During the COVID-19 pandemic, the researchers in Associate Professor of Biological Sciences Joel McManus's lab were finding new ways to further their work when they couldn't come into the lab due to social distancing restrictions.

With the lab closed, they connected by Zoom, where they discussed published reports from other research groups related to the lab's work studying how mRNA produces proteins for gene expression. One paper they discussed involved an unusual RNA that was thought to control protein expression through an exotic mechanism. The RNA supposedly folded into a structure that drove the expression of a developmentally critical gene called Hoxa9. But McManus's team noticed that the data and the conclusions just didn't seem to match. The structure could be mutated without affecting protein expression.

McManus Lab Research Associate Gemma May then went to a genome browser to compare the Hoxa9 gene annotation with its mRNA sequence. Genome annotations are like a book's index that can guide researchers to the area of a genome that is responsible for certain functions. She found the annotation didn't make sense. The part of the gene's mRNA that was supposed to contain the regulatory structure wasn't being made. This was particularly important, because Hoxa9 has important roles in development and childhood acute myeloid leukemia.

"The genome annotation says that 'this gene is located on a specific chromosome and starts in one place and ends in another,'" said McManus. "But Gemma noticed that the annotation was wrong, such that the gene was actually shorter than the public annotation. This meant that the part of the gene that was thought to control protein synthesis really just controlled RNA synthesis."

McManus said that his group didn't originally do much with their discovery, until several new reports emerged touting that over a hundred genes contained similar unusual regulatory sequences and, as a result, shared this exotic way of translating proteins. Biological Sciences graduate student Christina Akirtava then set out to check this larger set of genes for evidence of similar annotation errors using computational analyses. At the same time, May tested the regulatory sequences of these genes to see if they did in fact control RNA synthesis in laboratory cells. Together, they found compelling evidence that the published research had been misinterpreted.

"Mouse studies suggested that the gene was being produced by an unusual mechanism where ribosomes bind directly to the inside of the mRNA," said McManus. "And this was really exciting; it changed the way people thought about how mRNA are translated. But then my lab did some control experiments, and the findings weren't supported."

McManus' group found that Hoxa9 started at a much different position than was recorded in the genome annotation. Part of the gene that was purported to be part of the RNA wasn't — it was part of the DNA. Their findings, along with those of other labs working on the same problem, overturned at least five high-profile published studies.

"This doesn't typically happen in our field, where a new model is accepted, then proven to not be what it had claimed to be. We call it paradigm unshifting," said McManus. The result could have a significant impact on current research on Hoxa9 and efforts by pharmaceutical companies to use the gene to develop new treatments for leukemia. The work also brings into question the accuracy of genome annotations.

"There are big errors in the mouse genome annotation and in the human genome annotation," said McManus. "The main goal for us was to help other researchers know about these issues so that these problems don't reoccur in the future."