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Posts Tagged ‘PKA’

Hey junk people, I accept your challenge (part I)

In Uncategorized on September 23, 2008 at 1:42 pm

Some say that almost a half of the human DNA is “junk”, I say that the evidence indicating otherwise is strong enough to allow for speculation on (and research into) possible functions. In a recent discussion with Professor Larry Moran over at Sandwalk, on this topic, I received this challenge:

If you enjoy speculation so much then speculate on this [list of 8 questions]…

And I’ll answer each question as thoroughly as I can (please, find the first 4 below) but first, as an introduction, I would like to explain one of the reasons I take interest in the subject of “junk”-DNA. Bear with me, it’s an interesting story….

Back when I did my PhD, most of us PhD-students would get at least two projects to work on. One would be a “safe” project that would be reasonably easy to publish and the other one would be a high risk project where the outcome was more uncertain, but could potentially be published in ha higher impact journal (this is an approach I think all labs should follow !!). My “safe” project was to clone and characterize a gene for an isoform (of cAMP-dependent protein kinase) where the mRNA (cDNA) was already published (yes,believe it or not, this was actually publishable back in the days 10 years ago).  So, I cloned and I started sequencing looking out for exon/intron boundaries and intron sequences as was standard procedure. Problem was that I didn’t find any intron sequences. The genomic sequence was identical to the mRNA (cDNA) sequence.  At the ends where the cDNA and genomic sequences stopped being homologous, I found  direct repeats. These are the hallmarks of a retroposon. A retroposon is an mRNA that has been reverse transcribed and inserted into the genome. To add to this peculiar finding, my gene, the PKA C-gamma gene, it turned out, was a retroposon inserted into the intron of another gene (see illustration).

From my C-gamma paper: “genomic mapping of the STM7 gene demonstrated that the C-gamma open reading frame was situated within the intron separating exon 16 and 17 in the STM7 gene. In addition, part of the 3’-untranslated region of C-gamma (nucleotide 1478 to 1538 in fig. 2) constitutes an alternately spliced exon (exon 17) in the STM7 gene. The two genes are transcribed in opposite directions in the human genome, ……”

The common perception is that a retroposon is not functional. But, the circumstances in this case, I believe points towards some kind of function. C-gamma translates into mRNA in vivo (as is the case with a handful of other retroposon’s), and since it has an intact reading frame this mRNA can be translated into protein in vitro and in cell lysates. So is this protein detected in vivo ? ….and what if anything, does the protein do………?

This I’ll answer after returning to speculations on Larry Moran’s list of questions:

1. Why do pseudogenes and most of the transposon-related sequences look so much like broken genes?

By a broken gene I guess he means a broken reading frame that seems not to code for a protein product. The genetic sequence may still constitute a regulatory element influencing other genes (as was the case with the finding that led to the original discussion, see here for reference). Also, any trancribed RNA from parts of this DNA may have function either as long RNA molecules, miRNA’s or siRNA’s.

2. Why is the DNA sequence in most of our DNA not conserved?

Shorter segments may be conserved interspersed with unconserved regions. Also there has to be a great deal of non-conserved sequence to allow for evolution. This DNA serves a buffering capacity in that it is a reservoir for evolution (that does not make it “junk” !!, see below). Another point on conservation comes from the fact that miRNA’s function on the basis of their structure rather than a conserved sequence (many different sequences can give rise to similar two/three dimensional RNA structures). Thus conserved DNA sequences may not give any information on the function of such sequences and new approaches are needed to study their function.

3. Why can we delete large segments of mammalian DNA with no observable effect?

If you were an alien scientist examining a modern car you would find essential stuff like wheels, steering and breaks. You would also observe parts that seemed not to have any apparent function like the seat-belts, car-radio, air-conditioning, anti-spin system, ashtray, maybe a fire extinguisher and so on. These parts have a distinct function only under certain conditions and unless you are observing the car the instant these items are used, you might, -if you are of the “junk”-people, think that these parts are “junk”. Your conclusion would be strengthened by the fact that you could remove most of these items and the car would still be performing it’s basic functions (start, forward, reverse, turn, stop). Same could be true for the possible functions of large parts of our DNA.

4. Why is there so much variations in repetitive DNA within a species? Some people have segments that are ten times longer than segments found in other people. Are all of the nucleotides in the longer segments functional?

Repeat variation like STR’s are common variations that sometimes leads to functional differences between people, thus they are not “junk”. Similarly, Copynumber variation is a relatively newly discovered phenomenon that may have profound effects. That there is copynumber variation also in DNA yet to be ascribed a function, comes as no surprise and is not an argument for “junk”.

Then again, professor Moran might be right, it may all be junk. This may also be true for my C-gamma gene. because, the protein has never been found in vivo and consequently no function is known to date. Many have tried to find the protein (and/or a function) and some have suffered under the lack of results.

And I should mention in all fairness, that for me personally, it was wise to stop working on this and move on to other research projects and ventures.

Thus, I do understand the position of the “junk” people……..but, and here’s where my disagreement with them starts…..

That we haven’t found a function so far, does not mean that there isn’t any. Reports on new functions of DNA sequences are frequent and one of them was the ….. “opposing thumb” regulatory element….

This belief that there’s hidden function to be found, treasures to unearth if you will, is the difference between those advocating these parts of DNA as “junk” and me. In my opinion, It’s not the details of what is junk and what isn’t, ..- and how much, that bothers me…..

It’s the attitude. To dismiss something as junk is contrary to my idea of science being driven out of curiosity and the need to explore. Curiosity may kill a cat every now and then, but I’ll take that risk and continue to praise the scientist who recognize possibilities in the junk rather than dismissing it.

Answers to the last 4 points made by professor Moran to follow….

Was it all in vain ? The scientific method tale

In Uncategorized on June 23, 2008 at 9:34 am

post to news.thinkgene.com

I did my PhD on one of the most studied enzyme-systems of all time, the cAMP-dependent protein kinase (PKA). One would think that the tools we used were accurate when the enzyme was as thoroughly characterized as it was. But alas!, now we learn they had major flaws. The endless kinase assay’s lasting until the wee hours, were they all in vain ?

“….nonspecific effects are
widespread; they include actions on other protein kinases and signaling
molecules and also on basic cellular functions, such as transcription.”

We had two teams in my group, one was the gene characterizing team – we did cloning, sequencing, expression and transcription analyzes mostly. The other was the protein activity and interaction team who did a lot of enzyme activity (kinase) assays, protein-protein interaction and immunoblots. Members frequently crossed team borders to learn the necessary methods. I was in the gene-group, but I did my occasional kinase assay and cell-culture experiment as well. As a rule, the kinase assays (as well as cell culture assays) always included inhibitors of the kinase and very often those inhibitors were H89 and/or  KT5720. These inhibitors were thought to be specific so that any decrease in enzyme activity one would see upon adding them, was attributed too a loss of PKA activity.

A recent review by Andrew J. Murray in Science puts a serious dent in that assumption. These inhibitors seems to act on a range  of other signaling systems. Their targets seems to be very diverse indeed. Specific they most certainly are not. That means that a lot of the inhibition we would see and base our conclusions on, was wrong.

“…a substantial
body of evidence has now accumulated
that indicates that both H89 and KT 5720
can have effects independent of PKA inhibition.
These actions are extremely varied;
some of the most worrisome actions are the
substantial effects on the MAPK and calcium
signaling pathways, which interact with
the PKA pathway and mediate multiple
cellular functions.”

and even more worrying:

“Furthermore, many of
these non–PKA-based actions of H89 and
KT 5720 occur at concentrations that have
been widely used to investigate PKA function.”

The final conclusions and the cellular mechanisms we unraveled, one can only hope were not wrong, which would be fortunate for me and others with a history in the field. But, one can never be sure and reevaluation may be in place for some of the past studies

“the molecular bases of some
cellular processes attributed to PKA solely
through the use of these compounds may
have to be reevaluated.”

Any experiment with H89 in the future needs to take this new information into account. And in the review, the author outlines a number of alternative approaches to inhibit PKA activity in a specific manner.

The above outlined studies indicate that neither
KT 5720 nor H89 should be used alone
to study the function of PKA. As these compounds
are so commonly used, it will therefore
be necessary to devise strategies that
can overcome their shortcomings.

Although annoying and possibly detrimental to papers I have published in the past, I cannot help having this proud feeling. Because, this is the beauty of the scientific method: not only do we admit we’re wrong (and publish it in high impact journals), we have efficient methods to prevent the wrongdoings from continuing. Any responsible and updated reviewer will now dispute all conclusions based on experiments involving these inhibitors. Then scientific results in the field will become more accurate. In addition, new conclusions and insight may come from this increased understanding of the limitations on previous results.

Science involves unrestricted sharing, regardless of the nature of the information. Add peer-review and you have  the scientific method working at its best.