Proteins are the building blocks of the cell. They do most of the work and are essential for the structure, function and dynamic regulation of the cell and body's tissues and organs. Proteins rarely work alone, they interact, form protein complexes or bind DNA and RNA to control what a cell does. These complexes are key pieces of many important reactions within the cell, such as energy metabolism or gene regulation. Any change in those interactions, which can for example be caused by a mutation, can make the difference between health and disease. Hence, for understanding how cells operate, or what might go wrong in ill cells, it is essential to know how their building blocks interact.
New technologies allowed scientists during the last decades to understand the genetic information an organism possess, which of this information is actively used and which proteins are made by the cell in different circumstances. Now it is a big challenge to understand how biomolecules such as proteins and RNA messenger molecules combine to form the complexes required for a functional cell. In other words, we know the ten thousands of parts a cell is build off, but we don't know how they belong together.
In a paper published in Nature Communications, scientists describe the development of a new method, named "rec-YnH", which was designed to understand the complexes formed between hundreds of proteins and RNAs at the same time.
The method, whose development is the first technique that allows the detection of interactions between a large number of proteins and RNA molecules at the same time. The researchers put emphasis on the development of a doable and affordable method which is widely applicable.
rec-YnH is a new yeast two and three-hybrid-based screening pipeline capable of detecting interactions within protein libraries or between protein libraries and RNA fragment pools. rec-YnH combines batch cloning and transformation with intracellular homologous recombination to generate bait–prey fusion libraries.
By developing interaction selection in liquid–gels and using an ORF sequence-based readout of interactions via next-generation sequencing, authors eliminate laborious plating and barcoding steps required by existing methods.
They use rec-Y2H to simultaneously map interactions of protein domains and reveal novel putative interactors of PAR proteins. Authors further employ rec-Y2H to predict the architecture of published coprecipitated complexes. They also use rec-Y3H to map interactions between multiple RNA-binding proteins and RNAs—the first time interactions between protein and RNA pools are simultaneously detected.
"Our method reliably measures interactions between many proteins or many proteins and RNA fragments without the need for expensive, specialized equipment," explains the author. "This methodology can be used by any standard biomedical research laboratory and will be useful for studying a particular process in the cell but also for researchers having to explore millions of protein interactions at a time to look for a complex involved in a particular disease," the author concludes.
A new screening method to detect protein-protein and protein-RNA interactions
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