Nucleo-cytoplasmic shuttling of snoRNAs in Saccharomyces cerevisiae
by Gianluca Zaccagnini
Date of Examination:2022-06-23
Date of issue:2022-07-19
Advisor:Prof. Dr. Heike Krebber
Referee:Prof. Dr. Heike Krebber
Referee:Dr. Oliver Valerius
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EnglishSmall nucleolar RNAs (snoRNAs) are a group of small non-coding RNAs involved in the post-transcriptional modification of RNAs, such as rRNA and tRNA. The importance of their role is evidenced by the rRNA regions subject to modification that are highly conserved throughout evolution. In Saccharomyces cerevisiae, snoRNAs are transcribed in the nucleus and processed by the activity of the nuclear ribonucleases Rnt1, Rrp6, and Rat1. Thus, it was always assumed that snoRNAs never leave the nucleus. However, recent work has revealed the binding between snoRNAs and proteins atypical for this group, including the Mex67 export factor. Therefore, in the present work, we investigated the possibility of a cytosolic phase of snoRNAs by in vivo RNA co-immunoprecipitation (RIP) and fluorescent in situ hybridization (FISH) experiments. We confirmed that snoRNAs bind the export factor Mex67 and that they use it to be exported from the nucleus. Furthermore, we showed that the Sm-like2-8 (Lsm2-8) complex, a heteroheptameric complex involved in the processing of different non-coding RNAs, is able to bind different snoRNAs and not just U3 snoRNA as reported in the literature. We found that the Lsm2-8 complex binds snoRNAs in the nucleus, with the exception of U3, which appears to bind the Lsm-ring in the cytosol, after export. Following export, the Lsm2-8 complex turned out to be crucial for the nuclear re-import of the snoRNAs via the karyopherins Mtr10 and Cse1. Indeed, cytoplasmic fractionation and FISH experiments revealed a cytosolic mislocalization of snoRNAs in the mutant strains of MTR10 and CSE1 that cooperate in the nuclear re-import of snoRNAs. Moreover, the same cytosolic mislocalization was observed in the mutant strain for LSM8, confirming the importance of the Lsm-ring for the snoRNA re-import, potentially by connecting the snoRNA with the importins Mtr10 and Cse1. Finally, we investigated the role of the snoRNA second transcription termination site (site II). Using the cytoplasmic fractionation experiment in the trf4Δ mutant, which is known in the literature to bypass the first termination site (site I), we showed that when snoRNAs terminate transcription at the second site, they are more inclined to be exported into the cytosol. Furthermore, through a FISH experiment, we reinforced this result by observing a higher cytosolic localization of a snoRNA (snR65) in a strain where we mutated the sequence of the first transcription termination site, thus forcing termination at site II. Therefore, our data show the presence of a cytosolic pool of snoRNAs in S. cerevisiae, and we propose that snoRNAs are exported as a step of their maturation pathway. Moreover, we suggest that the cytosolic localization of snoRNAs may depend on the transcription termination site used.
Keywords: snoRNA; Saccharomyces cerevisiae; nucleo-cytoplasmic transport; Mex67; Lsm2-8; Mtr10; Cse1; transcription termination; Fluorescence in situ hybridization; RNA Co-Immunoprecipitation; Nucleo-cytoplasmic fractionation; Quantitative Real time PCR