Laura Ranum to the Rescue

I previously mentioned that several different C9ORF72 ALS-FTD groups have published that there is an antisense transcript at the repeat region, but only used FISH targeting the antisense transcript.  I was surprised that the claims made it through the review process, given my experience in the trinucleotide repeat field.  More specifically, my most recent grants were returned with harsh reviews suggesting that antisense transcripts at repeat regions are spurious and unimportant.  But the ALS-crowd embraces the prospect whole heartedly.  Additionally, the ALS-crowd embraces Repeat-Associated Non-ATG (RAN) translation, a story that should be solely credited to Laura Ranum.  OK, I am certain there are others who suggested non-ATG translation is possible.  However, Laura realized it was a critical feature of trinucleotide repeat expansion diseases.  As the repeat expands, there is a longer transcript generated from the repeat region.  This transcript can generate longer or more polypeptides, in multiple frames. Dr. Ranum took a great deal of heat for this line of research, but she opted to move through the critiques rather than give up.  Here is a link to a great review by John Cleary and Laura Ranum.

Why is RAN-translation important?  It opens a window to a new field of study.  An important window.  There have been mumblings about "aberrant" peptides that are generated by our cells.  Of course, they are passed off as unimportant.  After all, if something does not adhere to the central dogma:

DNA->RNA->Protein

it cannot be real/important/possible/likely/useful (pick your favorite word).

What is wrong with scientists?  It seems as though there is a great population of scientists who cannot think outside of the core classes they took as undergrads or graduate students.  I am always puzzled.  In a discipline with a history of searching for the novel, the current "research-leaders" shun the brilliant observations and celebrate the popular studies.  Of course, as a scientist, it is as much my responsibility to highlight good science as it is to recognize the bad.  On that note: here is another paper on C9ORF72 ALS-FTD, this time from the Ranum lab.  Because it is from a veteran repeat-expansion disease lab, there is actual analysis of the sense and antisense transcripts.  Note: while my work on the antisense transcript at the C9ORF72 locus is still not published, I will point out that I have different results. Of course, I am fairly sure I know why and I am looking forward to addressing the question myself.

Laura's paper is a reminder of what I find interesting about bidirectional transcription, as well as a reminder to not give up on a pursuit that is important.

Focusing on the Important Things in Life

I planned to write about duons and John Stamatoyannopoulos, at the University of Washington. A UW press release this past week set off quite a discussion on twitter (#duons).  More haters than supporters, and the hate is directed at the premise that the Stam lab was the first to discover duons.  Of course, this is not true on many levels.  I suspect John Stam did not claim to be the first.  Instead, this is a genome-wide approach to unify what many other researchers have demonstrated.  Is this the real reason for the outcry? Numerous researchers feel slighted when a big project does not effectively acknowledge the history of a project.  Press releases are meant to be hype, as there is a great deal at stake to make your University seem like it has the most brilliant and innovative scientists.  Want to poke fun at the most hyped press releases? Visit Eisen's blog.  Perhaps there is more to the outcry than I appreciate.  Is there something about UW Genome Sciences or John Stam himself?  If there is, spit it out. If not, talk about the science.  Anything in between is troll-like, which is more harmful to science than a hyped press release.

Unfortunately, I was distracted this week by a struggle of a family in the midwest.  A heartbreaking death of a child from cancer.  No, they are not the first family to cry for a child, nor will they be the last. And I know that there are many causes of childhood death, including infectious disease, accidents, as well as inherited disorders.  Like so many other researchers, I entered this field to try to understand and ultimately help those who are suffering.  During the past few years, I have been distracted by the fraud and egos in biomedical research.  After reading about this family's loss, I realized that I am no longer focused on the important aspect of science, namely, discovery.

Breakthroughs are still needed for metastatic cancers, drug-resistant infections, and degenerative disease.  Working towards these breakthroughs are more important than discussions about a press release.  New motto: read the report, evaluate the science, glean important knowledge, move forward.

Can you remember your original goals?

I have a hobby, knitting.  I have been knitting since I was an undergrad at Indiana University, School of Business.  One of my marketing professors suggested that we teach ourselves something new.  Part of the goal was to demonstrate that we had reached a point in our academic careers that we could teach ourselves. Of course, he suggested that we continue to teach ourselves something new for the rest of our lives.  The objective is to increase our ability to converse with our customers.  As we broadened our experiences, we had greater subjects to be able to discuss.  Ultimately, this approach resulted in my returning to school to become a scientist.  I actually only intended to take 1-2 courses to learn some chemistry.  My first chemistry course was so interesting that I read the entire chemistry book by the middle of the semester.  My professor took be aside and asked me whether I was working in the right field, reminding me that life is more interesting if we are passionate about our work.

Fast forward, and I have a PhD in Biochemistry.  I love the work that I have done and I want to do more.  But a few years ago, I started knitting again.  At first I thought my desire to knit was to create things for my young son, but I did finally realize that something was missing in my work life.  I was not asking the questions that I wanted to ask. Actually, let me clarify, my then boss had a habit of using my projects as the platform for recruiting new students or postdocs. Countless times, when I interviewed a postdoc or grad student for the lab, the person would say: "I was told to get the details of what you are working on right now, as I will take over the project". Thus, I was never able to complete a story. So I turned to knitting, where I could control the project.

I joined an online knitting community "Ravelry".  It is a fantastic community where you can join sub-groups based on your interests.  One of the groups I joined was "Scientific Knitters".  We discuss knitting, but mostly we discuss science, career, co-workers, experiments, etc.  Currently, we have a very young member of the group who is just learning about chemistry, biology, physics, and experimentation.  This member is actually a high school student, thus very young.  For the rest of the post, I will call this person "Y" for young.  Y wants to be the first person to create a eukaryotic cell, from scratch.  Seems that Y is enamored by synthetic biology (and knitting).  But what has made Y so interesting is the pursuit.  Like any new student, Y has just enough information to ask great questions, but not enough experience to understand how to perform the work.  This is typical of a student.  I was at that stage once.  But we change during training.  We learn to dig deeper to fully understand to science behind our results.  We question everything, so that we can provide well informed answers to reviewers.

However, there is another change that takes place.  During graduate school, we have requirements that must be met, course work, qualifying exams, committee meetings, and more.  A labmate called these things "hoops" and suggested to all graduate students: "just jump through the hoops" "do not protest, as the hoop will remain until you finally jump, so jump and get it over with".  This was the best advice I was given in grad school.  I did as I was told and advanced to graduation fairly easily.  But I was surprised that during my postdoc, I was expected to jump through "postdoc-hoops" AND additional, non-training hoops.  My mentor would throw out this odd hoops that were not informative, instead, obstacles to slow me down.  I realized that most of the postdocs had these extra hoops thrown in the way, as did new faculty.  Not everyone jumped, some resisted, some found ways around the hoops.  It seemed that postdoc and beyond was more of a lesson in learning which hoops were important.  Great lesson, but it changes our thinking.  We expect our trainees to jump through hoops to satisfy our requirements.  Ultimately, we spend too much time thinking about hoops rather than about science.

As a result, I am ignoring hoops.  Fortunately, I joined a great lab, with talented, goal-oriented individuals and a PI insatiable curiosity.  It is refreshing and a wonderful reminder of why I am a scientist.  Of course, Y reminds me of my original goal to become an experimentalist.  I want to understand how alterations in a cell's transcriptional identity leads to disease and how we can characterize and monitor these changes.  I want to touch the transcripts, read the sequence, and see the changes in function.  I want to understand how the transcriptome is regulated, temporally, spatially, and developmentally.  There is so much to do, way to too much work to be bothered by extraneous hoops.

Hazy Dreams, Productive Thoughts

There has been a bug in my household, not surprising when there is a 7 year old living in the same space.  He seems to bring home new viruses each week.  This one did not hit me as hard as some of the others, but I have been tired, very tired.  The downside of each new bug is the time lost in "recovery".  I hate giving up this time to a virus, especially if I have viruses at work that need my attention.  Of course, I am referring to actual viruses in the -80°C freezer that are waiting for me to interrogate.

The upside of sleeping a little bit more each morning (instead of working out) is that I have let my mind wander to what I really want to accomplish professionally.  A few years ago, I attempted a search for a tenure track position.  To my surprise, I received several personal notes from search committee members expressing some interesting points.  What was particularly interesting is that these were notes that were in addition to the rejection letter and generally unsigned.  The notes were complementary, and pointed out what I would have needed to advance in that round.  Almost always, the missing item was NIH funding and obvious independence.  Neither is a big revelation.

As a problem solver, I have thought about this issue a great deal, not only for myself, but for future scientists.  The reality is that not every postdoc has the opportunity to submit grants to the NIH.  Seriously.  Sometimes, it is the PI who is the roadblock, other times it is a dynamic of the lab.  In the end, the actual issue is that a postdoc lives in two worlds.  One world is the lab setting based by a PI, who has a particular agenda.  The other world is the research community, which has "committee" based criteria for the postdoc deemed hirable.  Many others have discussed the inequality of academic hiring, including fantastic, well spoken, bloggers, tweeters, etc.  I cannot do this topic justice.  Instead, I will point out the math: if there is one job and 300 applicants, 299 people are not hired.  Many of them are qualified, but some criteria, fair or not, stood in the way.  This does not mean the other 299 applicants are not hard-working, accomplished scientists. So why do we treatment as losers?

I am hungry for a change, another path for the career scientist.  The biggest hurdle is knowing how to exit the current academic setting and still maintain health insurance and income.  Of course, every self-employed individual struggles with that hurdle!  The difference is that a scientist does not always have a "sellable" product.  At least, we do not understand what the sellable product really is.  In hindsight, I now realize that the postdoc effort was the sellable product.  It is the relationship between PI, postdoc, and University that needs to change, and postdocs need to demand greater ownership of their data.

Just consider what a postdoc does for the PI.  In a typical lab, the postdoc does the manual labor, trains grad students, writes protocols, research updates, papers, and lab maintenance.  This is not to say the PI does not work, on the contrary, the PI is working on administrative aspects of the lab.  Having a postdoc in the lab means more data is generated, more students are trained, and projects run smoothly.  However, there is no safety net under the postdoc, who is bound by the constraints of the PI when it comes to grants and papers.  A better system is one where the postdoc has a 3 yr contract.  Year one, the postdoc contributes 80-90% effort on the PI's project and 10-20% effort on a personal project.  This could be a literature search, small grant or crowd funded proposal, or basis of a larger project.  The second year, this changes to less effort on the PI's project and more effort on the personal project, perhaps a 60-40 split.  Finally, the third year is flipped, where the postdoc spend greater than 50% effort on a personal project.  This is what "payment" means.  Postdocs should not be service providers as much as they should be scientists.

Now, most PIs and postdocs would say that they need to give 110% for 2-3 years just to get a project started.  In that case, the payment is ownership of the data generates, meaning that as a postdoc advances each year, the postdoc acquires greater ownership of the project.  For those who say that we already have this system, I love that there are already PIs who think this way.  But it is not the norm, at least not in my experience.

After 3 years, a postdoc really is independent and should be treated as such.  Does this mean a 4th year cannot be a staff scientist?  No, it means that we need to make distinctions much earlier.  At the 4th year, academia needs to be responsible for all of the researchers in the department, meaning that postdocs are hired by the University rather than the lab.  This is a very important change that serves to add respect for a postdoctoral position.  Give all postdocs equal footing in the academic landscape and allow all to be responsible for their scientific portfolio. Open the door to allow cross effort on another PI's grant, a bit of internal freelancing.

Finally, allow these postdocs freelance space.  There are so many ways for a University to capitalize on the independent scientist, without providing them long term contracts.  The greatest reward to a University is the great science that is produced by highly creative and intelligent people.  It makes no sense to discard superdocs at the current rate.  Why not recoup the investment?

Bidirectional Transcription, Tandem Repeats, ALS, and Competition

When I started my postdoc position at the Fred Hutchinson Cancer Research Center in 2004, I had the great opportunity to identify an antisense transcript at the FMR1 CGG repeat region (HMG, 2007).  Better yet, I was able to interact with Diane Cho as she finished her story on antisense transcripts at the DMPK locus (Mol Cell, 2005).  The only other group actively looking at antisense transcripts was the Ranum lab, who was in Minnesota at the time (Nat Gen, 2006).  We had so much to do as we navigated the skeptical mRNA-centric world.  As a consequence, we had to characterize the antisense transcripts using the tools and questions that we would use to analyze coding transcripts.  Were the antisense transcripts polyadenylated? Capped? In the nucleus or cytoplasm? Spliced or unspliced? RNA pol II or III?  There were so many preconceived notions about the extra transcription exhibited by a gene locus that we had to overcome with careful characterization.  Ultimately, we defined the appropriate way to characterize antisense transcripts at repeat regions.

But, publications and grant applications were not easy to obtain. Most reviewers hated the notion of antisense transcript. A major assumption was that the transcripts we identified were "spurious", a term that I have come to realize is tossed around to mean: NOT REAL.  My other favorite critique was the statement "you do not know if the antisense transcript causes disease".  All of the genes we characterized had a repeat expansion associated with disease.  I find it incredibly short sighted to think that the antisense transcript somehow was independent of the sense "disease-causing" transcript when both harbored an expansion from the same region.

A representation of strand-specific interrogation of transcripts at a CAG repeat region

As a consequence, it is difficult to publish noncoding transcriptional activity at a locus.  Basically, the transcriptome is an uncharacterized frontier, largely due to lack of interest by genomics field and lack of research funding for the transcriptional regulation field.  The tenuous relationship between the transcriptomics field and what seems like the rest of science is best depicted by the public distain for the ENCODE Project.  I would rather not spend time on discussing the merits of either side of the argument, rather I want to focus on what I have seen, touched, sequenced, characterized, and understood about bidirectional transcription at unstable tandem repeat loc.

To date, only a few gene regions have been characterized well enough to understand the consequence of a repeat expansion.  This includes the FMR1, SCA8, DMPK, FRDA, SCA7, HTT, loci, plus a few more.  However, there are ~30 known unstable tandem repeat regions associated with neurodegenerative, developmental disorders, and muscular dystrophy.  The repeats are tri, tetra, penta, hexa, and dodeca repeat regions found near promoters, introns, and exonic regions of gene loci.  A quick glance of any human gene of interest on the UCSC browser reveals higher transcriptional activity near repeat regions as demonstrated by deposited ESTs.  Note, this activity is in both directions.  Comparison of the human genome and other organisms, such as mouse, reveal that in many cases, there are overlapping genes at the repeat regions.  A huge question is whether the antisense transcripts we have identified are actually remnants of genes no longer in play due to the formation of a true repeat region or whether the antisense transcripts represent regulatory elements, not yet fully appreciated.  

Ideally, I would like to continue to explore bidirectional transcription with the goal to provide a clearer picture of the transcriptional activity at tandem repeat gene loci.  This is primarily due to the number of diseases associated with repeat expansions.  When a repeat region expands, additional and potentially toxic RNAs are generated.  These transcripts could also be utilized as locus-specific biomarkers for the associated disease pathogenesis. With an increased focus on therapeutic agents, such as ASOs that target expanded sense transcripts for degradation, it is imperative that we understand what transcripts are generated from expanded disease-associated alleles.   

Unfortunately, as a senior scientist in some one else's lab, I did not have my own funding.  The years of work to break down the barriers to even recognize antisense transcripts are perhaps wasted for my career, as I could not hang on long enough to acquire funding.  However, there is some light at the end of the tunnel.  The Amyotrophic Lateral Sclerosis (ALS), community seems to have embraced bidirectional transcription, as well as another controversial topic, Repeat-associated Non-ATG (RAN) translation.  RAN translation was first described by Laura Ranum, another pioneer in bidirectional transcription at repeat regions.   In fact, the ALS community is incredibly competitive about the sense, antisense, coding, non-coding, toxic, non-toxic, RNAs and transcripts and the C9ORF72 gene.  It is a thrill to read each paper, each perspective, and each interpretation.  There seems to be little room of critique of antisense transcripts in their world, instead they seem to be focused on using every bit of evidence they can find to characterize the molecular mechanisms underlying this disease.  

Make no mistake, I do not support some of the underhanded competition that has occurred in this field.  Even I have been taken advantage by a few members of the C9ORF72 community, but I stand firm that the outcome will benefit the patient community, so it is worth it.  I have faith that the researchers are close to having viable and realistic therapeutics for ALS, something that is truly needed.  But I chuckle at how easily authors have claimed to "demonstrate" bidirectional transcription at C9ORF72, such as the Petrucelli Lab, where the evidence is basically in vitro analysis of transfected constructs or Edbauer Lab, where evidence of antisense translation is the proof.  This is liberating for the field, where scientists with expertise in disease characterization do not have to spend 3-5 years characterizing the transcript boundaries, localization, and processing prior to addressing impact on disease.  

There is time and space for researchers such as myself, who can more quickly address the RNAs generated at the locus.  I hope the recent body of work at the C9ORF72 locus will make it easier to address bidirectional transcription at the remaining 20+ unstable tandem repeat genes, all associated with equally devastating disease. Nonetheless, I wish that I could have a conversation with the reviewers of my grants, papers, and research proposals and ask them why they rejected the validity of bidirectional transcription.  What did they gain by blocking progress?  There was never a consequence for them, instead the ultimate burden is on the individuals who harbor repeat expansions and suffer from disease.  These individuals continue to wait for someone to be interested in their gene mutation.  It pains me that I came so close to being able to advance understanding of the gene loci associated with 20+ unstable repeat regions, but the road block of grant review did not see these diseases as important.