Dynamics of RNA transcription and transcript stabilisation in wild-type and mutant Saccharomyces cerevisiae

Ribonucleic acids (RNAs) are a diverse and highly dynamic class of molecules. Coding RNAs facilitate the production of proteins from the heritable information stored in deoxyribonucleic acid (DNA), while pervasively transcribed non-coding RNAs have catalytic, scaffolding and regulatory functions.
Since cellular requirements vary with time, cell type and environmental condition, RNA pools are highly dynamic. Steady-state RNA levels are balanced through the production of RNAs via transcription and the removal of RNA via degradation. Both RNA transcription and degradation rates are highly regulated and can vary over several orders of magnitude even among closely related or overlapping RNAs. What is more, these rates frequently correlate with transcript translatability, i.e. how efficiently individual transcripts are being translated into proteins.
This means that the cell fine-tunes protein levels indirectly via RNA pools through different processes to coordinate cell growth and the response to various stimuli using interconnected feed-back loops. RNA transcription is the most studied aspect of the regulatory machinery that determines cellular RNA concentrations. However, the absence of important components of RNA degradation pathways also lead to a global deregulation of steady-state RNA levels.
The aim of this thesis is to link different aspects of RNA metabolism in the budding yeast Saccharomyces cerevisiae. This is achieved by characterising steady state RNA concentrations, transcription rates and nucleosome occupancy, that is DNA packaging, which is inherently linked to transcription, in wild-types and two degradation pathway mutants.
The study of wild-type yeast conducted in this thesis shows that 3’ end formation of stable RNAs is linked to a slowdown of RNAP II and a depletion in nucleosomes. Moreover, the analysis of 3’ ends reveals a novel class of intragenic transcripts that are constitutively transcribed and terminated, at least in part, via non-canonical termination pathways.
The absence of Rrp6p, a nuclear exoribonuclease, causes the stabilisation of unstable RNAs in the nucleus. In spite of previous reports linking Rrp6p to transcriptional read-through, RNA stabilisation does not appear to be linked to wide-spread changes in transcription or nucleosome occupancy. Stabilised RNAs have diverse 3’ ends that primarily map to the gene body of non-coding genes and are likely to be terminated by the Nrd1p-Nab3p-Sen1p termination pathway. The observed 3’ ends are associated with distinct sequences and generally map to the end of nucleosome depleted regions.
In xrn1Δ defects in RNA degradation lead to a global stabilisation of all RNAs and specific secondary changes in chromatin and RNA transcription. These changes are likely to be driven by altered concentrations of transcription factors and chromatin remodellers. Binding of these factors to DNA leads to nucleosome depletion at promoters in the absence of an external stimulus, ultimately causing an altered responsiveness to acute environmental stress and a distinct growth phenotype.
Hence, RNA degradation pathways are a major determinant of levels of specific RNA isoforms. Changes in the activity of RNA degradation pathways result in altered cellular signalling and modified transcriptional responses. RNA iv

Advisor: Mellor, J
Schuster-Böckler, B