Opposing forces in cells could hold clues to treating disease

UNIVERSITY PARK, Pa. — A newly revealed molecular tug-of-war may have implications for better understanding how a multitude of diseases and disorders — including cancers, neurodegenerative diseases and immune disorders — originate, as well as how to potentially treat them, according to researchers at Penn State. The team made the finding in a study of cellular messengers called mRNAs that carry the DNA blueprints needed to make proteins, the building blocks of life that keep cells functioning. After delivering the instructions, the messengers are cleared out by a protein complex called CCR4-NOT. The proteins in CCR4-NOT were thought to operate in harmony, but the researchers said that’s not the case: one protein destabilizes mRNA while the other steadies it.

The team made the revelation in human colorectal cancer cells with a tool to precisely and temporarily turn off specifically targeted proteins. By essentially removing one protein, called CNOT1, CCR4-NOT slowed mRNA removal, but eliminating another protein from the cell, CNOT4, increased the clean-up process. Details of the tool and their analyses are available online now ahead of publication in an upcoming issue of the Journal of Biological Chemistry.

“Traditionally, subunits are expected to work together toward a common function, but our results show that CNOT4 has unique roles beyond RNA degradation or catalysis,” said first author Shardul Kulkarni, assistant research professor of biochemistry and molecular biology at Penn State. “Our study shows that not all subunits of a ‘degradation’ complex act the same way — some can have distinct and even opposing roles. Understanding these opposing forces gives us a clearer picture of how cells maintain balanced gene expression and could point to new ways to intervene when that balance is lost.”

This balance is critical to gene regulation, which Kulkarni described as a dimmer dial that precisely increases or decreases light to control when, where and how much each gene is used, or how often instructions for specific proteins are carried out. Kulkarni said.

“The study of gene regulation is essential for understanding cellular differentiation, the progression from a single embryonic cell to a multicellular organism, and the mechanisms by which organisms adapt to environmental stimuli,” Kulkarni said, explaining that, in health, genes provide the blueprints for every biological component of an organism, including mRNA, but that doesn’t mean the process is precisely the same all the time. “Our finding provides more information about how the molecules involved in gene regulation balance or even challenge each other as cells respond to stress, nutrition, temperature and other environmental changes. When that regulatory system fails, it can lead to diseases such as cancer, developmental disorders or metabolic problems.”

Kulkarni works in the laboratory of Joseph C. Reese, distinguished professor of biochemistry and molecular biology and corresponding author on the study, in the Penn State Center for Eukaryotic Gene Regulation, where they focus on CCR4-NOT, the molecular machine that regulates multiple stages of the RNA lifecycle. This refers to the entirety of RNA’s existence in cells: a protein transcribes instructions from DNA to mRNA; the mRNA is processed and packaged so that it can move in the cell and provide the information needed to make a new protein; and, finally, the RNA is broken down. That final step, when the RNA is broken down and the message is erased, is primarily governed by CCR4-NOT.

CCR4-NOT was first discovered in yeast in the early 1990s and appears in almost all eukaryotic cells — the cells that make up all animals, plants, fungi and many other organisms. While much is known about the complex from yeast studies, Kulkarni said, comparatively less is understood about CCR4-NOT's role in human cells. To address this gap, the team developed an experimental system to uncover previously uncharacterized functions of CCR4-NOT in human cells.

Called the auxin-inducible degron (AID) system, the tool allows scientists to rapidly and reversibly “switch off” a specific protein inside a cell, Kulkarni explained. It works by introducing a tag to a protein of interest that tells the cell to destroy that it.  

“By being able to quickly destroy proteins of interest, the AID system allows precise control over protein levels in human cells, letting us observe what happens when a specific protein is temporarily removed,” Kulkarni said.

To test the AID system, the team focused on two proteins in CCR4-NOT in a commercially available cell line of human colorectal cancer cells called DLD-1. The first protein, CNOT1, serves as the central scaffolding of CCR4-NOT, while the second, CNOT4, is a less-understood protein involved in gene regulation.

With the AID system, the researchers could completely reduce either protein of choice within 60 minutes. Depleting CNOT1 altered thousands of transcripts — the single strands of RNA that are transcribed to mRNA — and slowed mRNA decay. Depleting CNOT4, on the other hand, did little to alter transcripts, but it promoted mRNA degradation.

“Understanding the intricacies of the opposing effects CNOT1 and CNOT4 have on mRNA stability has several implications,” Kulkarni said, explaining that such knowledge could help identify disease contexts in which either subunit is dysregulated, inform the development of biomarkers based on characteristic mRNA decay patterns and provide insights for therapeutic strategies that target mRNA stability and fine-tune gene regulation. “With our new system, we’ve opened the door to learning even more.”

Other co-authors affiliated with the Penn State Center for Eukaryotic Gene Regulation are Courtney Smith and Oluwasegun T. Akinniyi, both graduate students in the biochemistry, microbiology and molecular biology program. Smith; Akinniyi; Belinda M. Giardine, programmer/analyst; and Cheryl A. Keller, research professor, are affiliated with the Penn State Department of Biochemistry and Molecular Biology. Keller is also the director of the Penn State Genomics Core Facility in the Huck Institutes of the Life Sciences at Penn State. Alexei Arnaoutov, Division of Molecular and Cellular Biology in the National Institutes of Health’s (NIH) National Institute of Child Health and Human Development, also contributed to this work.

This research was made possible with several core facilities managed by the Huck Institutes of the Life Sciences at Penn State, including the Huck Proteomics and Mass Spectrometry Core Facility, the Genomics Core Facility, the Flow Cytometry Core Facility and the Genomics Research Incubator.

The NIH funded this research.