Benchmarking RNA sequencing for more accurate alternative splicing analysis
Alternative splicing allows a single gene to produce multiple RNA transcripts by combining different portions of the original RNA message. This process greatly expands the diversity of proteins that cells can make, and abnormal splicing has been linked to many diseases. Short-read RNA sequencing is widely used to study alternative splicing, but researchers have not always agreed on which experimental and computational approaches produce the most accurate results. Differences in sequencing depth, data quality, software, and analysis strategy can all affect which splice junctions and transcript isoforms are detected. A large benchmarking effort by researchers at the Key Laboratory of Geriatrics of NHC, Chinese Academy of Medical Sciences has now compared these approaches using standardized reference materials. Comparing hundreds of RNA sequencing workflows The researchers evaluated short-read RNA sequencing data generated across 42 laboratories and analyzed the results using 207 different pipelines. They used two established reference systems, the Quartet ...
A functionally integrated cross-tissue alternative splicing program during short-term calorie restriction
Aging is a major risk factor for chronic diseases, and calorie restriction (CR) is a robust non-pharmacological intervention that can extend health span in multiple species. Alternative splicing (AS) generates multiple RNA isoforms from a single pre-mRNA and becomes dysregulated with age; intriguingly, prior work suggests that CR can attenuate age-associated splicing noise. What has remained unclear is whether AS responses to CR are coordinated across tissues and whether they scale with the level of restriction. In a new study researchers at the University of Aberdeen, Scotland analyzed multi-tissue RNA sequencing (RNA-seq) data from mice undergoing graded CR and show that a 3-month CR intervention induces a largely tissue-specific, yet functionally convergent, AS program that is largely independent of transcriptional (gene expression) changes. Male C57BL/6J mice were maintained for 3 months on graded CR (10%, 20%, 30%, or 40% restriction) and compared with a 12-h ad libitum feeding control. The ...
Long-read RNA sequencing reveals how alternative splicing shapes immune responses
Our immune system relies on many different cell types working together to detect and respond to threats. One important group of immune cells is monocytes, which help recognize invading microbes and trigger inflammatory responses. Although scientists have long known that gene expression changes during immune activation, the role of alternative splicing in this process is still being uncovered. Researchers from University Medical Center Utrecht and Utrecht University in the Netherlands used native long-read RNA sequencing to examine how alternative splicing changes when human monocytes become activated. Alternative splicing is a process that allows a single gene to produce multiple RNA transcripts, known as isoforms. These different isoforms can generate proteins with distinct structures and functions, greatly increasing the diversity of molecules that cells can produce. To investigate this process, the researchers used Oxford Nanopore native RNA sequencing to analyze primary human monocytes before and after stimulation with lipopolysaccharide, or ...
Understanding alternative splicing with long read sequencing
Alternative splicing is a process that allows a single gene to produce multiple RNA transcripts, called isoforms. This greatly increases the diversity of proteins that cells can make, and it plays an important role in many biological processes. When alternative splicing is disrupted, it can contribute to diseases such as cancer and neurological disorders. Traditional sequencing methods, often referred to as short read sequencing, can measure RNA levels very accurately but struggle to capture full-length RNA molecules. This makes it difficult to determine which isoforms are present, especially when transcripts are complex. Long read sequencing technologies help solve this problem by reading entire RNA molecules in one continuous sequence, allowing researchers to directly observe different isoforms. In this work, led by researchers at the University of Melbourne, researchers describe how long read sequencing provides a clearer view of alternative splicing. By capturing full-length RNA sequences, scientists can better understand ...
A functionally integrated cross-tissue alternative splicing program during short-term calorie restriction
Calorie restriction, reducing calorie intake without causing malnutrition, has long been studied for its effects on metabolism, aging, and overall health. Scientists have observed that lowering calorie intake can influence many biological pathways, but the underlying molecular mechanisms are still being explored. In new research led by researchers at the University of Aberdeen, investigators examined how calorie restriction affects a process called alternative splicing. Alternative splicing is a natural process that allows a single gene to produce multiple versions of RNA and protein. By rearranging how segments of RNA are joined together, cells can create different protein variants from the same gene. This process helps cells adapt to changing conditions and regulate complex biological systems. The researchers investigated how short term calorie restriction influences alternative splicing across multiple tissues in the body. Rather than affecting a single organ, the results showed that splicing patterns change in a coordinated way ...
Longcell – single cell and spatial alternative splicing analysis with Nanopore long read sequencing
A team led by researchers at the Perelman School of Medicine, University of Pennsylvania, have developed a powerful new tool called Longcell to improve the analysis of alternative splicing using Nanopore RNA sequencing. Alternative splicing is a biological process that allows a single gene to produce multiple RNA variants or “isoforms,” which can have different functions in the cell. Understanding how these isoforms vary between individual cells or across different regions of tissue is important for learning how genes are regulated in health and disease. Long-read RNA sequencing, such as that offered by Nanopore technology, is especially useful because it reads entire RNA molecules from start to finish. This allows scientists to see the full sequence of each isoform, rather than piecing together short fragments. However, long-read sequencing comes with challenges. Nanopore reads tend to have higher error rates compared to short-read methods, making it difficult to correctly identify ...
RNA sequencing reveals novel antigens from alternative splicing for cancer immunotherapy
UCSF scientists find a trove of new targets for cancer immunotherapy in cancer’s unique version of RNA splicing. Immune therapy has transformed how cancer is treated, but many tumors continue to evade these treatments, thanks to their resemblance to healthy tissue. Now, researchers at UC San Francisco have found that some cancers, like brain cancer (glioma), make unique, jumbled proteins that make them stand out. These newly recognized cancer-specific proteins, or antigens, could speed the development of potent immunotherapies that recognize and attack hard-to-treat tumors. The study, which was supported through grants from the National Institutes of Health, appears in Nature on Feb. 19. The newly discovered antigens were the product of mistakes in RNA splicing, which controls how RNA molecules – the blueprints for proteins – are pieced together from smaller parts. The study found that in cancers of the brain, prostate, liver and colon, among others, the tumors spliced ...
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