The transcripts of some genes can be alternatively spliced to produce more than one biologically functional product (e.g. proteins). There are several well-documented examples in the scientific literature but they are not common. There are probably fewer than 500 human genes (2.5%) that exhibit true alternative splicing where the alternate gene product has been conclusively shown to exist and be biologically functional.
However, it's easy to detect multiple examples of unusually spliced transcripts of humans genes. The vast majority of these splice variants are present at less than one copy per cell, are rapidly degraded, and not conserved in closely related species. That has led to the idea that they are simply the result of splicing errors, a conclusion that's reinforced by solid evidence that splicing is error prone.
In recent years, many scientists have come to realize that the role of alternative splicing has been greatly exaggerated. If you're interested in learning more, I cover the controversy on pages 154-169 in my book and in numerous blog posts (see below).
Unfortunately, there's a Nature editor who didn't get the message so they perpetuated the standard misinformation in a recent (May 21, 2025) editorial in Nature Reviews Genetics.
Anonymous (2025) RNA splicing — a central layer of gene regulation. Nat Rev Genet 26:369–370 [doi: 10.1038/s41576-025-00846-x]
... Splicing is essential for the accurate translation of DNA sequence information and comes with the added perk of generating transcriptomic and proteomic diversity in the form of alternative splicing — that is, the regulated inclusion or exclusion of exons. Alternative splicing greatly expands the coding potential of the genome; more than 95% of human multi-intron genes undergo alternative splicing, producing mRNA isoforms that can differ in coding sequence, regulatory elements or untranslated regions. These isoforms can influence mRNA stability, localization and translation output, thereby modulating cellular function.
... The ability of a single gene to produce several, functionally distinct protein isoforms through alternative splicing could enable organisms to rapidly adapt to changing environments. By enabling the sequencing of full-length transcripts, long-read sequencing data have yielded a more complete picture of alternative splicing. Subsequent comparative transcriptomic studies have revealed striking differences in the extent of alternative splicing between eukaryotes. Indeed, recent studies suggest that heritable variation in patterns of alternative splicing contributes to adaptive evolutionary chang.
Wouldn't it be nice if some leading researchers in the field wrote a scathing letter to Nature about the propagation of such misinformation? Does anyone know who to contact at Nature if you want to register a complaint?
Blog posts on alternative splicing
- The number of splice variants in a species correlates inversely with the population size - what does that mean?
- Splicing errors or alternative splicing?
- Alternative splicing and evolution
- Alternative splicing: function vs noise
- The frequency of splicing errors reflects the balance between selection and drift
- Alternative splicing in the nematode C. elegans
- The persistent myth of alternative splicing
- The textbook view of alternative splicing
- Are splice variants functional or noise?
- Debating alternative splicing (part I)
- Debating alternative splicing (part II)
- Debating alternative splicing (Part III)
- Debating alternative splicing (Part IV)
Larry, just send a letter to Nature. There are sections 'Comment' and 'Correspondence'.
ReplyDeleteunreproductive transcripts isoforms have nothing to do with splisome inefficiency, there are part of a regulatory mecanism. A particular subtype of this mechanism is referred to as regulated unproductive splicing and translation (RUST) or simply unproductive splicing (9,10), in which the cell employs alternative splicing (AS) to produce a premature termination codons containing transcript in order to post-transcriptionally down-regulate the expression level of the gene
ReplyDelete@Mehrshad: "unreproductive transcripts isoforms have nothing to do with splisome inefficiency"
DeleteNobody claims they do. You're deeply confused and ignorant about the subject.
"A particular subtype of this mechanism is referred to as regulated unproductive splicing and translation (RUST)"
And how many alternatively spliced genes have been shown empirically to have a well-characterizied function through that mechanism? One? Two?
By the way is that a quote from some paper? You seem to be making citations to numbered references but don't list any. If you're copy-pasting material from some paper you should properly reference it.
Tissue-specific regulation of gene expression via unproductive splicing
ReplyDeleteAlexei Mironov 1, Marina Petrova 2, Sergey Margasyuk 3, Maria Vlasenok 4, Andrey A Mironov 5, Dmitry Skvortsov 6, Dmitri D Pervouchine 7,✉
This is the paper the transcript could not be noise because they are tissue specific
@Mikkel Rumraket Rasmussen
Delete@Mehrshad: "This is the paper the transcript could not be noise because they are tissue specific"
DeleteThat literally doesn't make sense. Noise can absolutely be tissue-specific. Your reference demonstrates a function for exactly zero USEs. All they show is that some USEs affect tissue-specific transcription levels for some genes. Whether those are functional or noise is still entirely up in the air.
Noise can definitely be tissue-specific. The proteome of cells vary by tissue and as a result their non-specific transcription profiles (i.e. noise) vary as well.
Delete@Anonymous: I suggested that there are probably fewer than 500 human genes that exhibit real alternative splicing. This is my personal opinion. It's a highball estimate because that's all I need to make the point.
ReplyDeleteI'm only aware of one paper that did a systematic review of the literature and the authors found only 23 examples meeting their minimal requirements. Please let me know if you have any evidence demonstrating that there are more than 500 genes that produce multiple biologically relevant products by alternative splicing.
Bhuiyan, S.A., Ly, S., Phan, M., Huntington, B., Hogan, E., Liu, C.C., Liu, J. and Pavlidis, P. (2018) Systematic evaluation of isoform function in literature reports of alternative splicing. BMC Genomics 19:637. https://doi.org/10.1186/s12864-018-5013-2
ABSTRACT: "Although most genes in mammalian genomes have multiple isoforms, an ongoing debate is whether these isoforms are all functional as well as the extent to which they increase the functional repertoire of the genome. To ground this debate in data, it would be helpful to have a corpus of experimentally-verified cases of genes which have functionally distinct splice isoforms (FDSIs)."
A very quick look at Bhuiyan et al.reveals that is doesn’t include an obvious and very well-known example of functionally-significant alternative splicing, namely that involving the IGHM gene. I don’t have much confidence in this report, and it is not very good support for the argument that functionally-important alternative splicing is rare.
Delete@Mikkel Rumraket Rasmussen... I wonder what kind of noise is, that can be specifically adjusted to a particular tissue type... Isn't random trascript a by product of stochastic transcription or splicing? So why they are so biased to specific tissue?
ReplyDeletePretty simple. Random transcripts can be due to binding of transcription factors to accidental promotor sites, which of course only occurs in those cells that express the transcription factor in quesiton.
Delete@John Harshman ... but trascription is never a haphazard phenomena. Multiple regulatory element and almost 6 general transcription factor plus remodelar enzymes and epigenetic modification are needed for an normal transcription.... Don't you think how can 6 transcription factors plus another regulatory factor elements associate randomly to provide a setting for noisy trascript?
ReplyDeleteRemember that we're talking about extremely rare transcripts for the most part. Doesn't take much.
Delete@Mehrshad
DeleteTranscription absolutely can be a haphazard phenomenon:
Xu H, Li C, Xu C, Zhang J. Chance promoter activities illuminate the origins of eukaryotic intergenic transcriptions. Nat Commun. 2023 Apr 1;14(1):1826. doi: 10.1038/s41467-023-37610-w. PMID: 37005399; PMCID: PMC10067814.
@Mehrshad said, "... but transcription is never a haphazard phenomena."
ReplyDeleteIt can be. Spurious transcription initiation has been known for decades. We also know that if you inset random DNA sequences into cells a significant fraction will be transcribed at a low but detectable level.
It may take a complex array of transcription factors to get high frequency transcription initiation but any of the RNA polymerases can initiate a few transcripts on their own whenever they bind to a DNA sequence.
How RNA polymerase binds to DNA
https://sandwalk.blogspot.com/2008/09/how-rna-polymerase-binds-to-dna.html
but even assuming that, we can not ignore recent evidences regrading function for transcriptional noise. Which have an important role in regulating cell fate and cell decision - making.... Here is the paper
DeleteLiving in a noisy world—origins of gene expression noise and its impact on cellular decision-making.
Sampriti Pal, Riddhiman Dhar
The cited article doesn't seem to support your claim of a function for transcriptional noise, and is even talking about an entirely different meaning of the term from the one we're talking about here.
Delete@John Harshman. Does Gene Expression Noise Play a Functional Role in Plants?
ReplyDeleteSandra Cortijo et al in Trends in Plant Science vol. 25 (10) pp. 1041-1051
Can low level stochastic gene expression serve a functional role? Sure. Does that mean we should conclude that all low-level stochastic gene expression is therefore functional? Of course not.
DeleteI suggest you count the occurrence of the words may, might, could and would in the article and the try to find any primary data that corroborate the claims you make above. As the authors themselves state:
Delete"However, we are still missing a comprehensive understanding of: (i) the source of gene expression noise; (ii) whether there is a link between cell-to-cell, tissue-to-tissue, and plant-to-plant gene expression variability; and (iii) how transcriptional and phenotypic interindividual variability are related in plants. Recent optimisations in experimental methods, analysis tools, and modelling approaches in plants now allow the exploration of these questions."
@Mikkel Rumraket Rasmussen__ could I know based on what evidence you are certain that much of the transcripts and genome are non_ functional?
ReplyDeleteDespite all the ongoing evidences against neutrality you are keeping your faith strongly, maybe that's because of fear of intelligent design
That would be all the same evidence for junk DNA elucidated on this blog countless times before, in Larry's book, and in the many references provided in these and other threads. The fact that variations in genome size comes from the sort of DNA most prone to vary in amount all by itself through well elucidated mechanisms such as transposons activity, and duplications of repetitive DNA, that most of this excess DNA are degraded retroelements of various sorts (if an intelligent designer needed more regulatory elements or transcription factor binding spots, why create them from decaying transposons all the time, which frequently are much bigger than they need to be for som transcription factor to bind somewhere within them? You don't need mutation-decaying copies of GAG, POL, and ENV flanked by decaying remnants of LTRs to have a transcription factor binding spot somewhere within it, so the simpler explanation is that it's there because transposons can copy and insert themselves and we are seeing the leftovers of this process having occurred millions of times over evolutionary history, this is so obvious it's a form of insanity to deny it), that it is being poorly conserved both between speces and within the same population (so is unlikely to have any sequence-based function), that experiments have shown large chunks can be removed with no observable effect (megabase deletions in mice), and so on and so forth.
DeleteI'm afraid it sounds to me like you're the one who has tied your faith to the idea that there should be little to no junk DNA.
Does Gene Expression Noise Play a Functional Role in Plants?
ReplyDelete@Mehrshad, you're getting closer. This at least offers a claim for function. But the meaning of "gene expression noise" is still entirely different from what we're discussing here. Do you even read more than the titles of the stuff you cits?
Sorry, could you elaborate a bit more what point I had missed.... I assume the papers are exactly taking about recent relevant functional entity (cell specification) for noisy trascript....
DeleteRather than assuming based on titles, try reading the actual papers and thinking (this step is crucial) about what they mean and how it's connected to the current discussion, if at all.
ReplyDeleteSo, I am a bit late to this one, but I agree with Larry except for one point,. I would put 500 genes with functionally important alternative isoforms as the lower bound. The Bhuiyan et al paper is quite right, there are very few papers that demonstrate the function of alternative isoforms (IGHM doesn't count, it is an Ig gene), but conservation is a good substitute and off the top of my head I could give you a list of 250 genes that have AS isoforms that are conserved across jawed vertebrates. If I looked hard enough I think I could probably find another 250 conserved at least across mammals.
ReplyDelete19,000 coding genes are too many for a detailed count, but we have a method that predicts the functional importance of isoforms (Pozo et al. NAR Genom Bioinfo. 2021) and if we apply it to the whole genome and use the cuf-off for functional importance we usually use, the method predicts 3,633 genes with functionally important isoforms. I think our method probably overpredicts (various technical reasons), so I would use this as an upper bound.
So, my estimation is that there is somewhere between 500 and 3,500 genes with functionally important alternative isoforms.
Overall alternative exons are not under selection pressure though, so the vast majority of AS is still biological noise.
As an aside, the number of coding transcripts in the human gene set is set to double in the next couple of months.
@Michael Tress, I don’t understand why an example of alternative splicing that results in either membrane-bound or secreted antibodies “doesn’t count” when it comes to enumerating authentic instances of alternative splicing with functional outcomes.
DeleteBecause it is defined as an IC C gene and not a protein coding gene in the databases. Similarly, I have had people asking why I don't include the protocadherins as obvious examples of alternative splicing. Protocadherin transcripts are alternatively spliced , but for historical reasons each transcript is defined as a separate gene within a "gene cluster".
ReplyDelete@Michael Tress says, "... off the top of my head I could give you a list of 250 genes that have AS isoforms that are conserved across jawed vertebrates."
ReplyDeleteI'd love to see that list and the evidence for "conservation."
Sorry Larry, I only just saw your email. Most of them are in two criminally overlooked papers by Laura Martinez Gomez (Nucleic Acids Res. 2021 and Genome Biol Evol. 2022), and are genes that have transcripts that swap one tandem duplicated exon for another. The rest are in a paper that I am writing at the moment.
ReplyDeleteAnd (by the way) most of these are examples of REAL tissue specificity.
You've probably seen this already. But just in case: https://whyevolutionistrue.com/2025/06/13/michael-lynch-takes-apart-two-attempts-to-forget-new-evolutionary-laws
ReplyDeleteLove your book.