Genetic variants that can act as switches directing structural changes in RNA molecules that code for plant proteins have been validated experimentally in plants for the first time. Changes in RNA structure can affect the stability of the molecule, how it interacts with other molecules, and how efficiently it can be translated into protein, which can impact its function and the characteristics of the plant. These genetic switches could be an important genetic mechanism that allowed plants to adapt to their microclimates in the past and could be vital for future adaptation and the development of resilient crops as climates continue to change.
An article describing the research, conducted by Penn State scientists, appears in the journal Genome Biology.
“Proteins, which are one of the major structural and functional molecules of life, are encoded by RNA, which is in turn encoded by DNA,” said Sarah M. Assmann, Waller Professor of Biology at Penn State and research team leader. . “Changes in DNA sequence can therefore lead to changes in proteins through an RNA, but not all changes in DNA affect the protein. Recently, genetic variants that do not necessarily change the encoded protein, but rather alter RNA folding, have been associated with human disease.We investigated whether similar mechanisms exist in plants and whether they may depend on environmental variables.
DNA is a double-stranded molecule – it looks like a twisted ladder, with the side rails representing the two strands and the rungs representing the bonds that hold them together. RNA, on the other hand, is single-stranded – imagine the ladder split in half in the middle of the rungs. However, single-stranded RNA is usually not just one long linear molecule. It folds back on itself forming short double-stranded sections between loops and bubbles of single-stranded RNA. This secondary folded structure is determined by RNA sequence in conjunction with the cellular microenvironment and is important for RNA function.
The folded structure of an RNA molecule can therefore be altered by genetic variants known as “single nucleotide polymorphisms” or SNPs – places in the genome where a single letter of the DNA alphabet differs between two or more individuals or groups . These SNPs modifying the structure of RNA are known as “riboSNitches”, combining “ribo” of the R in RNA, “SNPs” and “switches”.
“We are studying Arabidopsis, a model organism for plant biology,” said Ángel Ferrero-Serrano, assistant research professor of biology at Penn State and first author of the paper. “Over the past decade, the advent of high-throughput technologies has generated vast amounts of genetic data and physical descriptions of Arabidopsis varieties collected from the species’ native range. Specimens of Arabidopsis from locations around the world have had their entire genomes sequenced.We have recently developed a set of computational tools, CLIMtools, which allows us to determine associations between genetic DNA variation between Arabidopsis varieties collected in their native range and a large set of climatic variables that define the local environments of these varieties.We used these tools to find SNPs associated with temperature variables, and then tested whether any of the SNP acted like riboSNitches.
The team took the set of SNPs associated with temperature changes and narrowed it down further by looking for SNPs that were also associated with changes in RNA abundance, which often result from changes in the folding of RNA. They then applied an RNA structure algorithm to see if any of the SNPs should lead to structural changes, and chose two genes to validate experimentally.
“We tested the stability of short synthetic RNA molecules that include the potential riboSNitches against the standard reference sequence of that same stretch of RNA, over a range of temperatures,” Ferrero-Serrano said. “Based on our experiments, the SNPs of both genes appear to act as riboSNitches, one of which in particular alters the stability of RNA structure in a temperature-dependent manner. We suggest the term ‘conditional riboSNitches’ to refer to riboSNitches that depend on environmental variables.”
After experimentally demonstrating the existence of riboSNitches in Arabidopsis, the team then performed a massive computational study to predict potential riboSNitches across the genomes of hundreds of different sequenced Arabidopsis varieties. Of the more than 3.8 million SNPs that have been evaluated, more than one million, or approximately 27%, have the potential to act as riboSNitches.
“Plants cannot move, so adaptation to their local environment promotes survival,” Assmann said. “We now know that riboSNitches are another arrow in the quiver of genetic tools available to plants for this adaptation and that they may be conditional on environmental variables.”
With CLIMtools, the team created a set of resources to study the relationships between genetic variation in Arabidopsis and the environment. They hope this will give the scientific community a better understanding of how plants have adapted to local environments and how they can continue to thrive as climates change.
“Easy access to data is going to be key to solving today’s challenges in agriculture in a sustainable way,” said Dr. Doreen Ware, USDA ARS scientist at Cold Spring Harbor Laboratory. “We are delighted to host CLIMtools within Gramene, our online portal for comparative functional genomics, as it contains new resources for accessing information on permanent genetic variation that will be useful for climate adaptation, as well as information that can be used for gene editing. approaches to create new alleles in agriculturally important crops.
The techniques used for this study range from biophysics to molecular biology and ecology.
“We have assembled a diverse team of researchers at Penn State, ranging from an undergraduate to a graduate student to a research professor, and it has been a pleasure to work with them and Professor Assmann as well as with our colleagues at Cold Spring Harbor Laboratory. said Philip C. Bevilacqua, distinguished professor of chemistry, biochemistry and molecular biology at Penn State. “The finding that sequence variation can manifest itself in variation in RNA structure has long-term implications for modulating crop yield in the face of adverse climatic conditions.”
In addition to Assmann, Ferrero-Serrano, Ware, and Bevilacqua, the research team includes Plant Biology graduate student Megan M. Sylvia and Biochemistry and Molecular Biology undergraduate student Peter C. Forstmeier at Penn State; and Principal Engineer Andrew J. Olson at Cold Spring Harbor Laboratory in New York. The research was funded by Penn State, the US National Science Foundation and the US Department of Agriculture.
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