Researchers at the Sylvester Comprehensive Cancer Center have developed and applied a nascent RNA sequencing technique that captures real-time transcriptional activity using far fewer cells than traditional methods. Called Rapid Precision Run-On Sequencing (rPRO-seq), the approach addresses long-standing limitations in existing nascent RNA profiling methods, which typically require large cell numbers and extended processing times.
The method, published in Molecular Cell across two companion papers, reduces sample input to just 5,000 cells and delivers results in under 12 hours. In contrast, existing technologies often require millions of cells and several days of processing.
“Standard RNA sequencing looks at 'steady-state' RNA—the accumulation of what's been made,” said Pradeep Kumar Reddy Cingaram. “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.”
In the first study, the team used rPRO-seq to study the Integrator complex, specifically its catalytic subunit INTS11, which had not been traceable by prior nascent RNA techniques. Removing INTS11 in neuronal differentiation models caused widespread dysregulation of genes linked to neurodevelopmental and psychiatric disorders.
The second study extended these findings to early embryonic development. The researchers showed that INTS11 is involved in regulating pluripotency by day two, placing the Integrator complex at the earliest steps of transcriptional initiation. “The work is paradigm shifting,” said Ramin Shiekhattar, senior author on both studies, “as it places Integrator complex at the earliest steps in transcriptional cycle, known as ‘initiation,’ compelling a revision of current theories for transcriptional initiation.”
Given its speed and low sample requirement, the team suggests rPRO-seq could also be used clinically, such as analyzing tumor responses to therapy in near real time.
Publication Details
Cingaram PKR, Beckedorff F, Yue J, et al. Enhancing transcriptome mapping with rapid PRO-seq profiling of nascent RNA. Mol Cell. 2025;85(15):2988-3001.e4. doi:10.1016/j.molcel.2025.06.029
Edupuganti RR, Beckedorff F, Cingaram PKR, et al. Integrator promotes the association of TFIID and RNA polymerase II to maintain pluripotency during development. Mol Cell. 2025;85(15):2937-2955.e10. doi:10.1016/j.molcel.2025.07.006
The method, published in Molecular Cell across two companion papers, reduces sample input to just 5,000 cells and delivers results in under 12 hours. In contrast, existing technologies often require millions of cells and several days of processing.
“Standard RNA sequencing looks at 'steady-state' RNA—the accumulation of what's been made,” said Pradeep Kumar Reddy Cingaram. “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.”
In the first study, the team used rPRO-seq to study the Integrator complex, specifically its catalytic subunit INTS11, which had not been traceable by prior nascent RNA techniques. Removing INTS11 in neuronal differentiation models caused widespread dysregulation of genes linked to neurodevelopmental and psychiatric disorders.
The second study extended these findings to early embryonic development. The researchers showed that INTS11 is involved in regulating pluripotency by day two, placing the Integrator complex at the earliest steps of transcriptional initiation. “The work is paradigm shifting,” said Ramin Shiekhattar, senior author on both studies, “as it places Integrator complex at the earliest steps in transcriptional cycle, known as ‘initiation,’ compelling a revision of current theories for transcriptional initiation.”
Given its speed and low sample requirement, the team suggests rPRO-seq could also be used clinically, such as analyzing tumor responses to therapy in near real time.
Publication Details
Cingaram PKR, Beckedorff F, Yue J, et al. Enhancing transcriptome mapping with rapid PRO-seq profiling of nascent RNA. Mol Cell. 2025;85(15):2988-3001.e4. doi:10.1016/j.molcel.2025.06.029
Edupuganti RR, Beckedorff F, Cingaram PKR, et al. Integrator promotes the association of TFIID and RNA polymerase II to maintain pluripotency during development. Mol Cell. 2025;85(15):2937-2955.e10. doi:10.1016/j.molcel.2025.07.006