Harvard scientists discover entirely new gene regulation system in mammals

For decades, scientists have understood that each of the roughly 20,000 genes in the human body carries the instructions for a single kind of protein. Now, researchers at Harvard Medical School have discovered that instructions from different genes - even those on different chromosomes - can combine to create chimeric mRNAs that produce previously unknown, functional proteins.

The findings, published Sept. 2 in Nature, reveal the existence of potentially thousands of new chimeric proteins and demonstrate that at least some of these play important roles throughout the body.

Nobody knows these exist. Medicine doesn't know they exist, the pharmaceutical industry doesn't know they exist. We've discovered an entirely new gene regulation system that could expand the known genome and proteome dramatically."

Ruaidhrí Jackson, senior author, assistant professor of immunology, Blavatnik Institute at HMS

While the researchers don't yet know how widespread the phenomenon is, chimeric proteins could be involved in a variety of systems and may contribute to disease processes that are poorly understood at the moment. This overlooked aspect of biology could offer new insights into challenging diseases and provide a new avenue for finding drug targets to treat them.

"There are suddenly many new possibilities for the kind of molecules and proteins that cells can create," said Harry Kane, co-first author and HMS and Gene Lay Institute research fellow in immunology in the Jackson Lab. "If it is possible to leverage chimeric RNAs for drug discovery and medicine, then this is very exciting."

A "dark genome" library

While some single-celled and invertebrate organisms, such as trypanosomes and nematodes, can combine genes for regulatory purposes, these systems don't seem to create proteins and have not been found in mammals. The best-known examples of chimeric mRNA in humans came from cancer-causing abnormalities where DNA breaks into pieces and some of the scattered genes fuse together.

RNA sequencing results occasionally suggested that chimeric mRNA could exist in healthy tissue, but traditional sequencing techniques had difficulty finding them and may have even created some artificially.

"This project was high risk, high reward from the very beginning," said co-first author Olivia Venezia, a Harvard Kenneth C. Griffin Graduate School of Arts and Sciences PhD student in immunology in the Jackson Lab. "We didn't know how many chimeric mRNAs we would find or if they would be biologically relevant."

Using a new technology called direct RNA sequencing, the researchers were able to compile a list of over 30,000 chimeric mRNAs that have been observed at least once in mammalian cells - the "dark genome" library, Jackson calls it. So far, they have been able to profile how almost 400 of these are regulated by inflammatory signals, including chimeric mRNAs conserved in both human and mouse immune cells.

The team found that healthy chromosomes can loop together in mouse cells as part of the immune response, bringing the normally distant genes into proximity. The newly adjacent genes transcribe a chimeric mRNA, which takes part of its sequence from each gene and produces a protein that is a hybrid of the two. 

"We thought we had a blueprint of every mRNA that is made in the body, and now we're saying that was just page one," Jackson said. "There are all these other combinations that can occur."

But the existence of chimeric mRNA doesn't necessarily mean that it creates functional proteins or plays a significant role in the body.

A functional chimera

To determine whether a chimeric mRNA could actually serve a biological purpose in mammals, Jackson and his team chose to investigate one they found in mice: a combination of the genes encoding GSDMD and TMEM106A. The protein created by GSDMD alone is responsible for opening up cell membranes during pyroptosis, a process in which immune cells burst open to summon a large inflammatory response.

First, the team confirmed that the chimeric protein, GSDMD-TMEM106A, occurred naturally in mice. Indeed, it was produced as part of the immune response in cells from two different strains of lab mice as well as a wild-derived strain.

Then the researchers developed a genetic tool to stop the production of the chimeric protein without interfering with the production and function of the standard GSDMD and TMEM106A proteins.

They found that mice without the chimeric protein had a slower immune response than normal mice. When the mice were infected with Salmonella, they were less able to fight off the bacteria.

"They can't control the bacterial infection," Jackson said. "Nearly 50 percent of the immune process has been reduced without our chimeric protein, even though the normal GSDMD is still present. GSDMD needs our GSDMD-TMEM106A to function fully."

The researchers also tested the mice with an endotoxin that causes sepsis - an extreme immune response that can be deadly. Seventy percent of mice without the chimeric protein survived and recovered from what would normally be a lethal dose, because their immune response was milder than normal.

The researchers worked with the biotechnology company Moderna to engineer an mRNA that would boost the production of GSDMD-TMEM106A in the mice. Mice with elevated levels of the chimera did not survive even a mild endotoxin dose.

However, mice that lacked the GSDMD gene and also had elevated levels of the chimera (courtesy of the engineered mRNA) had no response to the endotoxin at all. This demonstrated that while GSDMD-TMEM106A significantly speeds up the process of pyroptosis, it cannot open the cell membranes without GSDMD.

The findings confirmed that this chimeric protein is vital for the inflammatory response in mice.

"GSDMD-TMEM106A was initially discovered based on a single read in one of our samples. We were bracing ourselves for this to turn out to be nothing," Kane said. "It was very exciting when we found that GSDMD-TMEM106A was not only a real protein but also functional - it could modulate release of inflammatory molecules from cells."

Exploring the potential of chimeric RNA

There is still a lot to learn about chimeric mRNA. Jackson, Kane, Venezia and their colleagues are continuing to explore the molecular cues behind its formation - why the two partial gene sequences are always connected at the same point, why chimeric proteins take certain shapes, and what triggers the chromosomes to bring specific genes together.

But they are most excited about evaluating a variety of chimeric mRNAs for potential uses in medicine.

"We want to find out which ones are operative in currently incurable diseases," Jackson said. "We think we can find new players that have been completely overlooked by medicine, by pharma, by biomedical science in general."

The Jackson Lab is currently investigating several chimeric mRNAs that could be involved in cancer, inflammatory disease, and neurodegenerative disease. They have shared additional chimeric mRNA sequences with colleagues interested in a variety of other processes.

"It will take years of research and effort from many different scientists to figure out the best ways to study chimeric RNAs and characterize their biological relevance," Kane said. "If this pattern turns out to be widespread and lots of chimeric RNA molecules are doing interesting things in cells, this opens up a ton of possibilities for discovering new biology and finding new targets for drug discovery."