Debating alternative splicing (part I)

I recently had a chance to talk science with my old friend and colleague Jack Greenblatt. He has recently teamed up with some of my other colleagues at the University of Toronto to publish a paper on alternative splicing in mouse cells. Over the years I have had numerous discussions with these colleagues since they are proponents of massive alternative splicing in mammals. I think most splice variants are due to splicing errors.

There's always a problem with terminology whenever we get involved in this debate. My position is that it's easy to detect splice variants but they should be called "splice variants" until it has been firmly established that the variants have a biological function. This is not a distinction that's acceptable to proponents of massive alternative splicing. They use the term "alternative splicing" to refer to any set of processing variants regardless of whether they are splicing errors or real examples of regulation. This sometimes makes it difficult to have a discussion.

In fact, most of my colleagues seem reluctant to admit that some splice variants could be due to meaningless errors in splicing. Thus, they can't be pinned down when I ask them what percentage of variants are genuine examples of alternative splicing and what percentage are splicing mistakes. I usually ask them to pick out a specific gene, show me all the splice variants that have been detected, and explain which ones are functional and which ones aren't. I have a standing challenge to do this with any one of three sets of genes [A Challenge to Fans of Alternative Splicing].

1. Human genes for the enzymes of glycolysis
2. Human genes for the subunits of RNA polymerase with an emphasis on the large conserved subunits
3. Human genes for ribosomal proteins

I realize that proponents of massive alternative splicing are not under any obligation to respond to my challenge but it sure would help if they did.

Here's my position.

1. Most splice variants are present at very low concentrations. They are most easily explained as splicing errors since we know the spliceosome is error prone (Hsu and Hertel, 2009). As is so often the case in these discussions, the null hypothesis should be no function.
2. The term "alternative splicing" should be reserved for those cases where a biological function of the splice variants has been demonstrated.
3. Hyperbole about the prevalence and importance of presumed alternative splicing should be avoided unless you can back it up with solid evidence.
4. All publications concerning presumed alternative splicing should mention that there's a controversy and discuss why they prefer to treat their data as biologically relevant. They should describe what steps they have taken to eliminate spurious splicing errors from their data.

I've discussed these issues many times on this blog ....

• Using conservation to determine whether splice variants are functional
• Splice variants of the human triose phosphate isomerase gene: is alternative splicing real?
• Do you believe what's written in the introduction to this paper?
• Questions about alternative splicing
• The most important rule for publishing a paper on alternative splicing
• A Challenge to Fans of Alternative Splicing
• Splicing Error Rate May Be Close to 1%
• Two Examples of "Alternative Splicing"
• The Frequency of Alternative Splicing

At the present time, the overwhelming opinion among workers in the field of alternative splicing is that most splice variants are biologically relevant. The "splicing error" view that I support is definitely the minority position. There aren't many papers in the scientific literature that challenge the current dogma. Here's a short list of the most important ones.

Hsu, S.-N., and Hertel, K.J. (2009) Spliceosomes walk the line: splicing errors and their impact on cellular function. RNA biology, 6:526-530. [doi: 10.4161/rna.6.5.986]

Melamud, E., and Moult, J. (2009) Stochastic noise in splicing machinery. Nucleic acids research, gkp471. [doi: 10.1093/nar/gkp471]

Pickrell, J.K., Pai, A.A., Gilad, Y., and Pritchard, J.K. (2010) Noisy splicing drives mRNA isoform diversity in human cells. PLoS Genet, 6:e1001236. [doi: 10.1371/journal.pgen.1001236]

Tress, M.L., Abascal, F., and Valencia, A. (2016) Alternative Splicing May Not Be the Key to Proteome Complexity. Trends in Biochemical Sciences. [doi: 10.1016/j.tibs.2016.08.008]

Zhang, Z., Xin, D., Wang, P., Zhou, L., Hu, L., Kong, X., and Hurst, L. D. (2009) Noisy splicing, more than expression regulation, explains why some exons are subject to nonsense-mediated mRNA decay. BMC biology, 7:23. [doi:10.1186/1741-7007-7-23]

I especially like the Pickrell et al. paper because it presents the controversy in the correct scientific manner. The abstract is great ....

While the majority of multiexonic human genes show some evidence of alternative splicing, it is unclear what fraction of observed splice forms is functionally relevant. In this study, we examine the extent of alternative splicing in human cells using deep RNA sequencing and de novo identification of splice junctions. We demonstrate the existence of a large class of low abundance isoforms, encompassing approximately 150,000 previously unannotated splice junctions in our data. Newly-identified splice sites show little evidence of evolutionary conservation, suggesting that the majority are due to erroneous splice site choice. We show that sequence motifs involved in the recognition of exons are enriched in the vicinity of unconserved splice sites. We estimate that the average intron has a splicing error rate of approximately 0.7% and show that introns in highly expressed genes are spliced more accurately, likely due to their shorter length. These results implicate noisy splicing as an important property of genome evolution.

Let's me make my position very clear. I'm a strong supporter of the idea that most splice variants are due to splicing errors. I'm a strong supporter of the idea that the proper way to do science is to provide evidence for any claims of functionality. (Don't just assume it.) I strongly believe that if a field is controversial—and this one certainly is—then you have a ethical obligation to mention the controversy and point out why you disagree with your opponents. This criticism is directed mainly at proponents of massive alternative splicing; they must demonstrate that they understand the criticism of their position.

Here's the paper that Jack Greenblatt and his colleagues published in February.

Han, H., Braunschweig, U., Gonatopoulos-Pournatzis, T., Weatheritt, R. J., Hirsch, C.L., Ha, K.C., Radovani, E., Nabeel-Shah, S., Sterne-Weiler, T., Wang, J., O'Hanlon, D.O., Pan, Q., Ray, D., Zheng, H., Vizeacoumar, F., Datti, A., Magomedova, L., Cummins, C.L., Hughes, T.R., Greenblatt, J.F., Wrana, J.L., Moffat, J., and Blencowe, B.J. (2017) Multilayered control of alternative splicing regulatory networks by transcription factors. Molec. Cell, 65:539-553. e537. [doi: 10.1016/j.molcel.2017.01.011]

Networks of coordinated alternative splicing (AS) events play critical roles in development and disease. However, a comprehensive knowledge of the factors that regulate these networks is lacking. We describe a high-throughput system for systematically linking trans-acting factors to endogenous RNA regulatory events. Using this system, we identify hundreds of factors associated with diverse regulatory layers that positively or negatively control AS events linked to cell fate. Remarkably, more than one-third of the regulators are transcription factors. Further analyses of the zinc finger protein Zfp871 and BTB/POZ domain transcription factor Nacc1, which regulate neural and stem cell AS programs, respectively, reveal roles in controlling the expression of specific splicing regulators. Surprisingly, these proteins also appear to regulate target AS programs via binding RNA. Our results thus uncover a large “missing cache” of splicing regulators among annotated transcription factors, some of which dually regulate AS through direct and indirect mechanisms.

The idea is to identify all those factors that contribute to producing splice variants. It turns out there are many factors involved, including known transcription factors. The data says nothing about biological function since splicing errors are just as likely to be causing by spurious binding of factors as true alternative splicing. The authors are assuming that these factors are involved in "regulation" but they don't discuss the alternative explanation; namely, splice errors and artifacts.

Let's look at the introduction to see if it meets the sugested standards for papers on this subject.

Alternative splicing (AS) is the process by which different combinations of splice sites in precursor mRNA (pre-mRNA) are selected to generate structurally and functionally distinct mRNA and protein variants. It acts widely to expand the functional and regulatory capacity of metazoan genomes (Irimia and Blencowe, 2012; Licatalosi and Darnell, 2010 ; Nilsen and Graveley, 2010). For example, nearly all transcripts from human multi-exon genes are alternatively spliced, and a substantial fraction of these splice variants are differentially expressed in a cell- and tissue-specific manner (Pan et al., 2008 ; Wang et al., 2008). AS has critical roles in diverse biological processes, including cell-fate determination, and misregulation of AS is associated with numerous diseases (Daguenet et al., 2015; Jangi and Sharp, 2014 ; Kalsotra and Cooper, 2011). An important challenge is to understand how networks of AS events are coordinately regulated to impart their biological roles in different cellular contexts.

The good news is that they associate alternative splicing with functionally distinct mRNA and protein variants. The bad news is that they don't present any evidence that the variants they look at are truly functional.

The introduction contains the standard dogma about alternative splicing. They assume, without proof, that "nearly all" human protein-coding genes are alternatively spliced to produce functional isoforms. This is a true example of begging the question since the real question is not what causes alternative splicing but whether it actually exists. You shouldn't be investigating coordinate regulation until you can establish that there really is coordinate regulation in any meaningful sense. The authors ignore any controversy that challenges their claims even though they are certainly aware of it.

The authors make the standard implicit assumption that cell- and tissue-specific splicing is evidence of function. In fact, this assumption is wrong since the alternative explanation—incorrect splicing due to inappropriate binding of various factors—will also be tissue-specific.

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