Maturation and nucleo-cytoplasmic shuttling of snRNAs in Saccharomyces cerevisiae
by Daniel Becker
Date of Examination:2018-04-24
Date of issue:2019-01-24
Advisor:Prof. Dr. Heike Krebber
Referee:Prof. Dr. Ralf Ficner
Referee:Prof. Dr. Ralph Kehlenbach
Referee:PD Dr. Wilfried Kramer
Referee:Prof. Dr. Stefanie Pöggeler
Referee:Prof. Dr. Jörg Stülke
Files in this item
Name:PhD Thesis Daniel Becker eDiss.pdf
Size:3.44Mb
Format:PDF
Abstract
English
While nucleo-cytoplasmic shuttling of snRNAs is a well known process in higher eukaryotes, it is still unknown whether snRNAs shuttle in Saccharomyces cerevisiae. Furthermore, it is unclear why snRNA shuttling evolved in general. In yeast, snRNAs are transcribed in the nucleus and processed by nuclear endonuclease Rnt1 and exonuclease Rrp6. Additionally, snRNAs are trimethylated by the nucleolar methyltransferase Tgs1 before beeing integrated into spliceosomes. Therefore, a cytoplasmic phase seems unnecessary. However, in the present work we investigated a potential cytoplasmic phase of the snRNAs by fluorescent in situ hybridizations (FISH) and in vivo and in vitro RNA co-immunoprecipitation (RIP) experiments. We found that the snRNAs are exported from the nucleus by Mex67 and Xpo1 and determined the order of the subsequent maturation events. After export, the Sm-ring assembles on the snRNAs in the cytoplasm, which is an essential step for the nuclear import of the snRNP via the karyopherins Mtr10 and Cse1. In fact, FISH experiments revealed a cytoplasmic mislocalization of snRNAs after partial depletion of the Sm-ring, showing the importance of correct snRNP assembly. To answer the question if snRNAs are processed and TMG-capped before their export or after nucleo-cytoplasmic shuttling, we blocked snRNA shuttling. Interestingly, we detected increasing amounts of unprocessed U1 via qRT-PCR analysis in all transport mutants. In addition, we identified reduced amounts of TMG-capped snRNAs in import mutants, suggesting that snRNAs are processed and capped after shuttling and that they rather require the shuttling for subsequent processing steps. Furthermore, we performed a two step TMG-RIP analysis with the spliceosomal proteins Prp40 and Prp17 as well as with Mex67 and Xpo1 and identified that TMG-capped snRNAs preferentially bind to the late splicing factors and not to Mex67 and Xpo1. Thus, we suggest a model in which trimethylation of snRNAs prevents further snRNA shuttling. Importantly, we show that if shuttling is prevented, unprocessed snRNAs are incorporated into the spliceosomes, as the binding of Prp40 to immature U1 is significantly increased in mex67-5. Thus, spliceosomes cannot distinguish between mature and immature snRNAs and incorporate these immature snRNAs when they are not eliminated from the nuclear compartment. This in turn leads to severe genome wide splicing defects as confirmed by qRT-PCR and RNA-Sequencing analysis.
Keywords: snRNAs; Nucleo-cytoplasmic transport; non-coding RNAs; Mex67; Cse1; Splicing; Spliceosome; Xpo1