A Structural Analysis of LARP6 in Complex with the 5’ Stem-Loop of Collagen 1α1
Additional Funding Sources
This work was supported in part by the National Institute of General Medical Sciences of the National Institutes of Health under Award No. R15GM141770. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The project described was also supported through a summer research fellowship from the Boise State University Department of Chemistry and Biochemistry.
Abstract
Fibrosis is often attributed to the overproduction of type I collagen in an affected organ. One of the molecular interactions that activate this excessive synthesis is La ribonucleoprotein 6 (LARP6) binding to the 5' stem-loop (5’SL) of collagen 1α1 mRNA. Such an interaction has been of interest for developing antifibrotic drugs. Thus, this project aims to study the structure of this protein-RNA mechanism using wet-lab and computational techniques. In order to efficiently produce the high volumes of mRNA required for this analysis, experiments were done to determine the optimal time and DNA concentration for in vitro transcription. Agarose gel analyses showed that doing a transcription reaction for 16 hours with 50 nM of DNA would likely produce the highest yield of mRNA. Additionally, an artificial intelligence system called AlphaFold was used to predict the structure of LARP6. Based on the resulting predictions, it was found that most of LARP6 is disordered except for the La motif and the RNA recognition motif, the parts that directly interact with the mRNA. It was also seen that the linker between the two motifs is highly flexible. Using computational tools to combine these pieces of information with future SEC-MALS, SAXS, and NMR data can narrow down the possible structures for the LARP6-5’SL complex.
A Structural Analysis of LARP6 in Complex with the 5’ Stem-Loop of Collagen 1α1
Fibrosis is often attributed to the overproduction of type I collagen in an affected organ. One of the molecular interactions that activate this excessive synthesis is La ribonucleoprotein 6 (LARP6) binding to the 5' stem-loop (5’SL) of collagen 1α1 mRNA. Such an interaction has been of interest for developing antifibrotic drugs. Thus, this project aims to study the structure of this protein-RNA mechanism using wet-lab and computational techniques. In order to efficiently produce the high volumes of mRNA required for this analysis, experiments were done to determine the optimal time and DNA concentration for in vitro transcription. Agarose gel analyses showed that doing a transcription reaction for 16 hours with 50 nM of DNA would likely produce the highest yield of mRNA. Additionally, an artificial intelligence system called AlphaFold was used to predict the structure of LARP6. Based on the resulting predictions, it was found that most of LARP6 is disordered except for the La motif and the RNA recognition motif, the parts that directly interact with the mRNA. It was also seen that the linker between the two motifs is highly flexible. Using computational tools to combine these pieces of information with future SEC-MALS, SAXS, and NMR data can narrow down the possible structures for the LARP6-5’SL complex.