Structural and biochemical characterization of interactions centered on RNA decay factors: MTR4 and SMG1

Lingaraju, Mahesh (2020): Structural and biochemical characterization of interactions centered on RNA decay factors: MTR4 and SMG1. Dissertation, LMU München: Fakultät für Chemie und Pharmazie

Abstract

The nuclear exosome is the central 3'-5' RNA degradation machinery that performs a myriad roles critical for the health of a cell. The exosome associates with the MTR4 helicase, which binds and unwinds RNA substrates that are threaded through the exosome barrel for degradation. In several cases, MTR4 is targeted to specific RNA substrates via its association with adaptor proteins. Since MTR4 is a component of several exosome adaptor complexes, it was hypothesized that it might be recognizing the adaptor proteins via a common motif. The first results section of this thesis presents a study in which I identified and characterized the interactions of the MTR4 helicase with a pre-ribosome processing adaptor, NVL and the scaffolding and MTR4 activating component of the nuclear exosome targeting complex, ZCCHC8. I identified that the N-terminal regions of NVL and ZCCHC8 contain conserved sequences resembling the arch interacting motif (AIM) of the yeast rRNA processing factors. The structural and biochemical analysis indicate that these AIM-like motifs bind the MTR4 arch domain in a manner similar to that of the AIMs described earlier in the literature. Overall, the results suggest that nuclear exosome adaptors have evolved canonical and non- canonical AIM sequences to bind to human MTR4 and demonstrate the versatility and specificity with which the MTR4 arch domain can recruit a repertoire of different RNA- binding proteins. Recognizing RNA substrates for degradation is not only important in the nucleus but also in the cytoplasm. Nonsense mediated decay (NMD) is a cytoplasmic RNA decay mechanism which recognizes and degrades aberrant mRNA containing premature stop codons. It has also been shown to function in the regulation of physiological gene expression. SMG1, a 410 kDa PI3K related kinase, plays a crucial role in metazoan NMD by phosphorylating the UPF1 helicase. The phosphorylation of UPF1 was shown to be essential for the execution of NMD and represents the committed step of the NMD pathway. Although earlier low-resolution electron microscopic structures of human SMG1 along with some of its interacting partners were useful in gaining insight into the domain architecture of SMG1, the mechanism and regulation of SMG1 phosphorylation activity by SMG8-SMG9 remain poorly understood and are a subject of current research. The second results section presents a study, where I contributed to the characterization the C. elegans SMG8-SMG9 structurally and biochemically in an attempt to gain insights into the architecture of the complex and its possible biochemical role in NMD. The structure of the SMG8-SMG9 complex revealed that the complex exists as G-domain heterodimer with nucleotide binding capabilities. In a later study, presented as the third part of the results section, I contributed to understanding of the architecture of the SMG1-SMG8-SMG9 complex. The results not only recapitulate the findings of the SMG8-SMG9 complex but also provide structural basis for the SMG8-SMG9 interaction with SMG1. The structure also revealed that inositol-6-phosphate is a constitutive component of SMG1 and seems to play a role as a critical structural co-factor. The high- resolution structure of SMG1-SMG8-SMG9 provides a basis for several follow-up structural and biochemical studies centered on the early steps of NMD.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Themengebiete: 500 Naturwissenschaften und Mathematik ; 500 Naturwissenschaften und Mathematik > 540 Chemie
• Fakultäten: Fakultät für Chemie und Pharmazie
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 21. Januar 2020
• 1. Bericht­erstatter:in: Conti, Elena