Project Details
Projekt
High-throughput structure probing of RNA based on specific ligation of cyclo-phosphate ends combined with deep sequencing
Subject Area
Biochemistry
Term
from 2019 to 2024
Project identifier
Deutsche Forschungsgemeinschaft (DFG) - Project number 431361947
Similar to proteins, RNA molecules fold up into manifold and complex structures in order to fulfill their tasks in the cell. While highly defined folds in rRNAs, tRNAs, ribozymes, riboswitches and terminators are well-known for a long time, an increasing amount of defined structures are also found in mRNAs, correlating with stability and especially translatability. Hence, it is of great interest to identify the structures of all transcripts within a cell to better understand their functionality and regulation. While in vitro approaches to characterize RNA structures are straightforward and well established, the in vivo investigation is rather laborious. Currently, there are only a few robust methods available, like in cell SHAPE, DMS-Seq and their variants SHAPE-MaP und DMS-MaPseq. These approaches are based on the read-out of RT stop signals or specific error signatures generated during RT. A disadvantage is that RT stops are also generated by spontaneous termination or stable RNA secondary structures and that not all single-strand specific chemical modifications lead to RT signatures. We want to establish a novel method that reads ligation positions as single stranded regions. 2’3’-cyclo-phosphate and 5’OH ends induced by lead cleavage in single-stranded RNA are fused to adapters by specific ligases and identified by deep sequencing. RT-based cDNA terminations are not registered. A further reduction of the background signal is achieved by the analysis of ligation products of both generated RNA ends. In parallel to the experimental approach, we will develop a precise bioinformatic tool for qualitative and quantitative recognition of single-strand signals to achieve RNA structures as accurate as possible. As a direct measure of the structure at a certain position requires a high read coverage (10-15 reads per position), we will also develop methods that aggregate the recorded signal along small intervals so that reliable conclusions can be drawn even with lower coverage.Combined with ribosome profiling analyses, we will use this novel orthogonal strategy to clarify how mRNA structure correlates with translational efficiency in E. coli, as recently published investigations show highly conflictive results. In addition, we want to analyze in psychrophilic microorganisms how mRNA structures are regulated at extremely low temperatures to allow efficient translation.Taken together, we want to develop a novel high throughput analysis of RNA structures to address important questions concerning the impact of RNA folding on stability, translational efficiency and their regulation in different cellular systems. Expression plasmids for the optimized ligases will be deposited in the non-profit vector collection addgene, so that the scientific community has access to this method.
DFG Programme
Research Grants
