Debating alternative splicing (Part IV)

In Debating alternative splicing (Part III) I discussed a review published in the February 2017 issue of Trends in Biochemical Sciences. The review examined the data on detecting predicted protein isoforms and concluded that there was little evidence they existed.

My colleague at the University of Toronto, Ben Blencowe, is a forceful proponent of massive alternative splicing. He responded in a letter published in the June 2017 issue of Trends in Biochemical Sciences (Blencowe, 2017). It's worth looking at his letter in order to understand the position of alternative splicing proponents. He begins by saying,

It is estimated that approximately 95% of multiexonic human genes give rise to transcripts containing more than 100 000 distinct AS events [3,4]. The majority of these AS events display tissue-dependent variation and 10–30% are subject to pronounced cell, tissue, or condition-specific regulation [4].

Ben references two papers. The first one, Pan et al., (2008) (ref. 3), is from his own lab. It documents the existence of abundant splice variants and estimates that, "95% of multiexon genes undergo alternative splicing." This is misleading because the results only show the existence of splice variants. Whether these can be explained as splicing errors or alternative splicing (AS) is what's being debated. Nobody questions the existence of abundant splice variants but that doesn't mean they are produced by AS.

The second paper is Wang et al. (2008). It was also published nine years ago. The authors looked at splice variants in different tissues and found that the number and the amount of these splice variants varied from tissue to tissue. This is the result predicted for both the splicing errors explanation and the massive alternative splicing explanation so it doesn't distinguish between these conflicting explanations. Nevertheless, Wang et al. conclude, "... most alternative splicing events are regulated between tissues, providing an important element of support for the hypothesis that alternative splicing is a principle contributor to the evolution of phenotypic complexity in mammals."

The conclusion is doubly flawed because they have not demonstrated that they are dealing with true alternative splicing and they have not demonstrated that they are viewing regulation. The word "regulation" is not an observation; it's a conclusion. It is a loaded word. "Regulation" implies that the variation observed has biological relevance. But not all variation is due to regulation. This is the same problem we encounter in the pervasive transcription debate. Just because a transcript is tissue-specific doesn't mean it's biologically relevant. Similarly, just because some splicing errors are restricted to certain cells doesn't mean they are regulated.

Bencowe's opening arguments are seriously flawed. I don't think he's making as strong a case as he thinks.

The latter events are significantly enriched for evolutionary conservation and frame-preserving potential.

The Wang et al. paper showed that there were several classes of transcript variants. Some of them showed lots of evidence of being real examples of alternative splicing. In that class, about 60% of skipped exon events showed preservation of the reading frame. In the class that looked the least promising—a class that included most events—only 41% of the skipped exon events preserved reading frame. (You expect 33% by chance alone.)

As for "conservation," the results in the Wang et al. paper do not say what you might think. There's no data on whether variants are present in other species. The evidence on conservation and preservation of open reading frame are far less impressive than Blencowe implies in his letter. In fact, most studies show that the production of splice variants is NOT conserved.

Moreover, dozens of independent studies have shown that subsets of differentially regulated AS events are significantly enriched in genes that function in common biological processes and pathways, and that, where characterized, exons in these splicing ‘networks’ have important functions [5,6]. For example, AS networks function extensively in the remodeling of cytoskeletal interactions, signaling cascades, and gene regulatory pathways, and the literature contains hundreds of examples in which translated splice variants contribute important roles in development, cell and tissue homeostasis, animal behavior, diseases, as well as other processes.

This is an important point. We would like to know the number of proven examples of alternative splicing in oredr to bring the debate into focus.

It's well known that most transcript variants have not (yet?) been shown to have biological relevance. This means that splicing errors is a viable explanation. On the other hand, it is well known that alternative splicing is a real phenomenon. There are many well-established examples.

However, the mere fact that splice variants are common in important genes is not evidence of alternative splicing because ALL multiexon genes have splice variants. Blencowe argues that "hundreds" of examples of real alternative splicing exist but I'm not aware of the data supporting that claim. Nevertheless, I'm willing to assume that several hundred human protein-coding genes are alternatively spliced. That's 1% of the total and it's a long way from 95%. This is not a debate about the mere existence of alternative splicing; it's a debate about its relative frequency.

Ben then raises certain technical issues with the Tress et al. paper. He wonders whether the inability to detect predicted protein isoforms can be due to the lack of sensitivity of the mass spec assays. The answer is "yes," that's a possibility. Such objections can always be raised when one is trying to demonstrate the absence of something. It's part of the problem with proving the negative.

However, there's a built in positive control for these experiments and that's their ability to detect the dominant predicted isoform for protein-coding genes. The results do detect these isoforms as Tress et al. point out in their response to Blencowe's letter (Tress et al., 2017b). Many of those isoforms are present in substantial amounts in the cells being assayed but many are relatively low abundance proteins. It doesn't mean that mass spec will detect all minor isoforms if they exist but it does establish that the assays detect what they are supposed to detect.

Blencowe then asks whether the conclusions in Tress et al., (2017a) are justified by the results. Here's the response from the authors ...

Question two is ‘are the conclusions justified based on the findings?’ In our opinion the available evidence leaves little room for doubt. Most protein coding genes have main protein isoforms, and most alternative exons are subject to neutral or near-neutral evolution. We believe our conclusions are well substantiated and invite readers to judge for themselves in the article and related papers.

I agree with Tress et al. Their conclusions are justified. They are not "proven." They are as tentative as most scientific conclusions should be. So far, I don't think Ben Blencowe has presented an adequate defense of his claim and I don't his conclusion is as well-justified as the one he is criticizing.

The third issue Blencoe raises is whether the splice variants are actually translated in spite of the fact that the protein products haven't been detected. He reviews data indicating that "tens of thousands of splice variant transcripts have been detected in polysome fractions." But there are just as many technical objections to those experiments as the ones he is criticizing. In fact, as Tress et al. point out in their response, all kinds of non-coding RNAs are detected in polysomes indicating that this is not a reliable indication of translation.

What we have is a conflict between direct assays for proteins in the mass spec experiments and indirect assays in ribosome profiling experiments. The results do not agree. You can't continue to argue for the existence of tens of thousands of protein isoforms unless you can actually prove they exist. But that's exactly what Blencowe does when he says,

In summary, when collectively considering multiple sources of false negative detection rates for splice variants in LC-MS/MS data, previous results demonstrating that protein abundance is predominately related to transcript abundance, and recent results from detecting splice variant sequences associated with ribosomes, it is apparent that most splice variants detected in transcriptome profiling data are likely translated. Therefore, it is possible that most splice variants contribute to cellular function. Unfortunately, the authors of [1] have unnecessarily dismissed as a ‘theory’ a large body of experimental data from numerous laboratories demonstrating extensive roles for AS in the remodeling of cellular networks that have diverse roles in critical biological processes.

I understand where Ben is coming from. He has a lot invested in the idea that massive alternative splicing is a real phenomenon. However, I think he could do a much better job of presenting his case if he would just acknowledge the alternative explanation (splicing errors) and discuss the results in light of that possibility. I think he is exaggerating the evidence for biological function and ignoring counter evidence such as lack of sequence conservation.

Tress et al. (2017b) demonstrate in their response to Blencowe that they understand the evolutionary significance of conservation and the lack of conservation. Here's what they say in defense of their view that most splice variants are due to splicing errors.

A wealth of genome-wide genetic variation data from human populations has recently become available, enabling us to test whether alternative exons are undergoing purifying selection (whether they really are innovations). The variant data we analyzed were only available from 2012. These show that most alternative exons are evolving neutrally: they have a much higher non-synonymous to synonymous substitution ratio, and a 10-fold higher proportion of potentially damaging high-impact variants. Most interestingly, these figures do not decrease with increasing allele frequency, as would be expected if alternative exons were under selection pressure.

The neutral or near-neutral selection pressures apparent in the current population are a very strong suggestion that most alternative variants have not been evolutionary selected to have important cellular roles.

It seems to me that this is strong evidence in favor of the splicing error explanation.

Blencowe closes his criticism with the following ....

Tress and colleagues have already shown promise for the more comprehensive detection of translated splice variants and will undoubtedly prove to be valuable in future studies [7,14]. Moreover, it is important to appreciate that it can take a single research group years of effort to determine the biological function of a single AS event. As such, an important goal for future studies will be to further develop high-throughput methods for interrogating the functions of splice variants. In the meantime, one should be mindful of the old aphorism, ‘absence of evidence is not evidence of absence’.

I agree with Ben that the real proof of the pudding lies in hard-core biochemistry and molecular biology. It takes a lot of work to demonstrate that predicted AS events are biologically relevant. But that's exactly what has to happen if we are ever going to be convinced that 95% of human genes are alternatively spliced to produce functional protein isoforms. I disagree with Ben that this controversy is going to be resolved in his favor by more genomics and proteomics. That hasn't worked so far. Ask ENCODE how it's working out for them.

Finally, the old aphorism is somewhat disingenuous. Ben is trying to shift the burden of proof onto his opponents. He is criticizing them for not proving that the protein isoforms do not exist. That's not fair. The burden of proof is on him and his supporters to show that alternative splicing is real. As more and more attempts to demonstrate the existence of abundant protein isoforms result in failure, it becomes increasing difficult to maintain they exist. At some point, the absence of evidence in support of massive alternative splicing should cause proponents to rethink their position.

---

Note: The front page story in The Toronto Star refers to a 2010 paper by Barash et al. That paper claims to have discovered an extensive regulatory code controlling alternative splicing. There's no mention of splicing errors.

Barash, Y., Calarco, J.A., Gao, W., Pan, Q., Wang, X., Shai, O., Blencowe, B.J., and Frey, B.J. (2010) Deciphering the splicing code. Nature, 465:53-59. [doi: 10.1038/nature09000]

Blencowe, B.J. (2017) The relationship between alternative splicing and proteomic complexity. Trends in biochemical sciences, 42(6), 407-408. [doi: 10.1016/j.tibs.2017.04.001]

Pan, Q., Shai, O., Lee, L.J., Frey, B.J., and Blencowe, B.J. (2008) Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing. Nature genetics, 40:1413-1415. [doi: 10.1038/ng.259]

Tress, M.L., Abascal, F., and Valencia, A. (2017a) Alternative splicing may not be the key to proteome complexity. Trends in biochemical sciences, 42:98-110. [doi: 10.1016/j.tibs.2016.08.008]

Tress, M.L., Abascal, F., and Valencia, A. (2017b) Most Alternative Isoforms Are Not Functionally Important. Trends in biochemical sciences, 42:408-410. [doi: 10.1016/j.tibs.2017.04.002]

Wang, E.T., Sandberg, R., Luo, S., Khrebtukova, I., Zhang, L., Mayr, C., Kingsmore, S.F., Schroth, G.P., and Burge, C.B. (2008) Alternative isoform regulation in human tissue transcriptomes. Nature, 456:470-476. [doi: 10.1038/nature07509]