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Biochemical method offers view into earliest stages of RNA production

When RNA molecules are synthesized by cells—a critical process in the creation of proteins and other cellular functions—they typically undergo a series of "folding" events that determine their structure and the role they will play in expressing genetic information in living organisms.

Until recently, however, not much was known about these folding processes that occur very early in the life of RNA molecules.

But Yale researchers have now developed a method to map and measure the structure of RNA as it develops, an advance that may help scientists design more effective treatments for a host of diseases. Their are described in the journal Molecular Cell.

An RNA's structure determines how the molecule functions, including whether it acts as a tRNA (transfer RNA), an rRNA (ribosomal RNA), or an mRNA (messenger RNA). Each of these molecule types performs crucial roles involving the expression of genetic information, like determining how much protein is made from an mRNA. In some cases, scientists have found, mutations that affect folding contribute directly to genetic disease.

(The process is called folding because the chemical nature of RNA—comprised of four nucleotides A, C, G, and U—allows individual nucleotides in one part of the molecule to interact with other nucleotides within the same molecule.)

"Our new method lets us visualize the earliest structures that RNAs form. Until now, we only knew the mature structures," said Leonard Schärfen, a Ph.D. candidate in Yale's Molecular Biophysics and Biochemistry Department and lead author of the study. "Early structures might have separate functions that could be targeted by therapeutics that don't work on mature RNAs."

Karla Neugebauer, senior author of the study and the R Selden Rose Professor of Molecular Biophysics and Biochemistry in Yale's Faculty of Arts and Sciences and Yale School of Medicine, called the work a "technical tour de force by Leo" with the potential to regulate gene expression very early on in an RNA's life. Neugebauer is also a professor of cell biology and director of the Yale Center for RNA Science and Medicine.

The method developed by the researchers, which they call co-transcriptional structure tracking (CoSTseq), is able to detect the earliest RNA folding activity in living cells.

The method, they say, combines two separate biochemical principles.

"First, we very specifically isolate RNA molecules at a time during their synthesis," said Schärfen, who is part of Yale's Graduate School of Arts and Sciences. "Then we add a chemical that modifies the RNA and allows us to study the structure as the molecules grow longer over time."

Among their findings, the researchers discovered that RNA molecules begin immediately folding as soon as they emerge from the RNA polymerase, the protein complex that synthesizes them. This discovery underscores how important early folding is for healthy gene expression and can shed new light on diseases caused by misfolded RNA, they say.

They also found that in mRNA, early folding dictates the final folded shape, so molecular structure is set from the start. In rRNA, however, early shapes are temporary and rearrange before a molecule is set. A protein called Dbp7, a helicase enzyme, plays a crucial role in the rRNA remodeling process, helping a molecule fold into its final structure.

Other authors of the study are Matthew D. Simon, an associate professor of molecular biophysics and biochemistry and a member of the Institute of Biomolecular Design and Discovery, and Isaac Vock, a graduate student researcher in molecular biophysics and biochemistry.