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When cells copy DNA to produce RNA transcripts, some stretches of the genetic material are included—these are known as exons—but the rest is thrown out. The resulting transcript is a fully mature RNA molecule, which can be used as a template to build a protein. One of the features of gene expression is that, through a process known as alternative splicing, a cell can select different combinations of exons to make different RNA transcripts. Researchers headed by a team at the Centre for Genomic Regulation (CRG), Barcelona Institute of Science and Technology, have discovered a set of tiny protein-coding gene fragments known as microexons that are spliced into pancreatic islet cell mRNA, and which appear to play important roles in islet function and blood sugar control, and may contribute to type 2 diabetes (T2D) predisposition.
The team, co-led by ICREA research professor Manuel Irimia, PhD, identified an RNA-binding protein, SRRM3, that regulates these IsletMICs, and found that both SRRM3 and IsletMICs were induced by elevated glucose levels. Their studies then showed that either depleting SRRM3 in human and rat beta cell lines and mouse islets, or repressing particular IsletMICs, impacted insulin secretion. Reporting on their findings in Nature Metabolism, the researchers discovered that human genetic variants that influence SRRM3 expression and IsletMIC inclusion in islets were associated with fasting glucose variation and T2D risk. The team suggests that these islet microexons may represent promising targets for treating dysfunctional beta cells in T2D. Their results also support the perception that pancreatic islet cells have evolved by borrowing regulatory mechanisms from neuronal cells.
In the published paper, titled “Pancreatic microexons regulate islet function and glucose homeostasis,” the authors stated, “Here, we found that a subset of neural microexons is also present in pancreatic islets, forming an evolutionarily conserved program that particularly affects genes involved in insulin secretory function and T2D risk … Our results thus uncover a post-transcriptional regulatory program necessary for mature islet cell function and glycaemic control.”
Living organisms use alternative splicing to enable complex functions. Akin to movie producers creating a regular and director’s cut of a film, including or excluding a single exon in a transcript can result in the production of proteins with different functions. So different types of cells in different kinds of tissues produce different RNA transcripts from the same gene. As the authors explained, “Alternative splicing regulates the differential processing of introns and exons to generate multiple mRNA isoforms from a single primary transcript, contributing to transcriptomic and proteomic diversity. It is estimated that over 95% of human multi-exonic genes undergo alternative splicing, a substantial fraction of which is regulated in a tissue-type and cell-type enriched manner.”
Understanding how this process works provides new clues about human development, health, and disease and paves the way for new diagnostic and therapeutic targets. “A plethora of studies have provided experimental evidence that alternative splicing regulation is essential for many biological processes, and unveiled the roles of several RNA binding splicing regulatory proteins in tissue-specific development and functions,” the team continued.
Microexons are a more recently discovered type of protein-coding DNA sequence. At just three to 27 nucleotides long, microexons are much shorter than the average exon, which more typically averages around 150 nucleotides. The existence of microexons across many different species ranging from flies to mammals suggests they have an important function because they have been conserved by natural selection for hundreds of millions of years.
In humans, most microexons are exclusively found in neuronal cells, where the tiny gene fragments play key roles. For example, recent studies show that they are crucial for the development of photoreceptors, a specialized type of neuron in the retina. Research has also shown that alterations to microexon activity are common in autistic brains, suggesting that these gene fragments play an important role in the clinical characteristics of the condition.
However, as the team further pointed out in their newly released paper, while alternative splicing is known to play crucial roles in the development and function of a variety of cell and tissue types, “its impact on the endocrine pancreas and its physiological relevance in the context of glycaemic control remain largely unknown.”
Irimia, a CRG researcher who explores the functional role of microexons, explained, “A microexon is a short fragment of DNA that codes for a few amino acids, the building blocks of proteins. Though we don’t know the exact mechanisms of action involved, including or excluding just a handful of these amino acids during splicing sculpts the surfaces of proteins in a highly precise manner. Therefore, microexon splicing can be seen as a way to perform microsurgery of proteins in the nervous system, modifying how they interact with other molecules in the highly-specialized synapses of neurons.”
The studies led by Irimia and ICREA research professor Juan Valcárcel, PhD, at the CRG found that microexons are also present in pancreatic islet cells, another type of cell that carries out highly specialized functions within a complex tissue and organ. Their work showed that microexon splicing is prevalent in the pancreatic islets containing the beta cells that make insulin.
The researchers made their discovery while studying the role of alternative splicing in the biology of pancreatic islets and the maintenance of blood sugar levels. They were studying RNA sequence data from different human and rodent tissues, specifically looking for exons that are differentially spliced in pancreatic islets, compared with other tissues.
Their data revealed that half the exons specifically enriched in pancreatic islets were microexons, almost all of which were also found in neuronal cells. “We found that a subset of neural microexons is also present in pancreatic islets, forming an evolutionarily conserved program that particularly affects genes involved in insulin secretory function and T2D risk,” they wrote. The findings are in line with the idea that pancreatic islet cells have co-opted regulatory mechanisms from neuronal cells.
The team’s studies showed that of the more than 100 pancreatic islet microexons identified, the majority were located on genes critical for insulin secretion, or linked to type 2 diabetes risk. “The research also revealed that microexon inclusion in RNA transcripts was controlled by SRRM3, a protein that binds to RNA molecules and is encoded by the SRRM3 gene. Experiments demonstrated that high blood sugar levels induced both the expression of SRRM3 and the inclusion of microexons, hinting at the possibility that the regulation of microexon splicing could play a role in maintaining blood sugar levels.
To further understand the impact of islet microexons, the researchers carried out functional experiments using human beta cells grown in the laboratory, as well as in vivo and ex vivo experiments with mice lacking the SRRM3 gene. They found that depleting SRRM3 or repressing single microexons lead to impaired insulin secretion in beta cells. In mice, alterations to microexon splicing changed the shape of pancreatic islets, ultimately impacting the release of insulin. “Consistently, misregulation of microexons in beta cell lines, either globally through SRRM3 depletion or individually using antisense oligonucleotides (ASOs), result in dysregulated insulin release. In line with this, Srrm3-knockout mice showed alterations in islet cell identity, increased insulin secretion, and persistent hypoglycemia,” the investigators wrote.
The researchers teamed up with the research group of Jorge Ferrer, PhD, also at the CRG, to study genetic and RNA transcript data from diabetic and non-diabetic individuals and explore possible links between microexons and human metabolic disorders. They found that genetic variants which affected microexon inclusion were linked to variations in fasting blood sugar levels and also T2D risk. They also discovered that type 2 diabetes patients have lower levels of microexons in their pancreatic islets. “Importantly, human genetic variants that influence SRRM3 expression and IsletMIC inclusion in islets are associated with fasting glucose variation and type 2 diabetes risk,” the team noted. “… we also observed that IsletMICs are broadly downregulated in islets from individuals with T2D, an effect also recently reported in a diabetic mouse model … Altogether, these data suggest that changes in the activity of SRRM3 and its microexon targets affect islet function and glucose homeostasis, potentially contributing to T2D predisposition.”
The findings of the study pave the way to explore new therapeutic strategies to treat diabetes by modulating splicing. “Here we show that islet microexons play important roles in islet function and glucose homeostasis, potentially contributing to type 2 diabetes predisposition,” explained CRC post-doc researcher Jonas Juan Mateu, PhD, who is first author of the study. “For this reason, microexons may represent ideal therapeutic targets to treat dysfunctional beta cells in type 2 diabetes. A wide range of splicing modulators are available to treat a variety of human diseases,” Mateau noted. “When I first started studying splicing in pancreatic islets eight years ago, I wanted to find out whether existing splicing modulators could be repurposed for diabetes. I think we’re one step closer to that.”
While the work shows microexons are important players in pancreatic islet biology, further research will be needed to determine their precise impact during the tissue’s development. Researchers also lack mechanistic insight into how each individual microexon alters protein function and affects key pathways in islet cells. Understanding this will shed light on their exact physiological role in diabetes and other metabolic diseases linked to pancreatic islets.
The study adds to a growing body of evidence that microexons play crucial roles in human development, health, and disease. “Less than 10 years after we first reported on their existence, we are seeing how microexons are key elements that modify how proteins interact with each other in cells with functions that require a high degree of specialization, such as neurotransmitter or insulin release and light transduction,” explained Irimia. “Consequently, we expect mutations in microexons to lead to diseases whose genetic causes we have not yet understood. We are beginning to search for these mutations in patients with neurodevelopmental and metabolic disorders as well as retinopathies, to then devise possible interventions to treat them.”
As the authors further concluded, “There is currently a broad range of strategies to manipulate RNA splicing therapeutically. Therefore, future research on the molecular function of IsletMICs and other islet-enriched exons and their role in islet dysfunction might provide valuable knowledge not only for islet biology but also for the development of novel therapies for T2D based on splicing modulation.”
