Sophia Zhao

A research lab at Yale led by Wendy Gilbert, associate professor of molecular biophysics and biochemistry, has published two papers illustrating crucial insights into the properties and behaviors of mRNA. 

In the first paper, published in the Jan. 17 issue of the Cell Systems journal, researchers focused on different mRNA sequences’ affinities for ribosomes, the macromolecules that synthesize proteins from mRNA strands. Once the mRNA is in the cytoplasm of a cell, a ribosome attaches to the mRNA molecule and synthesizes a protein based on the genetic sequence contained in the mRNA. The second paper, published in Molecular Cell on Jan. 19, describes the effects of pseudouridine synthases, which are amino-acid altering enzymes, on mRNAs. Once this work was published, Pfizer granted $1.7 million to the Gilbert lab to continue investigating mRNA properties and their implications in developing therapeutics.

“We’re still discovering new ways by which RNA and messenger RNA can be changed in order to change final protein production and cellular functions,” said Nicole Martinez, a former postdoctoral researcher in the Gilbert lab who led one of the studies and is now an assistant professor at Stanford. “I think it’s really exciting that we’re still finding new fundamental mechanisms of controlling gene expression in normal biology.”

However, certain mRNA sequences are better than others when it comes to recruiting ribosomes for protein synthesis, according to Gilbert. She attributes this variation to the cell’s need to maintain different levels of protein. Proteins that are necessary for the cell’s functions are encoded by mRNAs that effectively recruit ribosomes while proteins that are needed in small amounts are encoded by mRNAs that rarely recruit ribosomes, causing them to have decreased affinity for ribosomal recruitment.

“One idea in the field about why you might make RNAs that are capable of recruiting ribosomes, but at a low level, is to smooth out protein production.” Gilbert said. “If you just want to make a little bit of the protein, you can’t make fewer than one molecule of mRNA. You start and you make it or you don’t make it. If you want to make a steady low level, then frequently transcribing the DNA to make an mRNA that will slowly recruit just a few ribosomes is maybe a good way to do that.”

Rachel Niederer, a K99 postdoctoral fellow in the Gilbert lab, led the team for this paper. Her work focused on the 5’ untranslated region, or 5’ UTR, an area of the mRNA that serves as a “landing pad” for translation initiation. This is the point in the process where it is determined which specific mRNAs will be translated.

As a rate-limiting step, adjustments to translation initiation can result in an immense impact on the quantities of protein ultimately produced. Even changes to a single subunit can substantially influence protein output. The team developed a technique called direct analysis of ribosome targeting, or DART, in order to examine the relationship between the 5’ UTR and translation initiation.

According to Gilbert, DART employs a scoring system that can quantify how well the 5’ UTRs on various mRNAs recruit ribosomes and initiate translation.

“We could measure translation initiation scores on thousands of RNAs in parallel,” Niederer said. “So that’s been a really powerful approach. We can get these initiation scores both on endogenous mRNAs that the cell uses naturally, but we can also design in extra features to test hypotheses that we have about elements where we maybe already have an expectation about what they might do. It’s a really nice combination about the ability to discover new things that we hadn’t seen previously but also to test ideas that we have about old things.”

According to Martinez, RNA has chemical modifications, including ones to the canonical four nucleotides that make up the genetic alphabet. Gilbert’s lab studied a particular modification called pseudouridine, which is a chemical modification to a smaller subunit of RNA called uridine that is added by pseudouridine synthase.

Gilbert added that pseudouridine synthases are biochemically simple proteins and, therefore, their different biological functions are attributed to their specific targets. More specifically, each type of pseudouridine has a target among the different types of RNA. 

“At a biochemical level, pseudouridine affects some of the fundamental properties of RNA,” Gilbert said. “In particular, changing a uridine to a pseudouridine stabilizes RNA duplexes — base pairing between two strands of RNA — and for this reason, it makes sense that pseudouridine is very prevalent in structured RNAs in a cell.”

According to Martinez, it was essential to determine the point at which pseudouridine becomes prevalent in messenger RNA in order to understand pseudouridine’s function.

To do so, the team purified mRNA molecules that are attached to the DNA template, called pre-mRNA. Then, they searched the purified pre-mRNA for pseudouridine using its chemical properties and sequencing techniques. Finally, they were able to produce a map of pseudouridine in the purified mRNA. The team discovered that pseudouridine emerges very early in the production of mRNA and plays a significant role in splicing together the information that results in proteins.

“It influences the very earliest steps of gene expression and mRNA production, and the enzymes that put these decorations on the pre-mRNA are implicated with a number of diseases, including neurodevelopmental disorders,” Martinez said. “So now we really understand that this modification is installed in this class of RNA targets. Understanding where they are and what those modifications do is really important to understand how these enzymes are regulated in disease and why the process matters.”

Understanding more about mRNA could lay the groundwork for the development of new mRNA-based techniques for therapies and vaccines in the future. It could also lead to a greater understanding of how the dysregulation of RNA processing could affect diseases.

Gilbert’s lab moved from MIT to Yale in 2017.