| dc.contributor.advisor | Bohnsack, Markus Prof. Dr. | |
| dc.contributor.author | Aquino, Gerald Ryan | |
| dc.date.accessioned | 2021-02-25T13:23:48Z | |
| dc.date.available | 2022-02-13T00:50:08Z | |
| dc.date.issued | 2021-02-25 | |
| dc.identifier.uri | http://hdl.handle.net/21.11130/00-1735-0000-0005-158B-7 | |
| dc.identifier.uri | http://dx.doi.org/10.53846/goediss-8465 | |
| dc.language.iso | eng | de |
| dc.rights.uri | http://creativecommons.org/licenses/by-nc-nd/4.0/ | |
| dc.subject.ddc | 570 | de |
| dc.subject.ddc | 572 | de |
| dc.title | Molecular insights into the roles of RNA helicases during large ribosomal subunit assembly | de |
| dc.type | doctoralThesis | de |
| dc.contributor.referee | Ficner, Ralf Prof. Dr. | |
| dc.date.examination | 2021-02-15 | |
| dc.description.abstracteng | RNA helicases are enzymes present in all domains of life that are important in many aspects of RNA metabolism. They harbor a conserved helicase core that uses the energy of NTP binding and hydrolysis to change affinity to RNA, a biochemical property that provides RNA helicases mechanisms to unwind RNA duplexes. In the last years, there have been multiple lines of evidence that these specialized proteins can also displace proteins bound to RNAs and act as placeholders to promote intermediate RNA structures and folding. These RNA and ribonucleoprotein complex (RNP) remodeling functions underlie their involvement in many key cellular pathways and in the assembly of large RNPs, such as the ribosome. The synthesis of a ribosome is a very complex and dynamic processes consisting of the folding, processing and modification of the rRNA components coupled with the binding of ribosomal proteins (r-proteins). These events are highly orchestrated and involve numerous assembly factors (AFs), such nucleases, RNA helicases and RNA-binding proteins. Many RNA helicases belonging to DEAH and DExD subfamily act as molecular machines to drive structural rearrangements essential in the ribosome assembly process. However, the precise molecular functions of many RNA helicases implicated in this pathway and the underlying mechanisms of how they promote maturation events remain poorly understood. In this work, biochemical and molecular methods combined with transcriptome- and proteome-wide approaches were employed to gain insights into the molecular functions of the two DExD box RNA helicases Dbp3 and Dbp7 in yeast ribosome biogenesis. In vitro NADH-coupled ATP assays show that both RNA helicases are RNA-dependent ATPases. Pre-rRNA processing analyses implicate both in the large subunit (LSU) biogenesis. MS-based analysis of protein composition of purified Dbp7-containing particles identifies early-binding AFs and r-proteins, indicating a potential role of Dbp7 in the initial stages of LSU production. Moreover, compositional analyses of pre-60S from cells expressing and lacking Dbp7 suggests a role of Dbp7 in facilitating the recruitment of AFs and r-proteins. In vivo PAR-CRAC reveals crosslinking sites of Dbp7 on the 25S rRNA consistent with the known binding sites of these proteins. All these findings support a remodeling function of Dbp7 that leads to the compaction and stabilization of early pre-60S particles. Meanwhile, in the absence of Dbp3, many sites in the 25S rRNA were observed to have sub-stoichiometric 2’-O-methylation. Northern blot and qPCR analyses of snoRNAs guiding these affected sites reveal accumulation of several snoRNAs in the pre-ribosomal complexes when Dbp3 is lacking. Furthermore, overexpression of a snoRNA guiding several, non-proximal modifications recues the observed inefficient modification. Collectively, these data imply a role of Dbp3 in regulating snoRNP dynamics and pre-rRNA 2’-O-methylation during LSU biogenesis. Overall, this work provides important insights into the functions of Dbp3 and Dbp7 and therefore contributes to the functional characterization of RNA helicases involved in the early stages of LSU biogenesis. | de |
| dc.contributor.coReferee | Urlaub, Henning Prof. Dr. | |
| dc.contributor.thirdReferee | Stülke, Jörg Prof. Dr. | |
| dc.contributor.thirdReferee | Meinecke, Michael Prof. Dr. | |
| dc.contributor.thirdReferee | Richter-Dennerlein, Ricarda Dr. | |
| dc.subject.eng | RNA helicases | de |
| dc.subject.eng | Ribosome biogenesis | de |
| dc.subject.eng | ribosomal protein | de |
| dc.subject.eng | small nucleolar RNA | de |
| dc.subject.eng | RNA modifications | de |
| dc.identifier.urn | urn:nbn:de:gbv:7-21.11130/00-1735-0000-0005-158B-7-5 | |
| dc.affiliation.institute | Göttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB) | de |
| dc.subject.gokfull | Biologie (PPN619462639) | de |
| dc.description.embargoed | 2022-02-13 | |
| dc.identifier.ppn | 1749484161 | |