Chemical modifications occur on many types of biological molecules, including nucleic acids, proteins, sugars, and lipids. These modifications typically function as molecular switches. For example, many kinases are activated by phosphorylation and gene expression is silenced when its DNA sequence is methylated. More than 150 RNA modifications have been identified to date; however, the function of most RNA modifications remains elusive. Pioneering work by Drs. Katalin Karikó and Drew Weissman showed that mRNAs require modifications for robust translation of their encoded proteins and to prevent triggering of the innate immune responses when they are transfected into human cells. Karikó and Weissman’s findings paved the way for the creation of the COVID19 mRNA vaccines and they were awarded the Nobel Prize for Physiology or Medicine this year (2023).
Pseudouridylation was one of the first RNA modifications discovered and is one of the most abundant modifications found in many types of RNA molecules. Uridine residues within RNAs are converted to pseudouridine (Ψ) by pseudouridine synthase (PUS) enzymes (see figure). Specific PUS enzymes are known to target specific RNA sequences or structural elements. The function of Ψ has been mostly studied in transfer RNAs (tRNAs), which are the adaptor molecules that bring a specific amino acid to the ribosome for protein translation. In tRNAs, Ψ stabilizes critical RNA structures required for binding to the ribosome and anticodon-codon pairing during translation. These tRNA structures could not be achieved by the standard uracil base.
Ψ has more recently been shown to be abundant in protein-coding messenger RNAs (mRNAs). Little is known about the function of Ψ in mRNAs, the importance of the location of Ψ residues, and which PUS enzymes pseudouridylate mRNAs. Recent studies have pointed to PUS1, PUS4, and PUS7 as the major PUS enzymes to modify mRNAs. But how exactly are mRNAs identified for pseudouridylation by these PUS enzymes that were thought to recognize tRNA-specific sequences?
A recent paper in PLoS One from the Stoddard lab sheds light on this question. “We started working on this topic as a result of our own appreciation for the incredible diversity and number of PUS enzymes found across all kingdoms of life and via biochemical studies being conducted by our collaborators at New England Biolabs, led by Sebastian Grünberg, a former trainee in the Hahn lab here in the Hutch’s Basic Sciences Division,” said Dr. Barry Stoddard. Scientists at NEB had identified a “hot spot” within the firefly luciferase mRNA that is highly pseudouridylated by PUS1. They found that many of the uridines which subsequently get modified are found at the bottom of a stem-loop structure that resembles the known optimal PUS1 target. To better understand how PUS1 recognizes these sequences, Lindsey Doyle, a research technician in the Stoddard lab, co-crystallized PUS1 with a short RNA derived from the luciferase mRNA. The crystal structure confirmed that PUS1 requires a double stranded RNA stem for target recognition and revealed the PUS1 residues required for PUS1 to pseudouridylate its target RNAs.
Looking to the future, “the structure that our laboratory solved leaves open a number of interesting questions,” said Dr Stoddard. It remains to be determined whether the underlying base pair sequence within a given RNA stem loop is important for pseudouridylation. Is any such structure, regardless of sequence, sufficient for recognition by PUS1? “The broader question in the field remains what exactly is the biological role and importance of pseudouridylation is within the context of mRNAs.” Ψs are not randomly distributed within mRNAs and are enriched in coding regions and 3’ UTRs, suggesting a functional role. However, that function remains elusive for now.
The spotlighted research was funded by the National Institutes of Health.
Fred Hutch/University of Washington/Seattle Children's Cancer Consortium member Barry L. Stoddard contributed to this work.
Grünberg S, Doyle LA, Wolf EJ, Dai N, Corrêa IR Jr, Yigit E, Stoddard BL. 2023 The structural basis of mRNA recognition and binding by yeast pseudouridine synthase PUS1. PLoS One. 18(11):e0291267.