Researchers at the Centre for Genomic Regulation (CRG) have created the first blueprint of the human spliceosome, the complex molecular machine that edits genetic messages transcribed from DNA, allowing cells to create different versions of a protein from a single gene.
Their results could lead to new therapeutic approaches that target RNA processing in diseases, such as some cancers, that are linked to faulty RNA molecules produced by mistakes in splicing. The detailed map of the spliceosome, which the authors of the study have made publicly available, can help researchers pinpoint where the splicing errors are occurring in a patient’s cells.
Creating the resource took more than a decade. “We wanted this to be a valuable resource for the research community,” said Institució Catalana de Recerca i Estudis Avançats (ICREA) research professor Juan Valcárcel, PhD, a researcher at the CRG. “Drugs correcting splicing errors have revolutionized the treatment of rare disorders like spinal muscular atrophy. This blueprint can extend that success to other diseases and bring these treatments into the mainstream.”
Added Malgorzata Rogalska, PhD, co-corresponding author at the CRG, “Current splicing treatments are focused on rare diseases, but they are just the tip of the iceberg. We are moving into an era where we can address diseases at the transcriptional level, creating disease-modifying drugs rather than merely tackling symptoms. The blueprint we’ve developed paves the way for entirely new therapeutic approaches. It’s only a matter of time.”
Valcárcel is senior author of the scientists’ report in Science, “Transcriptome-wide splicing network reveals specialized regulatory functions of the core spliceosome,” in which they concluded, “Given the prevalence and functional relevance of the splicing process and its regulation, the resources, approaches, and insights provided in this and related studies have the potential to illuminate underlying physiological and pathological mechanisms and eventually inform the design of new therapeutic approaches.
Every cell in the human body relies on precise instructions from DNA to function correctly. These instructions are transcribed into RNA, which then undergoes a crucial editing process called splicing. During splicing, non-coding segments of RNA are removed, and the remaining coding sequences are stitched together to form a template or recipe for protein production. The vast majority of human genes are edited by the spliceosome. While humans have about 20,000 protein-coding genes, splicing allows the production of at least five times as many proteins, with some estimates suggesting humans can create more than 100,000 unique proteins.
The spliceosome is the collection of 150 different proteins and five small RNA molecules that orchestrate the RNA editing process, but until now, the specific roles of its numerous components were not fully understood. Errors in the splicing are linked to a wide spectrum of diseases including most types of cancer, neurodegenerative conditions and genetic disorders.
“The spliceosome is the complex molecular machinery that sequentially assembles on eukaryotic messenger RNA precursors to remove introns (pre-mRNA splicing), a physiologically regulated process altered in numerous pathologies,” the team explained. “Alternative patterns of intron removal (alternative splicing, AS) are observed in more than 90% of human genes and contribute to the regulation of cell differentiation and homeostasis.”
The sheer number of components involved and the intricacy of spliceosome function has meant the spliceosome has remained elusive and uncharted territory in human biology. “Despite the biological and pathological relevance of AS, the molecular mechanisms that regulate splice site selection remain poorly understood,” the author noted.
As part of their newly reported study the team at the CRG knocked down (knockdown; KD) the expression of hundreds of splicing-related protein-coding genes (SFs) in human cancer cells one by one, observing the effects on splicing across the entire genome. “We report transcriptome-wide analyses upon systematic knock down of 305 spliceosome components and regulators in human cancer cells and the reconstruction of functional splicing factor networks that govern different classes of alternative splicing decisions,” they wrote.
The resulting blueprint reveals that individual components of the spliceosome are far more specialized than previously thought. Many of the components have not been considered in the context of drug development before because their specialized functions were unknown. The newly reported discoveries might unlock new treatment strategies that are more effective and have fewer side effects.
Their results, they suggested, “disentangle intricate circuits of splicing factor cross-regulation …”. Valcárcel further stated, “The layer of complexity we’ve uncovered is nothing short of astonishing. We used to conceptualize the spliceosome as a monotonous but important cut and paste machine. We now see it as a collection of many different flexible chisels that allow cells to sculpt genetic messages with a degree of precision worthy of marble sculpting grandmasters from antiquity. By knowing exactly what each part does, we can find completely new angles to tackle a wide spectrum of diseases.”
The scientists’ work revealed that different components of the spliceosome have unique regulatory functions. “Collectively, our results demonstrate how systematic, large-scale interrogation of the spliceosome and its regulatory factors along with functional network reconstruction approaches can reveal mechanistic insights on the regulatory activities of SFs in different biological contexts,” they pointed out.
Crucially, the results indicated that proteins within the spliceosome’s core are not just idle support workers but instead have highly specialized jobs in determining how genetic messages are processed, and ultimately, influence the diversity of human proteins. “Notably, our findings uncover unexpected, specialized functions of components of the core splicing machinery, both in early- and late-acting complexes during spliceosome assembly,” the team further stated.
For example, one component selects which RNA segment is removed. Another component ensures cuts are made at the right place in the RNA sequence, while another one behaves like a chaperone or security guard, keeping other components from acting too prematurely and ruining the template before its finished.
The authors of the study compare their discovery to a busy post-production set in film or television, where genetic messages transcribed from DNA are assembled like raw footage. “You have many dozens of editors going through the material and making rapid decisions on whether a scene makes the final cut,” said Rogalska. “It’s an astonishing level of molecular specialization at the scale of big Hollywood productions, but there’s an unexpected twist. Any one of the contributors can step in, take charge, and dictate the direction. Rather than the production falling apart, this dynamic results in a different version of the movie. It’s a surprising level of democratization we didn’t foresee.”
One of the most significant findings in the study is that the spliceosome is highly interconnected, where disrupting one component can have widespread ripple effects throughout the entire network. For example, the study manipulated the spliceosome component SF3B1, which is known to be mutated in many cancers including melanoma, leukemia and breast cancer. It is also a target for anti-cancer drugs, though the exact of mechanisms of action have been unclear—until now.
The study found that altering the expression of SF3B1 in cancer cells sets off a cascade of events that affected a third of the cell’s entire splicing network, causing a chain reaction of failures which overwhelm the cell’s ability to fuel growth. The finding is promising because traditional therapies, for example those targeting mutations in DNA, often cause cancer cells to become resistant. One of the ways cancers adapt is by rewiring their splicing machinery. Targeting splicing can push diseased cells past a tipping point that cannot be compensated for, leading to their self-destruction.
“Cancer cells have so many alterations to the spliceosome that they are already at the limit of what’s biologically plausible. Their reliance on a highly interconnected splicing network is a potential Achilles’ heel we can leverage to design new therapies, and our blueprint offers a way of discovering these vulnerabilities” said Valcárcel.
“This pioneering research illuminates the complex interplay between components of the spliceosome, revealing insight into its mechanistic and regulatory functions,” pointed out Dom Reynolds, CSO at Remix Therapeutics, a clinical stage biotechnology company in Massachusetts who collaborated with the CRG on the study. “These findings not only advance our understanding of spliceosome function but also open potential opportunities to target RNA processing for therapeutic interventions in diseases associated with splicing dysregulation.”
Apart from cancer, there are many other diseases caused by faulty RNA molecules produced by mistakes in splicing. With a detailed map of the spliceosome, which the authors of the study have made publicly available, researchers can now help pinpoint exactly where the splicing errors are occurring in a patient’s cells.
“More generally, our dataset provides a distinct resource to explore targets of any component of the spliceosome and regulatory factors; to identify regulators of any individual, group, or class of AS events of interest detectable in this system; and to untangle the intricate regulatory relationships between components and conformational transitions of one of the most complex molecular machineries of eukaryotic cells,” the team concluded in their paper.