Structure determination of non-coding RNAs
EMSL Project ID
50912
Abstract
Non-coding RNAs (ncRNA) play many diverse but essential roles in biology. Among the first characterized ncRNAs are transfer RNAs, ribosomal RNAs, and the spliceosomal RNAs. Later on, researchers demonstrated that some RNAs even have catalytic function, such as the group I and II introns, hammerhead ribozyme, and RNase P. Many of these non-coding RNAs have defined three-dimensional structures associated with them that have been determined over several decades of research. Many of these structures revealed critical details about the RNA’s roles in biology. Recently, newer molecular biology tools such as deep sequencing revealed new types of ncRNAs. Many of them have been grouped together as long non-coding RNAs (lncRNAs), which are generally defined as non-coding RNAs that are greater than 200 nucleotides in length. Because of their broad definition, lncRNAs have many diverse functions in cells, including transcriptional regulation, nuclear organization, and protein or RNA regulation. Although the functions of several lncRNAs have been demonstrated, the precise role of many lncRNAs is unknown. They are difficult to study due to their low abundance in cells and large size. Another obstacle is the lack of structural information on lncRNAs. Because of their lengths and possible conformational heterogeneity, structure determination of lncRNAs has not been possible. Cryo-electron microscopy (cryo-EM) is currently not well-suited to studying lncRNAs because of difficulties in imaging naked RNAs because of their propensity to aggregate on vitrified grids. In this proposal, we are developing a high-throughput strategy for structure determination of lncRNAs by cryo-EM. We are testing this technique with two structured ncRNAs, the group II intron P.li.LSU2 and the GOLLD RNA. P.li.LSU2 will serve as a useful benchmark since we have published two crystal structures of it from our lab. In addition, we hope to capture additional conformational states not seen in our crystal structures. The GOLLD RNA is a lncRNA found in bacterial isolates associated with a prophage. It is well-conserved and through biochemical techniques it appears to have a defined tertiary structure. Though we are starting with these two RNAs, we envision that this technique can be generalized and used for many lncRNAs.
Project Details
Start Date
2019-06-15
End Date
2019-08-28
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members