In a previous post I mentioned that I spent the first year of graduate school working on a dead end project that was going nowhere.  I’m going to present and discuss the findings of the paper that changed the direction of my thesis project and ultimately lead to the completion of my PhD.

In the fall of 2006, I was finishing up the last few experiments on a project trying to link P53 (a very important protein in cells that helps to prevent cancer) to one of the most important transcriptional activators, P-TEFb.  I should probably pause for a second here to give a little bit of background on P-TEFb so that what follows is somewhat understandable.  Positive transcription elongation factor b (P-TEFb: Pee Tef bee) is a protein kinase (read as on/off switch) that regulates RNA polymerase II transcription of most genes.  RNA polymerase II is the protein responsible for messenger RNA (mRNA) transcription or the process of turning DNA into mRNA.  Without mRNA, the transfer form of DNA, proteins cannot be made by the cell, and life does not exist.  Essentially, P-TEFb tells RNA polymerase when it’s ok to start making RNA.  You can think of P-TEFb as the starter at a track meet, you know, the guy that holds a gun in the air and yells, “On your mark! Get Set! (long pause) GO!”

Transcription begins with initiation at the transcription start site (bent arrow).  The polymerase transcribes 30-50 bases before it comes under the negative regulation of DSIF and NELF causing the polymerase to pause.  P-TEFb can release the polymerase from this pause by phosphorylating NELF causing it to leave the complex, phosphorylating DSIF turning it from a negative factor into a positive one, and by phosphorylating the polymerase Credit: David Price

P-TEFb is inhibited in a complex composed of the small nuclear RNA 7SK and the protein HEXIM1/2 Credit: Brian Krueger

To make things even more confusing, the activity of P-TEFb is also regulated by its inclusion in an inhibitory complex.  Because P-TEFb regulates whether genes are transcribed, its activity must be highly regulated to be sure that the right proteins are being expressed at the right times.  The thing about P-TEFb though, is that it’s regulated by a small RNA, 7SK.  P-TEFb is the only protein kinase known to be regulated in this manner.  Simply put, P-TEFb is tied up in a rubberband ball of RNA and proteins and no one really knows how it is yanked out of this mess to regulate transcription.  The focus of my thesis work was to determine how P-TEFb is released.

Figure 1: Tap tagged protein isolated and associated proteins analyzed by protein gel elctrophoresis coupled with mass spectrometry Credit: Jeronimo et al

I was putting some of the final touches on my P53 experiments to try and show that P53 could indirectly activate P-TEFb when my mentor got an e-mail from a researcher in Canada that was having a very hard time trying to figure out the function of a new protein he had discovered.  This researcher had started a proteomic (relating to proteins) screen to determine how known proteins interact during the initiation stages of transcription and to find new proteins involved in this process.  This group had discovered a methylase (adds methyl groups to proteins).  In the context of transcription, methylases are really important because they can control whether or not proteins can “see” the DNA and transcribe it.  The researchers thought they had discovered a new protein involved in shutting down transcription, but it didn’t have that effect in any of their experiments.  The important key here is that these researchers showed that the methylase was somehow stuck to P-TEFb.  Since my boss had discovered P-TEFb, he was the go to guy to try to figure out what this new protein might be doing.  We had a pretty good idea about what was going on here and quickly became collaborators on the project.  It was known for a decade that the small RNA 7SK (the one involved in inhibiting P-TEFb) had a Methyl cap, but no one knew how it was added or what protein was responsible for adding it.

Figure 2: MePCE (BCDIN3) P-TEFb interaction network Credit: Jeronimo et al

Figure 3: 7SK methylation assay.  The methylase and 7SK RNA wer combined in a test tube in the presence of radioactive Methyl substrate in addition to non-radioactive competitor (AdoHcy), or Rnase A (chews up RNA) Credit: Jeronimo et al

The paper, “Systematic Analysis of the Protein Interaction Network for the Human Transcription Machinery Reveals the Identity of the 7SK Capping Enzyme” by Jeronimo et al Mol Cell 2007, describes the experiments that were done to show that the methylase was responsible for adding a stabilizing methyl cap to 7SK.  This discovery was made by tagging a bunch of proteins known to be associated with transcription, pulling them out of cells, and running the isolated complexes through a fancy machine to determine all of the different proteins that were stuck in the complex (see figure 1).   This technique was used on a number of different proteins and used to generate a protein interaction network (see figure 2).  The research went on to show that this protein, BCDIN3, renamed Methyl Phosphate Capping Enzyme (MePCE) could methylate 7SK RNA in the test tube (see figure 3) and that if the protein was eliminated from cells, that 7SK RNA was also lost indicating that MePCE is important for maintaining the stability of 7SK RNA (see figure 4).

Figure 4: The capping enzyme was knocked down using siRNA (left panel) and the effects of the loss of this protein on 7SK were determined after 48 and 72hr (right panel). Tubulin, U6, and U2 serve as controls. Credit: Jeronimo et al

So how does this work relate to my thesis project? Well, I was really interested in trying to understand how P-TEFb was released from its inhibitory complex.  It appeared, through this work, that methylation and stabilization of the key inhibitory RNA could play a role in shifting the balance between free and inhibited P-TEFb.  Additionally, if you squint really hard, you’ll see in the center of that interaction network that there’s a protein, LARP7, that is shown to be associated with the capping enzyme and both subunits of P-TEFb.  Characterizing LARP7 in both mammalian and insect cells was the focus of two chapters of my thesis.   Although LARP7 itself doesn’t appear to be extremely important for the regulation of P-TEFb, it served as the perfect tag for doing some really cool biochemistry.  Maybe one of these days I’ll take the time to summarize my two first author papers and how they explain one mechanism of P-TEFb’s release and activation from its inhibitory complex.

**

Jeronimo C, Forget D, Bouchard A, Li Q, Chua G, Poitras C, Thérien C, Bergeron D, Bourassa S, Greenblatt J, Chabot B, Poirier GG, Hughes TR, Blanchette M, Price DH, Coulombe B. Systematic analysis of the protein interaction network for the human transcription machinery reveals the identity of the 7SK capping enzyme. Mol Cell. 2007 Jul 20;27(2):262-74.