Researchers at the University of Chicago have developed a technology for identifying new cellular RNA targets of small nucleolar RNAs (snoRNAs), a common but overlooked group of guide RNA molecules that steer chemical modifications to cellular ribosomal RNA (rRNA) targets. The team’s work uncovered thousands of previously unknown targets for snoRNAs in human cells and mouse brain tissues, including many that serve functions other than guiding rRNA modifications. Some of the newly discovered interactions with messenger RNA (mRNA) facilitate protein secretion, an important cellular process that could be harnessed for potential therapeutics and biotechnology applications.
“Once you see so many targets for these snoRNAs, you realize there’s a lot more to be understood,” said Chuan He, PhD, John T. Wilson Distinguished Service Professor of Chemistry and professor of biochemistry and molecular biology at the University of Chicago. “We already see that they play a role in protein secretion, which has major implications for physiology, and it suggests a path forward to study hundreds of other snoRNAs.”
He is the co-senior author of the researchers’ published paper in Cell, titled “SnoRNA-facilitated protein secretion revealed by transcriptome-wide snoRNA target identification.”
Dynamic, reversible modifications of DNA and RNA regulate how genes are expressed and transcribed, which can influence cellular processes, disease development, and overall organismal health. There are more than 1,000 known genes for encoding snoRNAs in the human genome. Unlike other guide RNA molecules such as microRNAs that are all the same length, snoRNAs vary greatly in their length from 50–250 residues, suggesting that they can do many different things.
Scientists have only pinpointed the RNA targets for about 300 snoRNAs. These targets mostly involve guiding modifications for ribosomal RNA and small nuclear RNA involved in mRNA splicing. “Some snoRNAs regulate gene expression by affecting mRNA stability, editing, and splicing,” the authors noted. But snoRNA activities may not always rely on mRNA modifications, they continued. “Over 50 snoRNAs are dysregulated in more than 12 cancer types.” Others are linked to neurological disorders. “Approximately 80% of annotated snoRNAs in the human genome lack well-defined functions,” the authors further pointed out, noting “The mechanisms by which snoRNA deletions or mutations lead to disease remain poorly understood, largely due to the lack of tools for identifying snoRNA targets and target RNA modification status across the transcriptome.”
Over the past 12 years He’s lab has developed several biochemical and sequencing techniques for studying transcription, DNA modifications, and RNA modifications. For their newly reported study, He, together with co-senior author Tao Pan, PhD, Professor of Biochemistry and Molecular Biology, and colleagues, tested a new tool called snoKARR-seq that links snoRNAs with their target binding RNAs. Bei Liu, PhD, a Chicago Fellow postdoctoral scholar who is co-mentored by He and Pan, led the project.
“We recently invented KARR-seq that utilizes chemical crosslinkers to effectively capture physically proximal RNAs independent of local RNA-protein interactions,” the team explained, describing snoKARR-seq as “a method that integrates RNA chemical labeling, crosslinking of snoRNAs with their binding RNAs, and chimeric cDNA enrichment post-reverse transcription (RT).” The investigators claim the technology detects snoRNA targets with over 100-fold higher signal-to-noise compared with conventional approaches. “Using this method, we identified over 1,000 previously unknown snoRNA-mRNA interactions in human cell lines and mouse brain tissue.”
Their results indicated that most of the newly discovered snoRNA targets do not overlap with the known RNA modification sites, “… suggesting potential non-canonical snoRNA functions beyond RNA modification,” they stated. Added Pan, “Chuan’s lab developed this killer technology to look at exactly what RNA each snoRNA is interacting with at the transcriptome level … Now there’s a lot of open space for understanding comprehensively what these 1,000 human genes [that encode snoRNAs] are doing.”
One unexpected discovery was that a snoRNA called SNORA73 interacts with mRNAs that encode secreted proteins and cell membrane proteins. Protein secretion is a fundamental biological process by which proteins are transported from a cell into the extracellular space, which is crucial for various functions, including communication between cells, immune responses, and digestion.
The researchers saw that SNORA73 acts as a “molecular glue” between the mRNA and the protein synthesis machinery that helps facilitate this process. “SNORA73 targets mRNAs encoding secretory and membrane proteins, and its depletion impairs the secretion of target proteins,” they further noted. “This unexpected function of snoRNA in promoting protein secretion highlights the versatility and complexity of snoRNA-mediated regulation.”
Further analysis of how SNORA73 binds with mRNA suggested that synthetic snoRNA sequences can be engineered to affect protein secretion. The researchers tested this hypothesis by tweaking a green fluorescent protein (GFP) reporter to interact with SNORA73. GFPs are often introduced in cells to make them glow under certain conditions so scientists can see the effects of experiments. When the researchers expressed SNORA73 genes with the engineered GFP that can be secreted from cells, it increased protein secretion by 30–50% over controls.
These results showed the potential to make use of the snoRNA machinery to manipulate the secretion of a given protein, which might have relevance for developing therapeutics. For example, if a human disease involves a deficiency of secreted proteins, then bioengineers could hijack the system to deliver artificial snoRNAs to increase secretion of that protein.
While the technology for synthesizing and delivering snoRNAs to the right locations isn’t ready yet, both He and Pan feel confident those challenges can be solved, since it builds upon previous advances in technology using other forms of RNA. They also believe that since snoRNAs are specific to cell types, they could have much more diverse functions—and therapeutic possibilities—elsewhere.
“Think about neuronal cells, stem cells, or cancer cells,” He said. “There are just so many cell types one can study. So, I think the field is wide open. Tao and I have been working together for more than 15 years, and it’s a great showcase of collaboration between the Biological Sciences Division and Physical Sciences Division at UChicago. This paper is another example that this kind of collaboration leads to opening a new field of biology.”
