Experimental Biology 2016: Interactomic and Enzymatic Analyses of Distinct Affinity Isolated Human Retrotransposon Intermediates.

How does a RNA biologist end up studying the structure-function relationship of proteins? Dr. John LaCava moved from the world of nucleic acid to purifying and studying protein complexes. When he explained his path, it sounded like a natural transition. Dr. LaCava started out studying RNA biology—in a splicing lab. He started to become intrigued about the protein complexes in RNA. From there it was a quick hop, skip, and a jump to studying the structure-function relationship of proteins. However, the real gleam in his eye comes from when he talks about purifying protein complexes—but not just any protein complexes, the physiologically relevant for purifying protein complexes. 

The trick to protein complexes is keeping them, well, in the complex. It’s not uncommon to go through a very complicated protein purification protocol only to find out that a one protein with the complex has “fallen off” and is missing (I’m looking at you, delta subunit of the E. coli F1F0 ATP synthase). He and his lab have developed a method to determine under what conditions you can artificially prolong the life of the protein interaction.

While you might want to optimize the aforementioned complicated protein purification protocol, you just plain don’t have the time. What you really need is a high throughput way to test out a bunch of different versions of the protocol—but you just don’t have the time. This is exactly what Dr. LaCava figured out and is detailed in his Nature Methods paper last year (Hakhverdyan et al., 2015). I’ll do a quick summary here: You start with a wet pellet of cells that you then extrude into liquid nitrogen. This gives you beads of cells that you can “grind” into a fine powder using stainless steel beads that will get you down to the micron scale. One of the nifty parts of this method is that now the insides of the cells are now on the outside for easy access to protein complexes. Next, you can distribute this powder to different tubes and test out a different protocol in each tube to find the optimal conditions.

So, what does all of this have to do with RNA splicing? Well, RNA splicing is done in a, wait for it, protein complex. That’s where LaCava got his start. He’s now moved on to answering different questions. He got interested, through collaborations with colleagues, in retrotransposons—these so-called “selfish genes.” These are genes that are self-replicating and are present in the brain, in gametes, and in development.

His particular retransposon is in the LINE-1 family. It is bicistronic each with a 5’ UTR, promoter, and 3’ UTR that encodes a poly-A tail. It also displays a cis-preference. That is, its proteins prefer to associate with its own message (there’s that RNA biology again).

His lab purifies it using IDIRT—a spin on a metabolic tag in mass spec. To make a long story short, his lab tags the protein with heavy label and has an untagged version of the protein, which is mixed in a 1:1 ratio. You can read more about the general method in this article (Tackett et al., J Proteome Res, 2005).

The RNAs (either ORF1 or ORF2) that are engaged with tagged protein that are most likely true and stable under experimental conditions. He pulled out ORF2 from the supernatant and bound fraction and measured the average distance of the message. From this, he got a three node cluster (the chances of seeing any three nodes close together). 

For now, this measures the ability of ORF2 to replicate itself. However, it has opened the door for the future. You could use this method to do enzymatic assay on the intermediate structure, or genetic and biochemistry depletion assays, or even electron microscopy to examine the structure-function relationship. And now we’ve come full circle. This is how a RNA biologist ends up studying the structure-function relationship of proteins.

Interactomic and Enzymatic Analyses of Distinct Affinity Isolated Human Retrotransposon Intermediates.

J. LaCava, K.R. Molloy, D. Fenyö, M.S. Taylor, B.T. Chait, J.D. Boeke, M.P. Rout. The Rockefeller Univ.,NYU Sch. of Med. and Massachusetts Gen. Hosp