Alternative splicing (AS) is a genetic process that increases the diversity of proteins that can be generated from genes, by assembling sections of genetic code into different combinations. This is believed to enhance biological complexity by allowing genes to produce different versions of proteins—protein isoforms—for many different uses. The results of studies by researchers at the University of Chicago now suggest that the biggest impact of alternative splicing, other than creating new protein isoforms, may relate to its role in regulating gene expression levels by producing “unproductive” transcripts that are targeted for degradation through a process known as nonsense-mediated decay (NMD).
The investigators, headed by Yang Li, PhD, Benjamin Fair, PhD, and Carlos Buen Abad Najar, PhD, reported on their findings in Nature Genetics, in a paper titled, “Global impact of unproductive splicing on human gene expression,” in which they concluded, “Our findings suggest that much of the impact of AS is mediated by NMD-induced changes in gene expression rather than diversification of the proteome.”
Alternative splicing in human genes is widely viewed as a mechanism for enhancing proteomic diversity, the authors explained. During alternative splicing, different segments of genes are removed, and the remaining pieces are joined together during transcription to messenger RNA (mRNA). “Large-scale transcriptomics studies have confirmed that nearly all protein-coding genes generate multiple—sometimes dozens—of distinct mRNA isoforms,” the investigators wrote. However, they pointed out, multiple studies have suggested that the vast majority of isoforms are nonfunctional transcripts resulting from mis-splicing rather than regulated AS. “AS can also impact gene expression levels without increasing protein diversity by producing ‘unproductive’ transcripts that are targeted for rapid degradation by nonsense-mediated decay (NMD),” they stated, although the relative importance of this regulatory mechanism has been underexplored.
For their reported studies, Li et al. analyzed large sets of genomic data, covering various stages from early transcription to when RNA transcripts are destroyed by the cell. They discovered that cells produced three times as many “unproductive” transcripts—RNA molecules with mistakes or unexpected configurations—as when they analyzed steady-state, finished RNA only.
Unproductive transcripts are quickly destroyed by the cellular process nonsense-mediated decay (NMD). Li’s team calculated that on average, about 15% of transcripts that are started are almost immediately degraded by NMD; when they looked at genes with low expression levels, that number went up to 50%. “Through detailed analysis of molecular measurements that capture the major steps of RNA maturation, we found that aberrant splicing produces remarkably high levels of unproductive transcripts bearing a PTC [premature termination codon],” the authors wrote. “Unproductive mRNAs account for around 15% of all mRNA transcripts from the average human gene, even exceeding 50% for many long genes expressed at low levels.”
Li added, “We thought that was a huge breakthrough. It already seems wasteful to degrade 15% of mRNA transcripts, but no one would have thought that the cell is transcribing so much and getting rid of the errors immediately, seemingly without any purpose. Why would the cell fire up its genetic production machinery to immediately trash 15 to 50% of its output? And why would transcription make so many mistakes in the first place? We think it’s because NMD is so efficient,” said Li, who is associate professor of medicine and human genetics. “The cell can afford to make mistakes without damaging things, so there’s no selective pressure to make fewer mistakes.”
But Li suspected there must also be some purpose for such a widespread phenomenon. The team conducted a genome-wide association study (GWAS) to compare gene expression levels across different cell lines. They found many variations at genetic locations that are known to affect the level of unproductive splicing. These loci were just as often associated with differences in genetic expression caused by NMD as they were with differences in the production of multiple protein isoforms.
Li believes cells sometimes purposely select transcripts doomed for NMD to decrease expression levels. If the nascent RNA is destroyed before it gets fully transcribed, it will never produce proteins to execute biological functions. This effectively silences the genes, like deleting an email draft before its writer can press send. “Thus, our study suggests that the molecular impact of AS is largely shouldered by NMD, which regulates protein output by targeting unproductive transcripts for degradation,” they wrote. “Supporting this view, we identified nearly as many genetic variants that impact production of these unproductive transcripts as compared with those that tune the balance of stable mRNA isoforms.”
“We found that genetic variations that increase unproductive splicing often decreased gene expression levels,” Li said. “This shows that this mechanism must have some effect on expression because it is so widespread.”
The team found that many variants linked to complex diseases are also associated with more unproductive splicing and decreased gene expression. So, they believe that better understanding its impact could help develop new treatments that leverage the alternative splicing-NMD process. Drug molecules could be designed to decrease the amount of unproductive splicing, and thus increase gene expression. One approved drug for spinal muscular atrophy already takes this approach to restore proteins that are being shut off. Another approach could be to increase the NMD process to decrease expression, for example in rampant cancer genes.
“We think we can target a lot of genes because now we know how much this process is going on,” Li said. “People used to think that alternative splicing was mainly a way to make an organism more complex by generating different versions of proteins. Now we’re showing that it might not be its most important function. It could be simply to control gene expression.” The authors further commented, “… we posit that future research will reveal a preponderance of cases where regulated AS functions by tuning protein expression levels rather than by creating protein diversity, as the sheer abundance of AS–NMD events presents opportunities for evolution to co-opt AS–NMD as a functional regulatory mechanism.”
