positions is not a significant problem, despite having no apparent data to back up their position. As Polka et al. wrote earlier this year, NIH administrators don’t have data quantifying the total numbers of postdocs in the country at their disposal – calling into question whether they are prepared to address this fundamental problem [5].

A possible approach to mitigate this lack of opportunity would be to integrate permanent “superdoc” positions for talented postdocs who don’t have ambitions to start their own labs, but have technical skills needed to advance basic research.  The National Cancer Institute (NCI) has proposed a grant program to cover salaries between $75,000-$100,000 for between 50-60 of such positions [1,2], which might be expanded to cover the salaries of more scientists.  Additionally, a majority of the postdocs attending the meeting voiced their desire for more comprehensive career guidance.  In particular, while they are aware that PhD holders are viable candidates for jobs outside of academia – the career trajectory out of academia remains opaque to them.

This situation stands in stark contrast to the misconception that the US suffers from a shortage of STEM graduates.  While the careers of postdocs stall due to a scarcity of faculty positions, the President’s Council of Advisors on Science and Technology announced a goal of one million STEM trainees in 2012 [3], despite the fact that only 11% of students graduating with bachelor’s degrees in science end up in fields related to science [4] due in part, perhaps, to an inflated sense of job security.  While the numbers of grad students and postdocs have increased almost two-fold, the proliferation of permanent research positions hasn’t been commensurate [5]. So, while making science a priority is certainly prudent – the point of tension is not necessarily a shortage of students engaging the fields, but rather a paucity of research positions available to them once they’ve attained graduate degrees. 

Suggested Solutions

Ultimately, if the career prospects for academic researchers in the US don't change, increasing numbers of PhD students will leave basic science research in favor of alternatives that offer better compensation and career trajectories – or leave the country for international opportunities.  At the heart of the problem is a fundamental imbalance between the funding available for basic academic research and the growing community of scientists in the U.S [9,14], and a dysfunctional career pipeline in biomedical research [9].  Some ideas of strategies to confront this problem included the following suggestions.

Federal grant-awarding agencies need to collect accurate data on the yearly numbers of postdoctoral positions available.  This way, career counselors, potential students, rising PhD students, and the institutions themselves will have a better grasp of the apparent scarcity of academic research opportunities.

As the US National Academies have suggested, the postdoc salary ought to be increased.  One possible strategy would be to increase the prevalence of “superdoc”-type positions creating a viable career alternative for talented researchers who wish to support a family but not secure the funding needed to open their own labs.  Additionally, if institutions at which postdocs receive federal funding were to consider them employees with all associated benefits, rather than trainees, rising scientists might better avoid career stagnation and an inability to support families [11].

As the number of rising PhDs currently outpaces the availability of permanent faculty positions, one strategy may be to limit the number of PhD positions available at each institution to prevent continued escalation of postdocs without viable faculty positions to which they might apply.  One attendee noted that this could immediately halt the growth of PhDs with bleak career prospects.

Several attendees brought up the problems many postdocs encounter in particularly large labs, which tend to receive disproportionately high grant funding.  Postdocs in such labs feel pressure to generate useful data to ensure they can compete with their peers, while neglecting other elements of their professional development and personal life. As well, the current system funnels funding to labs that can guarantee positive results, favoring conservative rather than potentially paradigm-shifting proposals – translating to reduced funding for new investigators [9]. Grant awarding agencies’ evaluations of grant proposals might integrate considerations of the sizes of labs with the goal of fostering progress in smaller labs. Additionally, efforts like Cold Spring Harbor Laboratory’s bioRĪ‡iv might be more widely used to pre-register research projects so that postdocs are aware of the efforts of their peers – enabling them to focus on innovation when appropriate.

While increased funding for basic science research would help to avoid the loss of talented scientists, and private sources may help to compensate for fickle federal funds [6], some attendees of the meeting suggested that the current mechanisms by which facilities and administrations costs are funded might be restructured. These costs, also called “indirect costs” - which cover expenditures associated with running research facilities, and not specific projects - might be restructured to avoid over 50 cents of every federally allocated dollar going to the institution itself, rather than the researchers of the projects that grants fund [7,8].  This dynamic has been suggested to foster the growth of institutions rather than investment in researchers, and optimizing this component of research funding might reveal opportunities to better support the careers of rising scientists [9,12]

Additionally, if the state of federal funding could be more predictable, dramatic fluctuations of the numbers of faculty positions and rising scientists might not result in such disparities [9].  For example, if appropriations legislation consistently adhered to 5 year funding plans, dynamics in biomedical research might avoid unexpected deficits of opportunities.


From [5]

Career counselors ought to provide accurate descriptions of how competitive a search for permanent faculty positions can be to their students, so they don’t enter a field with a misconceived sense of security.  Quotes from a survey conducted by Polka et al. reveal a substantial disparity between expectations and outcomes in academic careers, and adequate guidance might help avoid such circumstances.

As shown in the NSF’s Indicators report from 2014, the most rapidly growing reason postdocs identify as their rationale for beginning their projects is “other employment not available” – suggesting that a PhD in fields associated with biomedical sciences currently translates to limited opportunities. Even successful scientists and talented postdocs have become progressively more pessimistic about their career prospects.  Accordingly - while there are several possible solutions to this problem - if some remedial action isn’t taken, biomedical research in the U.S. may stagnate and suffer in upcoming coming years.

Citations
 1.    Alberts B, Kirschner MW, Tilghman S, Varmus H. Rescuing US biomedical research from its systemic flaws. Proc Natl Acad Sci U S A. 2014 Apr 22;111(16):5773-7. doi: 10.1073/pnas.1404402111. Epub 2014 Apr 14.
 2.    http://news.sciencemag.org/biology/2015/03/cancer-institute-plans-new-award-staff-scientists
 3.    https://www.whitehouse.gov/sites/default/files/microsites/ostp/pcast-engage-to-excel-final_2-25-12.pdf
 4.    http://www.nationalreview.com/article/378334/what-stem-shortage-steven-camarota
 5.    Polka JK, Krukenberg KA, McDowell GS. A call for transparency in tracking student and postdoc career outcomes. Mol Biol Cell. 2015 Apr 15;26(8):1413-5. doi: 10.1091/mbc.E14-10-1432.
 6.    http://sciencephilanthropyalliance.org/about.html
 7.    http://datahound.scientopia.org/2014/05/10/indirect-cost-rate-survey/
 8.    Ledford H. Indirect costs: keeping the lights on. Nature. 2014 Nov 20;515(7527):326-9. doi: 10.1038/515326a. Erratum in: Nature. 2015 Jan 8;517(7533):131
 9.    Alberts B, Kirschner MW, Tilghman S, Varmus H. Rescuing US biomedical research from its systemic flaws. Proc Natl Acad Sci U S A. 2014 Apr 22;111(16):5773-7. doi: 10.1073/pnas.1404402111. Epub 2014 Apr 14.
 10.    National Science Foundation (2014) National Science and Engineering Indicators (National Science Foundation, Washington, DC).
 11.    Bourne HR. A fair deal for PhD students and postdocs. Elife. 2013 Oct 1;2:e01139. doi: 10.7554/eLife.01139.
 12.    Bourne HR. The writing on the wall. Elife. 2013 Mar 26;2:e00642. doi: 10.7554/eLife.00642.
 13.    Powell K. The future of the postdoc. Nature. 2015 Apr 9;520(7546):144-7. doi: 10.1038/520144a.
 14.    Fix the PhD. Nature. 2011 Apr 21;472(7343):259-60. doi: 10.1038/472259b.
 15.   Schillebeeckx M, Maricque B, Lewis C. The missing piece to changing the university culture. Nat Biotechnol. 2013 Oct;31(10):938-41. doi: 10.1038/nbt.2706.