Imagine you’re in a movie editing studio, trying to fix a film with some corrupted scenes. The film is vital, as it plays a crucial role in the development and function of an entire movie franchise. Suddenly, you find out that the corrupted scenes are scattered throughout the film, making it challenging to fix. Now, imagine that instead of fixing each corrupted scene individually, you could replace them all at once with perfect scenes from another source. This is the basic idea behind the RNA trans-splicing approach, which has been used in the paper we are explaining in this article, to correct mutations associated with a rare genetic disorder known as CTNNB1 syndrome.
CTNNB1 syndrome is caused by mutations in the CTNNB1 gene, which makes a protein called beta-catenin, a protein that is essential for brain development and function. These mutations can lead to difficulties with movement, learning, and behavior. Like corrupted scenes scattered throughout a film, CTNNB1 mutations are diverse and spread across the gene, resulting in the need for a universal and effective treatment.
In this paper, the researchers from the Kemijski Inštitut and CTGCT, in collaboration with other researchers, have explored an approach to treat CTNNB1 syndrome using a technique called RNA trans-splicing. This approach aims to fix the mutations associated with CTNNB1 syndrome by replacing the mutated parts with healthy ones at the RNA level, much like replacing corrupted scenes with perfect ones, ensuring that the “final film” plays smoothly without any unexpected glitches.
Key Words/Concepts to Understand
Before diving into the details of the study, let’s understand some key terms and concepts:
Gene: A segment of DNA that contains the instructions for making a protein.
Mutation: A change in the DNA sequence of a gene, which can be harmful, harmless, or even beneficial.
RNA: A molecule similar to DNA that helps carry out the instructions in a gene. When a gene is transcribed, its DNA code is copied into an RNA molecule called transcript. This RNA transcript is translated into proteins.
Spliceosome complex: The spliceosome is made up of several smaller components called small nuclear ribonucleoproteins, or snRNPs (pronounced “snurps”). Each snRNP consists of a small nuclear RNA (snRNA) molecule and several proteins. Together, these pieces act like an editing machine that cuts out the unneeded parts of RNA and joins the useful parts.
Ribozymes: RNA molecules that act like enzymes, most often cutting or joining RNA strands.
Antisense RNA: Are strands of RNA that are complementary to a specific messenger RNA (mRNA). When they bind to that mRNA, it can block protein production by preventing translation or marking that mRNA for degradation.
RNA Splicing and Trans-splicing: The Basics
To understand the innovative approach taken by the researchers, it’s important to grasp the concepts of splicing and trans-splicing.
RNA Splicing
RNA splicing is a crucial step in the process of gene expression. When a gene is transcribed into RNA, the initial RNA transcript, called pre-mRNA, contains exons (coding regions) that will be used to make final protein, and introns (non-coding regions) that need to be removed. The process of editing this pre-mRNA by removing the introns and joining the exons together into final mRNA is called conventional RNA splicing. This editing process is carried out by a complex called the spliceosome.
To help visualize this process, imagine you’re editing a movie. You have all the raw footage (like the pre-mRNA), which includes both the good scenes you want to keep (exons) and the unnecessary footage you want to cut out (introns). To create the final movie (the final mRNA that makes protein), you need an editor to cut out the unnecessary parts and join the good scenes together. In the world of cells, this editor is called the spliceosome. It’s a molecular machine inside the cell’s nucleus that edits RNA molecules to produce mature mRNA, which can then be used to make proteins. What makes this process even more remarkable is the ability of spliceosome to assemble exons in different combinations through a process called alternative RNA splicing. This allows a single gene to produce multiple protein variants, much like a movie that can have different endings depending on which scenes are included. This flexibility in RNA splicing is essential for the diversity and complexity of proteins in our bodies, enabling cells to perform a wide range of functions and adapt to different needs.
Trans-splicing
Continuing with our movie editing metaphor, now imagine you are a movie editor working on two different films. One film has some corrupted scenes (mutated mRNA), while the other film has perfect scenes (healthy mRNA). In normal editing (conventional RNA splicing), you only work within a single film, cutting out unnecessary footage and joining the good scenes together. But what if you could take a perfect scene from the second film and use it to replace a corrupted scene in the first film? This is the basic idea behind RNA trans-splicing.
In the world of cells, RNA trans-splicing is a special type of splicing where different RNA molecules are joined together. Unlike conventional RNA cis-splicing, which edits a single RNA molecule by removing non-coding regions and joining coding ones, RNA trans-splicing allows segments from separate RNA molecules to be combined into a single functional mRNA. Although this process is rare in nature, scientists can engineer it as a promising tool to potentially treat genetic disorders like CTNNB1 syndrome.
In the context of this study, RNA trans-splicing is used to replace the mutated parts of the CTNNB1 transcript with healthy ones. Here’s how it works:
- Design of PTM: Researchers design a pre-trans-splicing molecule (PTM) that carries a healthy version of the mutated part of the CTNNB1 transcript, along with binding sequences that can bind to the target RNA.
- Delivery of PTM: The PTM is introduced into the cells, where it can bind to the mutated CTNNB1 transcript.
- Trans-splicing: The spliceosome machinery recognizes the PTM and the naturally occurring CTNNB1 transcript, and joins them together, replacing the mutated part with the healthy part from the PTM.
- Protein Production: The resulting chimeric RNA (a mix of both RNAs, just like chimeras were a mix of animals) is then translated into a functional beta-catenin protein.
Why RNA trans-splicing is exciting for RNA-based therapy
One fix for many problems: Instead of needing a separate solution for every single mutation, RNA trans-splicing can swap out an entire section of RNA at once, allowing correction of many different mutations within the same gene. This means one treatment can work for a wide range of CTNNB1 mutations, making it a universal therapy option.
Keeps the cell in balance: Because the editing happens at the RNA level, RNA trans-splicing preserves the genes natural regulation. This is important for certain genes that need just the right amount of protein, as too much or too little can cause problems.
Works where other methods struggle: Some genes are too large for traditional gene therapy methods, which have limited space for delivering genetic material. RNA trans-splicing overcomes this by allowing the correction of both small and large genetic defects.
What’s New and Why It Matters?
The researchers designed and tested different PTMs targeting various parts of the CTNNB1 transcript. To measure trans-splicing activity, they screened various PTM molecules using a split Yellow Fluorescent Protein (YFP) reporter system. They split the fluorescent YFP protein into two parts: one part is included in a target intron reporter designed to to look similar to the target pre-mRNAfound naturally in the cells, while the other part is included in the pre-trans-splicing molecule (PTM), designed to bind to the target intron reporter and induce trans-splicing. When trans-splicing occurs, the two YFP parts join together, producing a fluorescent signal that can be detected and quantified by flow cytometry.
In the paper they explore different modifications to improve trans-splicing efficiency like: promoter selection, the addition of small antisense RNA molecules to block conventional cis-splicing, and ribozymes to increase nuclear retention of the PTM significantly enhanced trans-splicing efficiency.
Matea and the team then tested the most effective PTMs in cells that naturally express the CTNNB1 gene and confirmed successful trans-splicing of the endogenous transcript at the mRNA level. This represents the first demonstration of induced trans-splicing in the CTNNB1 transcript, highlighting its potential as a future therapeutic tool for CTNNB1 syndrome.
This research is important because it could be potential to treatment for CTNNB1 syndrome. By repairing the mutations at the RNA level while maintaining the natural regulation of the gene, this approach avoids the risk of overexpression, which can be harmful in the case of CTNNB1 gene. Moreover, this technique could potentially be applied to treat other genetic disorders.
In conclusion, the study by Matea Maruna and colleagues presents a promising method to treat CTNNB1 syndrome using RNA trans-splicing. They enhance RNA trans-splicing efficiency by promoter selection, incorporating ribozymes, and short antisense RNAs, demonstrating its potential as a therapy for CTNNB1 syndrome. As research in this area progresses, RNA trans-splicing could become an important tool for treating genetic disorders such as CTNNB1 syndrome.
Reference
Maruna, M., Sušjan-Leite, P., Meško, M., Miroševič, Š., & Jerala, R. (2025). RNA Trans-splicing to Rescue β\betaβ-Catenin: A Novel Approach for Treating CTNNB1-Haploinsufficiency Disorder. Molecular Therapy – Nucleic Acids. https://doi.org/10.1016/j.omtn.2025.102680
Written by Rodrigo Ortiz in collaboration with Matea Maruna first author of the paper.