Development of a ribosome profiling protocol to study translation in the yeast Kluyveromyces marxianus
University College Cork
During mRNA translation, the ribosome protects ~28 nt of mRNA within its mRNA tunnel. Ribosome profiling is a method which takes advantage of this protected mRNA fragment, commonly referred to as the ribosome protected fragment (RPF). This method uses endonucleases to digest unprotected mRNA, purifying the RPF and generating a cDNA library. Deep sequencing of these cDNA libraries can reveal the locations of all translating ribosomes in vivo. These data can be used to study aspects of mRNA translation including recoding (e.g. +1 frameshifting), ribosome stalling and differential gene expression between multiple conditions. In addition, as RPFs can be mapped back to the genome, novel translated ORFs may be discovered including novel protein coding genes and regulatory elements of mRNA translation such as upstream open reading frames present in the 5’ leaders (uORFs). Ribosome profiling was initially developed in the model yeast S. cerevisiae, but has since spread to human and bacterial models. The first results chapter of this thesis describes the development of a ribosome profiling protocol to study translation in the yeast Kluyveromyces marxianus, which has not previously be developed. This protocol includes detailed steps to carry out the wet lab protocol as well as some aspects on computational processing. This protocol is accompanied by the release of the K. marxianus genome and transcriptome to a publicly available genome (GWIPS-viz) and transcriptome browser (Trips-Viz) which are tailored specifically for ribosome profiling data. Together, these protocols and browsers will allow others in the K. marxianus community to generate ribosome profiling data and upload/analyse data via these genome browsers. The second results chapter describes the use of a combination of multiple methods including ribosome profiling, RNA-seq and transcript start site sequencing in what is described as “multiomics”. Using these multiomic data, the transcriptional and translational landscape is explored revealing a wide range of features including N-terminal extensions, upstream open reading frames and frameshifting. In addition, these data were used to generate a more complete and accurate genome annotation for K. marxianus by incorporating previously unannotated genes as well as a large number of gene annotation corrections such as splicing errors and start codon corrections. The third results chapter describes using the developed ribosome profiling protocol to study how K. marxianus adapts to a rapid increase in temperature in an effort to understand how thermotolerant yeast adapts to such a stress. These data include both ribosome profiling and RNA-seq with multiple timepoints. Interestingly, the response to heat shock included a large and rapid response whereby cellular respiration is immediately upregulated, supported by both ribosome profiling, RNA-sequencing and biochemical assays.
Ribosome profiling , Yeast , mRNA translation , Kluyveromyces marxianus , RNA-seq
Fenton, D. 2022. Development of a ribosome profiling protocol to study translation in the yeast Kluyveromyces marxianus. PhD Thesis, University College Cork.