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Researchers at Sylvester Comprehensive Cancer Center, part of the University of Miami Miller School of Medicine, have documented their use of a new RNA sequencing technology to uncover molecular drivers of cellular differentiation that could lead to better regenerative therapies.

In addition to being used in the lab, the technique, Rapid Precision Run-On Sequencing (rPRO-seq), has the potential to help doctors understand patients' disease states and response to treatment in real time.

The findings appear in two published articles in Molecular Cell.

"We saw a major bottleneck in the field of nascent RNA profiling," said Pradeep Kumar Reddy Cingaram, Ph.D., an assistant scientist at Sylvester and first author on one of the papers.

"Existing methods, while powerful, are simply too slow and require large amounts of biological material. Imagine needing tens of millions of cells and several days just to get started—that immediately ruled out crucial research on rare cell types or precious patient biopsies," he said.

Testing rPRO-seq

In the , the team used rPRO-seq to study the role of a protein complex called Integrator in regulating gene expression, which was previously untraceable with nascent RNA sequencing.

"INTS11, the catalytic subunit of the Integrator complex, was a compelling choice for us because we already knew it was a key player in gene regulation," said Cingaram.

Using cellular reprogramming models to induce neuronal differentiation, they found that when INTS11 was removed from neuronal cells, gene activity tied to brain development changed dramatically. Genes that needed to be active in order to prevent neurodevelopmental and psychiatric disorders were deactivated when the scientists removed INTS11.

"rPRO-seq allowed us to pinpoint a critical role for the INTS11 protein as a regulator of genes involved in neurodevelopmental disorders in neuronal cells," said Ramin Shiekhattar, Ph.D., senior author on both of the studies, co-leader of the Cancer Epigenetics Program at Sylvester and chief of the Division for Cancer Genomics and Epigenomics and the Eugenia J. Dodson Chair in Cancer Research.

The technique only required 12 hours and 5,000 cells. Existing technologies needed several days and millions of cells. Additionally, the scientists emphasize that rPRO-seq allowed them to understand not just when genes were turned on and off, but how.

"That is, rPRO-seq allows mechanistic understanding of gene expression changes," said Shiekhattar.

"Standard RNA sequencing looks at 'steady-state' RNA—the accumulation of what's been made. It's like seeing how many cars are on the road. But rPRO-seq reveals 'nascent' RNA—what's being made right now. It's like watching cars leave the factory. This gives us crucial, real-time insights into active gene transcription," said Cingaram.

INTS11 in regenerative medicine

Next, the team used rPRO-seq the role that INTS11 plays in stem cells' ability to differentiate into any kind of cell in the body, a characteristic called pluripotency. They found that the protein began playing a critical role in gene regulation as early as day two of embryonic development.

"This greatly expands our understanding of how pluripotency and differentiation are harmonized at the molecular level," said Shiekhattar.

Using a lab model, they found that the Integrator complex binds and regulates critical genes for stem cell identity essential for maintaining pluripotency. Additionally, "the work is paradigm-shifting as it places Integrator complex at the earliest steps in the transcriptional cycle, known as 'initiation,' compelling a revision of current theories for transcriptional initiation," said Shiekhattar.

While both findings regarding the role of INTS11 in early development are major steps in understanding cellular differentiation, the scientists point out that the rPRO-seq could be used for many more research and clinical purposes, such as sampling a tumor to see how it's responding to therapy.

"We're keen to apply rPRO-seq to a wider array of human clinical samples. This would allow us to validate its utility as a diagnostic or prognostic tool and potentially uncover novel, unstable RNA biomarkers that are currently invisible to existing technologies," said Cingaram.

"Overall, rPRO-seq could emerge as a valuable tool for both research and clinical settings, expanding the scope of transcriptomic analyses and enabling more precise, individualized health care," added Shiekhattar.