Analysis of small nucleolar (sno)RNA shuttling in Saccharomyces cerevisiae

by Fei Yu
Doctoral thesis
Date of Examination:2025-06-25
Date of issue:2025-08-07
Advisor:Prof. Dr. Heike Krebber
Referee:Prof. Dr. Heike Krebber
Referee:Prof. Dr. Kai Heimel
Referee:Dr. Oliver Valerius
Referee:Prof. Dr. Jörg Großhans
Referee:Prof. Dr. Ralph Kehlenbach
Referee:PD Dr. Wilfried Kramer
Persistent Address: http://dx.doi.org/10.53846/goediss-11412

 

 

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Abstract

English

Small nucleolar (sno)RNAs are a group of non-coding RNAs and can be classified into box C/D snoRNA and box H/ACA snoRNA based on their conserved sequences. They are further associated with snoRNA core proteins to form ribonucleoprotein complexes. The main function of snoRNPs is the post-transcriptional modification of rRNA. After transcription, the pre-snoRNAs are usually terminated by the Nrd1-Nab3-Sen1 (NNS) complex, followed by the core protein assembly and the 3’- and 5’-end processing. Finally, the mature snoRNPs are translocated to the nucleolus to fulfill their function. The whole biogenesis of snoRNA has been thought to occur exclusively in the nucleus. Recent studies suggest that some snoRNAs might be exported into the cytoplasm. However, it remained unclear for what reason they shuttle between the nucleus and the cytoplasm. This study used baker’s yeast Saccharomyces cerevisiae to analyze the shuttling. We show that some pre-snoRNAs are exported via Mex67. Moreover, we demonstrate that the transcription termination determines whether a pre-snoRNA is exported. Some snoRNAs contain a bipartite terminator and can therefore be alternatively terminated by the cleavage and polyadenylation (CPF-CF) complex as a fail-safe pathway. When the major NNS termination pathway is used, maturation takes place in the nucleus. We carried out systematic studies with the snoRNA snR13 in which we mutated the binding site of the NNS complex. When the CPF-CF pathway is preferentially used upon insertion of the NNS-mutation, the pre-snoRNA is polyadenylated and bound by Lhp1 and several mRNA guard proteins, such as Nab2 and Hrp1. These proteins recruit the export receptor Mex67 to facilitate the nuclear export. In the cytoplasm, Lhp1 and the Lsm2-8 ring, which are loaded onto the pre-snoRNA in the nucleus, protect the pre-snoRNA, until the Lsm2-8 ring recruits the import receptors Cse1 and Mtr10 for nuclear re-import. The assembly of the core proteins occurs partially after the re-import. Subsequently, final processing at both 3’- and 5’-ends proceeds to generate a mature snoRNP. Importantly, the CPF-CF terminated snoRNAs are fully functional to conduct the respective rRNA modifications. In conclusion, this study provides new insights into the snoRNA maturation and its shuttling.
Keywords: snoRNA / RNA modification / rRNA / transcription termination / NNS / CPF-CF