The complex formation of RNase P and RNase MRP in Saccharomyces cerevisiae
von Markus Röder
Datum der mündl. Prüfung:2024-02-29
Erschienen:2024-03-06
Betreuer:Prof. Dr. Heike Krebber
Gutachter:Prof. Dr. Heike Krebber
Gutachter:Dr. Oliver Valerius
Gutachter:Prof. Dr. Stefanie Pöggeler
Gutachter:Prof. Dr. Kai Heimel
Gutachter:PD Dr. Wilfried Kramer
Dateien
Name:Dissertation.pdf
Size:40.3Mb
Format:PDF
Description:Dissertation
Diese Datei ist bis 28.02.2025 gesperrt.
Zusammenfassung
Englisch
Ribonucleoproteins comprise a class of complexes that consist of RNAs and proteins. Riobozymes are catalytically active ribonucleoproteins that play a key role in the maturation of various RNAs. A prominent example is the essential ribozyme RNase P, which processes the 5’ leader sequence of transfer RNAs (tRNAs). The processing of the 5’ end of tRNAs is a necessary step for the maturation of the tRNA and the subsequent loading with amino acids. Another essential ribozyme is the closely related RNase MRP, which processes pre-ribosomal RNA. Mutations in the RNA component of RNase MRP can cause cartilage hair hypoplasia, a genetic disorder characterized by dwarfism. The complete functionalities of the RNases are therefore of great interest. However, the exact pathways of the complex assembly of both RNase P and MRP remain elusive. We characterized the step-wise complex assembly of RNase P and MRP in the model organism Saccharomyces cerevisiae. We show that the RNA component of RNase P RPR1, as well as the RNA component of RNase MRP NME1, are transcribed as precursors with 5’ and 3’ extensions. Pre-RPR1 is transcribed by RNA polymerase III and pre-NME1 is transcribed by RNA polymerase II. This causes the RNAs to be co-transcriptionally loaded with different moieties of messenger RNA (mRNA)-binding proteins and Lsm rings. These RNA-binding proteins mediate the binding to the export factors Mex67 and Xpo1 and thus, both RNAs shuttle into the cytoplasm after transcription. We show that the export of pre-RPR1 and pre- NME1 into the cytoplasm is a necessary step for the final assembly of the holoenzymes. In the cytoplasm, the importers Mtr10 and Cse1 bind to the co-transcriptionally loaded Lsm rings by direct protein to protein interaction. Both importers facilitate the re-import of the RNAs into the nucleus. After the re-import into the nucleus, the 5’ end of pre-NME1 is trimmed by the exonuclease Rat1 and pre-RPR1 is trimmed at the 3’ end by the exosome, Rex1, Rex2 and Rex3. Afterwards, a pre-RNase P complex is assembled with pre-RPR1, Pop6-Pop7, Pop1 and Pop5-Pop8-2xRpp1. We demonstrate that pre-NME1 is assembled into a pre-RNase MRP complex with the same protein subunits as the pre-RNase P complex. During the assembly of pre-RNase P, the 5’ end of pre-RPR1 is trimmed by Rat1. Finally, Pop4, Rpr2 and Pop3 bind to the pre-RNase P complex to form the mature holoenzyme. During the assembly of the pre- RNase MRP complex, the 3’ end of pre-NME1 is processed by Rnt1, RNase P and Rex3. Our findings suggest a step-wise assembly and RNA processing model for both ribozymes that include a necessary export step of the RNAs into the cytoplasm and the subsequent re-import into the nucleus for complex assembly.
Keywords: RNase P; RNase MRP; Saccharomyces cerevisiae