It’s Valentine’s season, which means love is in the air, and heart-shaped novelties are abundant. But new research from the labs of Trushar Patel (University of Lethbridge, Alberta, Canada) and Karissa Sanbonmatsu (Los Alamos National Laboratory, New Mexico, USA) has flipped the script: they are giving us the shape of Braveheart, a long non-coding RNA that is critical for heart cell lineage commitment.
For years, lncRNAs have been breaking hearts of structural biologists everywhere; their long and flexible configurations, low expression levels, and short half-lives make it difficult to determine 3-dimensional structure using crystallographic methods. This savvy team employed small-angle X-ray scattering (SAXS) to finally woo the Braveheart (Bvht) lncRNA, but the process they developed can be applied to other RNAs and RNA-based complexes.
Here’s the workflow that won our hearts:
- Transcribe the RNA of interest in vitro; snap-cooling can be used to promote stable RNA folding
- Isolate homogeneous, single-molecule RNAs using size-exclusion chromatography (SEC), which are then subjected to SAXS imaging
- Assemble the SAXS data into a low-resolution model; this generated model represents an average of the conformations
- Utilize ERNWIN software to predict the atomistic structure, using the SAXS data and known secondary structure elements as constraints
In this study, they report the Bvht SAXS-based structure at a resolution between 13.4 and 38.5 Å, and additionally generated a structure of Bvht bound to Cellular Nucleic acid Binding Protein (CNBP). They show that Bvht has well-structured domains connected by flexible regions and that this structure undergoes significant conformational change upon interaction with CNBP.
The authors were able to use the Bvht-CNBP model to identify structural features of Bvht that are required for CNBP binding, highlighting the importance of structure in understanding function. Senior author Karissa Sanbonmatsu shares, “Our work represents the first step in showing that these difficult-to-image RNAs do possess 3-D structures, and that these molecular structures may very well determine how they operate.”
Spread the love and read more in Nature Communications, January 2020, and make a date to get to know your favorite RNA more intimately.