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Rival Proteomic Analyses of the Spliceosome Show Importance of Validating Results

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In the academic world, it seems, the spliceosome is hot. Last week Nature published a proteomic analysis of the molecular machine responsible for removing introns from pre-messenger RNA, and at least three other groups are currently battling to receive recognition for their own analyses of the spliceosome via proteomics. The competing studies point to the importance researchers are placing on the topic, as well as the discrepancies that arise from multiple attempts to characterize a biological system – even when researchers employ proteomics, a theoretically comprehensive technique.

Although the most recent attempt to characterize the spliceosome appears in Nature, there is little consensus that the study is the most comprehensive to date. Robin Reed, a cell biologist at Harvard Medical School who led the study, found 145 distinct spliceosomal proteins, including all the known components of the multiprotein/RNA complex, and 58 newly characterized proteins associated with the spliceosome. In contrast, however, a recent report in Genome Research authored by Matthias Mann and other members of his group at the University of Southern Denmark described the identification of 311 proteins linked to the splicing complex, including 96 previously unknown components of the spliceosome.

So who’s right? The answer isn’t clear, because the number and type of proteins associated with the spliceosome varies with the type of splicing, and because the two groups used different methods both to purify their spliceosomal proteins and analyze them by mass spectrometry.

Mann defends his group’s discovery of 96 previously unknown components of the spliceosome by arguing that his group employed a more sensitive mass spectrometry technique, which could explain why he found a greater number of spliceosomal proteins. Mann used liquid chromatography coupled with Applied Biosystems QSTAR Q-TOF mass spectrometers, and employed a technique called pulse extraction that selectively enhances certain mass ranges typical of peptide fragments. In contrast, Reed’s group, in collaboration with Steven Gygi of the Taplin Biological Mass Spectrometry Facility at Harvard Medical School, relied on nanoscale LC-MS/MS techniques using a Thermo Finnigan LCQ-DECA ion trap mass spectrometer.

“We use the Sciex QSTAR instrument where we get quite high resolution and mass accuracy, so that probably accounts for why we actually identified more proteins even though we had much less material than [Reed’s group] had,” Mann said.

Reed, for her part, counters that she considers many of the proteins identified by Mann’s group to be contaminants, based on her opinion that about 20 are keratins, a class of proteins so ubiquitous that it is often ignored during analysis. She added that her group compared her putative spliceosomal proteins with those identified in a subsequent analysis of the H complex, which contains heterogeneous ribonucleoproteins that could potentially contaminate her purified spliceosome, and eliminated the proteins that overlapped. Both groups isolated and identified proteins from two separately purified spliceosome complexes, comparing the results to eliminate discrepancies.

Although neither group claimed to have performed an exhaustive analysis to compare the catalogs of protein complexes assembled in the studies, “he doesn’t have all of ours, and we certainly don’t have all of his,” Reed said.

But the two groups agreed in principle that Reed had found an effective and novel application for her purification strategy. Often, as in Mann’s case, researchers isolating a protein complex rely on the streptavidin/biotin interaction to tag certain proteins or constructs and fish them out once they form part of the complex under investigation. Reed, in her study, used a milder purification technique employing the interaction between maltose and maltose-binding protein under conditions that increase the likelihood of purifying intact complexes, she said. In fact, Reed’s purification technique allowed her group to collect more material for her analysis, and Mann has already expressed interest in adopting her method of purification.

“It’s a nice method to pull out the RNA with the spliceosome attached to it,” Mann said. “They’ve labeled with a little RNA sequence [so that] you don’t get any steric hindrance, [allowing] the spliceosome to assemble. You can pull it out, and you can elute it just by adding sugar.”

However, the researchers disagreed about the level of certainty required for a protein to be considered critical to the spliceosome’s function. In her study, Reed argued that her purification method and comparison to the H complex provide enough evidence to support her claim that the proteins she identified are “functional.”

On the other hand, Mann countered that the question of function can be answered only by colocalization studies or by preparing antibodies to remove specific proteins and observing whether the complex can still function normally.

“The proof in the field is that you have to develop an antibody against a new protein, and then you have to pull it out of the nuclear extract to [see] if the spliceosome can’t splice anymore,” Mann said. “That would be the splicing assay, but that’s obviously too much for a single paper.”

– JSM