Five questions on alternative splicing with Omar El Garwany

Cells use the alternative splicing process to get different proteins from the same gene. So, when your immune cells are under environmental stress and need to survive and multiply, they produce one version of a gene called BCL, but when they need to repress unchecked growth, they produce another version of the same gene.¹

To understand alternative splicing, we must revisit some key ideas in molecular biology. The central dogma describes how genetic information flows through the body – DNA is converted to RNA through transcription, then from RNA to protein via translation. Genes contain specific DNA sequences that code for proteins, or exons, and non-coding sequences, or introns.

When genes are transcribed, the DNA sequence is turned into an initial draft called pre-messenger RNA, or pre-mRNA. The cell processes the pre-mRNA in several steps before creating the final set of instructions – a mature mRNA molecule that leaves the nucleus, ready to synthesise proteins.

One of these processing steps is splicing, which takes place inside a molecular complex called a spliceosome. These ‘cellular scissors’ cut out introns and join exons to create mature RNAs. Alternative splicing happens when the cell chooses different combinations of introns and exons, creating proteins that differ in structure and function. This partly explains why humans produce a much larger number of unique proteins from just 20,000 protein-coding genes.²

Alternative splicing in action. Credit: Laura Olivares Boldú / Connecting Science. What is RNA splicing?

When alternative splicing goes wrong, it can have devastating results. Problems with alternative splicing, such as errors in exon removal or intron retention, are linked to many genetic diseases. Around 10–30 per cent of disease-causing variants are estimated to affect splicing.¹ Understanding alternative splicing and learning how to prevent or fix the errors could turn a potentially fatal diagnosis into a manageable condition. For example, SMA can be a serious condition, causing progressive loss of motor neurons, and Type 1 is a leading cause of infant death in the UK.³ The disorder is caused by incorrect skipping of an exon in the gene SMN1. FDA-approved therapies that correct this SMN1 splicing error, such as Nusinersen, have saved the lives of countless infants.⁴

But while we can correct splicing errors in rare diseases like SMA, common conditions such as IBD, which affects 10 million people worldwide, remain unsolved. We know little about the exact causes of these conditions, but there is a strong genetic link, with over 200 genomic regions associated with at least one type of IBD.⁵ Many of these genetic variants are located within introns and may disrupt alternative splicing.

Our research group at the Sanger Institute focuses on the two most common types of IBD: ulcerative colitis and Crohn's disease. We still have much to learn about how gene expression is regulated in IBD. To address this challenge, our team is sequencing RNA from single cells taken from gut and blood samples of hundreds of IBD patients and healthy volunteers. We combine these data with DNA sequencing information to identify genetic variants that regulate alternative splicing in the many cell types in these tissues. We are working with the pharmaceutical partners of Open Targets and the Crohn’s & Colitis Foundation to identify candidate drug targets for IBD.

Historically, alternative splicing has been difficult to study, but recent technological advances are making this easier. Short-read sequencing is a standard technique in most RNA studies, which involves breaking the RNA molecule into small chunks of 75 to 150 base pairs before sequencing and reassembling the fragments. However, most RNA molecules are over 1,000 base pairs long, so it can be difficult to accurately reassemble the fragments in the correct order and account for the complexities of genetic rearrangements caused by processes like alternative splicing. Researchers have to rely on computer algorithms to make educated guesses about which gene variant a given short read may have come from.

At the Sanger Institute, we are using long-read sequencing to overcome these limitations. This technology reads much longer stretches of RNA in a single pass, spanning thousands of base pairs and providing a more complete picture of RNA structures and variations. From this, we have greater certainty about which distinct version of the gene is expressed. Long-read sequencing lets us identify which genetic variants drive the expression of certain splicing variants, bringing us closer to targeted treatments.

Our research group uses the Sanger Institute’s advanced long-read sequencing machines from Pacific Biosciences to help us reliably measure these splicing variants across entire populations and advance our understanding of these processes. However, these data types are relatively new, so we use innovative approaches to extract meaningful insights from the data.

We work on two innovative projects that aim to help uncover how splicing differences drive disease. The first is the IsoIBD project, in which we compare RNA transcripts from IBD patients and healthy people at scale, for the first time, to identify how alternative splicing variants impact this disease. The project launched earlier this year, and you can read about it in Sanger’s collaboration news article.

We collaborate closely with the Trynka group at the Sanger Institute, who are leading Project JAGUAR, a collaboration that spans seven Latin American countries and explores how genetic diversity shapes immune responses. Our research group investigates how immune cells produce specific RNA transcripts to fight pathogens. Specifically, Project JAGUAR aims to chart the diversity of immune responses across ancestries with diverse genetic backgrounds. Long-read sequencing will allow us to understand which of these genetically regulated differences can be attributed to differences in alternative splicing patterns.

Through these projects, we are building the first population-scale maps of alternative splicing in disease-relevant tissues, providing a foundation for developing precision medicine.

Whilst we still have many unanswered questions about the biology of RNA, we are hopefully at a turning point in alternative splicing research. Our team is studying which gene transcripts are used in what contexts – which could bring us closer to understanding the different splicing choices cells make. The next challenge will be unravelling why cells make these choices and using this knowledge to predict how different splicing variants will function. But first, we must uncover the complexity of splicing regulation.

Once we have a more sophisticated understanding of alternative splicing and a detailed population-scale map of splicing variants, we could identify variants that would make effective drug targets to offset the harmful effects of disease-associated splicing variants. Recently, researchers have made significant progress in developing RNA-based medicines, with several now approved for clinical use,⁶ such as Moderna’s Spikevax COVID-19 vaccine and other RNA drugs that act by correcting splicing errors. This rapidly advancing field offers a promising opportunity to develop new therapeutics more quickly and efficiently.

Our research projects, like IsoIBD and Project JAGUAR, are building the foundations of our knowledge of alternative splicing biology. We look forward to a future where we can move from the laboratory towards real clinical applications, with new diagnostics and treatments for some of the most common genetic diseases.