RNA and why we love it
The cornerstone of our lab is RNA: we are fascinated by the functional diversity of this molecule and its importance for the cell today and during the evolution of life.
Currently, we assume that the earliest moment in molecular evolution that allowed for heredity is represented by an “RNA world”. This term describes an environment, where genetic information was passed on in the form of RNA, which also acted as the key catalyst, and highlights the unique role of RNA in the history of our existence. Nowadays, RNA has generally lost the function of information storage to DNA and the function of catalysis to proteins. Nevertheless, RNA is still critical for all cellular functions. It acts during multiple steps in the translation of genetic information into proteins. Furthermore, RNA can act as a ribozyme and through a plethora of small and long non-coding RNA (ncRNA) regulates gene expression and protein translation.
It is not surprising that the extremely diverse field of RNA biology has attracted increasing attention in recent years, leading to exciting new discoveries, which constantly continuously update our view of numerous cellular processes and their regulation. In the Leidel lab, we combine classical methods of molecular biology and biochemistry with modern sequencing technologies to study RNA and the role of various RNA species in translation – a crucial cellular process essential to building and maintaining life.
Our experimental models
Our methods
RNA modifications
Most RNA species carry numerous posttranscriptional chemical modifications, which regulate their function and/or stability. One of our key research directions looks deeply into the role of posttranscriptional modifications of tRNA, the most heavily modified RNA species. tRNAs are major players in translation. They act as adaptor molecules, physically connecting the triplets of the genetic code to the corresponding amino acids. The modifications in the tRNA anticodon loop are thought to be critical for codon-anticodon interaction during translation, while the modifications in the tRNA body mainly affect tRNA folding and stability. However, the exact mechanisms and in vivo roles of numerous tRNA modifications are not fully understood. In our lab, we investigate the roles of tRNA modifications in different model organisms and use ribosome profiling in combination with other advanced sequencing techniques to analyze how the absence of tRNA modifications influences translation dynamics at the codon level. We complement our experiments with a comprehensive analysis of the cellular proteome to investigate the effects that these codon-specific changes in translation have on protein homeostasis.
In addition to tRNA, we focus on mRNA modifications since both types of modifications can affect codon-anticodon interactions. We are particularly interested in m6A, the most abundant internal mRNA modification. m6A modification affects various steps of mRNA metabolism, including splicing, export, translation, and mRNA stability, and as a result, is critical for multiple cellular processes. Finally, we investigate noncanonical mRNA capping to understand the cellular consequences of perturbation of noncanonical mRNA cap turnover.
Small peptides
Ribosome profiling is an excellent tool for gene discovery. The method allowed us to discover actively translated short open reading frames (ORFs) on transcripts, which were previously annotated as non-coding. After confirming the presence of these short peptides in vivo, we characterize their roles in cellular homeostasis and vertebrate development.
Translational quality control
The process of protein synthesis is incredibly intricate and complex. It involves multiple key players: mRNA, ribosome, tRNAs, initiation, elongation, and termination factors. Every member of this “translation team” can become a potential reason for the change or even complete failure of the whole process: for example, mRNA can contain a defective nucleotide or a premature stop codon, the ribosome may be defective and inefficient at reading the mRNA sequence, tRNA may lack a crucial chemical modification or be underrepresented, etc. Naturally, as the process of protein synthesis is incredibly important in the cell, multiple quality control mechanisms make sure that the above-mentioned problems either don’t happen at all or help the cell deal with them when they eventually happen.
Moreover, as the ribosome moves along the mRNA, the newly synthesized peptide needs to be protected from aggregation and undergo folding in a step-wise manner to produce the functional protein. One of the directions of Leidel’s lab research focuses on ribosome-associated complex (RAC), consisting of co-translational chaperones, which associate with the ribosome and ensure the correct folding of the nascent proteins.
The ribosome can slow down while decoding a particular codon on the mRNA. If the slow-down is not resolved in time and the ribosome doesn’t continue the movement, the next-in-line ribosome can catch up with the slow ribosome, resulting in ribosome collision. Members of the Ribosomal Quality Control (RQC) system recognize the interface of collided ribosomes and resolve the situation by cleaving the mRNA, splitting and recycling the ribosomes while at the same time targeting the nascent peptide which didn’t have a chance to get fully synthesized to degradation.
We are particularly interested in the consequences of defective RQC system and co-translational chaperones on the proteome of the cell and how these quality control mechanisms contribute to the way the cell deals with codon-specific translational stress.
Multiomics
Over the years, we have produced an impressive collection of high-throughput and high-resolution RiboSeq, RNAseq, and tRNAseq libraries as well as protein and RNA mass spectrometry data. Moreover, we constantly continue refining these main well-established experimental techniques in our lab and adding new state-of-the-art methods to our toolkit. Having these quality data allows us to assess changes in all major players and processes related to protein synthesis: mRNA/tRNA transcription and modification, translation efficiency on the codon resolution as well as the quality and abundance of the resulting proteins. With the help of the computational members of the group, we are integrating these multilevel data to unravel the links between different processes in the cell and get closer to understanding how translation dynamics influence phenotypes.