Periodic Reporting for period 1 - TRANS-3 (Beyond the chromosome: unravelling the interplay between inter-chromosomal genome architecture and mRNA biogenesis)
Reporting period: 2023-09-01 to 2026-02-28
Inside the cell nucleus, the human genome is folded into a complex three-dimensional landscape. We have detailed maps of chromosomes, genes and regulatory elements, yet we still know little about how distant genes located on different chromosomes communicate to coordinate their activity. As an analogy, our world map does not indicate most natural corridors or man-made infrastructure that connect countries to one another. Worse, we barely understand how such connections function.
Recent discoveries have revealed many non-coding RNAs that act as structural molecules, helping to organise the genome. TRANS-3 challenges the traditional divide between “coding” and “non-coding” RNAs, asking whether even messenger RNAs (mRNA)—normally viewed only as transient templates for protein production—might also play non-coding structural roles while still in the nucleus.
The project is based on the hypothesis that mRNA molecules can shape genome architecture by forming “mRNA factories”: transient, membraneless condensates where genes that need to work together cluster in physical proximity to coordinate mRNA transcription and processing (e.g. splicing). By combining stem-cell models, live imaging, functional genomics, and physiological assays, TRANS-3 explores how these RNA factories assemble, function, and influence cell behaviour.
To make this concept experimentally tractable, the project uses the cardiac protein RBM20 as a paradigm. This RNA-binding factor and its target mRNAs form a factory that controls the splicing of key genes for heart contraction. Mutations in RBM20 cause severe forms of inherited cardiomyopathy, providing a disease-relevant model to study how disruption of nuclear organisation can lead to pathology. The project then examines whether similar mechanisms are pervasive for other mRNA factories, assessing how general the RBM20 paradigm may be across cell types and conditions.
The broader vision of TRANS-3 is to provide a new framework linking mRNA biology, nuclear structure, and human disease. Many conditions involve mutations in DNA- or RNA-binding proteins that may act as factory organisers; the same mechanism could also explain how genetic variants can unexpectedly influence the activity of genes located on other chromosomes—phenomena known as long-distance or trans-acting effects, which remain largely mysterious. By uncovering these hidden genomic “corridors,” TRANS-3 seeks to illuminate a fundamental yet unexplored layer of cellular regulation.
Recent discoveries have revealed many non-coding RNAs that act as structural molecules, helping to organise the genome. TRANS-3 challenges the traditional divide between “coding” and “non-coding” RNAs, asking whether even messenger RNAs (mRNA)—normally viewed only as transient templates for protein production—might also play non-coding structural roles while still in the nucleus.
The project is based on the hypothesis that mRNA molecules can shape genome architecture by forming “mRNA factories”: transient, membraneless condensates where genes that need to work together cluster in physical proximity to coordinate mRNA transcription and processing (e.g. splicing). By combining stem-cell models, live imaging, functional genomics, and physiological assays, TRANS-3 explores how these RNA factories assemble, function, and influence cell behaviour.
To make this concept experimentally tractable, the project uses the cardiac protein RBM20 as a paradigm. This RNA-binding factor and its target mRNAs form a factory that controls the splicing of key genes for heart contraction. Mutations in RBM20 cause severe forms of inherited cardiomyopathy, providing a disease-relevant model to study how disruption of nuclear organisation can lead to pathology. The project then examines whether similar mechanisms are pervasive for other mRNA factories, assessing how general the RBM20 paradigm may be across cell types and conditions.
The broader vision of TRANS-3 is to provide a new framework linking mRNA biology, nuclear structure, and human disease. Many conditions involve mutations in DNA- or RNA-binding proteins that may act as factory organisers; the same mechanism could also explain how genetic variants can unexpectedly influence the activity of genes located on other chromosomes—phenomena known as long-distance or trans-acting effects, which remain largely mysterious. By uncovering these hidden genomic “corridors,” TRANS-3 seeks to illuminate a fundamental yet unexplored layer of cellular regulation.
During the first phase of TRANS-3, the project’s diverse and interdisciplinary team produced compelling preliminary evidence that mRNAs can act as structural molecules helping to organise genome architecture. Using human stem cells differentiated into beating heart cells, the team visualised for the first time how RBM20 clusters behave in living nuclei, providing a biophysical model that can explain how they arise and are maintianed.
To explore the biological relevance of these findings, researchers engineered a collection of otherwise identical stem-cell models carrying disease-associated mutations in genes thought to regulate the RBM20 mRNA factory. Initial analyses in two-dimensional cultures supported the idea that the mRNA of the RBM20 target TTN initiates an mRNA factory connecting loci on other chromosomes, thereby promoting their RBM20-dependent splicing.
On the computational side, the team created a new network-based analytical tool to identify clusters of genes that interact across different chromosomes, and a complementary machine-learning algorithm that recognises DNA sequence features favouring such long-range associations. These analyses revealed that nearly half of transcription factors and about ten percent of RNA-binding proteins may participate in inter-chromosomal networks, suggesting that mRNA factories could represent a general organising principle rather than an exception.
In parallel, the project developed a suite of experimental technologies that make mRNA-factory biology directly testable in human cells. These include a biochemical method to catalogue the molecules bound to a specific mRNA, which was applied to dissect the TTN mRNA factory, uncovering unexpected additional players besides RBM20. Additional methods use single-cell genomics to modify gene expression at different stages of gene regulation: one modulates mRNA expression, the other impairs mRNA translation into protein. Together, these approaches allow researchers to separate the proximal-acting roles of specific mRNAs from the distant-acting effects of their protein partners, providing a versatile framework to probe cause-and-effect relationships in nuclear organisation.
The project has also established weekend-free home-mixed culture media to support scalable production of heart cells for studies using engineered tissues, and improved methods to control gene expression in heart cells to enable mechanistic studies in mRNA-factory biogenesis.
To explore the biological relevance of these findings, researchers engineered a collection of otherwise identical stem-cell models carrying disease-associated mutations in genes thought to regulate the RBM20 mRNA factory. Initial analyses in two-dimensional cultures supported the idea that the mRNA of the RBM20 target TTN initiates an mRNA factory connecting loci on other chromosomes, thereby promoting their RBM20-dependent splicing.
On the computational side, the team created a new network-based analytical tool to identify clusters of genes that interact across different chromosomes, and a complementary machine-learning algorithm that recognises DNA sequence features favouring such long-range associations. These analyses revealed that nearly half of transcription factors and about ten percent of RNA-binding proteins may participate in inter-chromosomal networks, suggesting that mRNA factories could represent a general organising principle rather than an exception.
In parallel, the project developed a suite of experimental technologies that make mRNA-factory biology directly testable in human cells. These include a biochemical method to catalogue the molecules bound to a specific mRNA, which was applied to dissect the TTN mRNA factory, uncovering unexpected additional players besides RBM20. Additional methods use single-cell genomics to modify gene expression at different stages of gene regulation: one modulates mRNA expression, the other impairs mRNA translation into protein. Together, these approaches allow researchers to separate the proximal-acting roles of specific mRNAs from the distant-acting effects of their protein partners, providing a versatile framework to probe cause-and-effect relationships in nuclear organisation.
The project has also established weekend-free home-mixed culture media to support scalable production of heart cells for studies using engineered tissues, and improved methods to control gene expression in heart cells to enable mechanistic studies in mRNA-factory biogenesis.
TRANS-3 is establishing a new conceptual bridge between genome organisation and RNA biology by showing that mRNAs can act as structural elements shaping the 3D architecture of the nucleus. This view challenges decades of dogma separating coding from non-coding RNAs, proposing instead that coding transcripts themselves may scaffold long-range gene networks while being processed in the nucleus.
Building on the example of RBM20 in cardiac cells, the project is now extending its analyses to identify additional mRNA factories. The combination of network-based genomics, biochemistry, and perturbation technologies enables systematic mapping of how mRNAs and their protein partners coordinate distant genes. These resources are being openly shared to accelerate discovery while, in parallel, intellectual property protection is being pursued for methods with translational potential, ensuring future exploitation opportunities.
The implications reach far beyond basic science. Many diseases are caused by mutations in RNA- or DNA-binding proteins that could act as mRNA-factory organisers — including cardiomyopathies, cancer, spinal muscular atrophy, amyotrophic lateral sclerosis, and retinitis pigmentosa. Understanding how these factors influence nuclear architecture may also clarify why certain mutations or non-coding variants affect genes located on other chromosomes, helping interpret genome-wide association and expression studies (known in technical jargon as GWAS and trans eQTLs) that remain mechanistically unclear.
TRANS-3 also provides tools with potential diagnostic and therapeutic value. The ability to isolate and perturb specific mRNA factories creates opportunities to identify biomarkers of disrupted nuclear organisation, while the project’s gene-control and single-cell platforms could facilitate targeted antisense oligonucleotide (ASO)-based therapies to correct misregulated transcripts.
Ongoing work aims to visualise in real time how RBM20 factories are formed and if they coordinate DNA folding and mRNA processing, test their physiological relevance in engineered human heart tissues, and explore their broader prevalence beyond RBM20. By coupling open science with strategic valorisation, TRANS-3 is paving the way from frontier molecular biology to potential biomedical applications, positioning its discoveries at the interface of basic research, precision diagnostics, and RNA-targeted therapeutics.
Building on the example of RBM20 in cardiac cells, the project is now extending its analyses to identify additional mRNA factories. The combination of network-based genomics, biochemistry, and perturbation technologies enables systematic mapping of how mRNAs and their protein partners coordinate distant genes. These resources are being openly shared to accelerate discovery while, in parallel, intellectual property protection is being pursued for methods with translational potential, ensuring future exploitation opportunities.
The implications reach far beyond basic science. Many diseases are caused by mutations in RNA- or DNA-binding proteins that could act as mRNA-factory organisers — including cardiomyopathies, cancer, spinal muscular atrophy, amyotrophic lateral sclerosis, and retinitis pigmentosa. Understanding how these factors influence nuclear architecture may also clarify why certain mutations or non-coding variants affect genes located on other chromosomes, helping interpret genome-wide association and expression studies (known in technical jargon as GWAS and trans eQTLs) that remain mechanistically unclear.
TRANS-3 also provides tools with potential diagnostic and therapeutic value. The ability to isolate and perturb specific mRNA factories creates opportunities to identify biomarkers of disrupted nuclear organisation, while the project’s gene-control and single-cell platforms could facilitate targeted antisense oligonucleotide (ASO)-based therapies to correct misregulated transcripts.
Ongoing work aims to visualise in real time how RBM20 factories are formed and if they coordinate DNA folding and mRNA processing, test their physiological relevance in engineered human heart tissues, and explore their broader prevalence beyond RBM20. By coupling open science with strategic valorisation, TRANS-3 is paving the way from frontier molecular biology to potential biomedical applications, positioning its discoveries at the interface of basic research, precision diagnostics, and RNA-targeted therapeutics.