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Characterization of RNA-modifying enzymes and their roles in diseases

dc.contributor.advisorBohnsack, Markus Prof. Dr.
dc.contributor.authorWarda, Ahmed
dc.titleCharacterization of RNA-modifying enzymes and their roles in diseasesde
dc.contributor.refereeGörlich, Dirk Prof. Dr.
dc.description.abstractengMore than 150 types of chemical modifications have been identified in cellular RNAs (collectively called ‘’the epitranscriptome’’), and such modifications have emerged as important regulators of gene expression. Despite the recent progress in studying RNA modifications, knowledge of the enzymes that install many individual modifications and the cellular functions of these modifications is lacking. Furthermore, an increasing number of diseases are linked to defects in RNA modifications or RNA modification enzymes but the molecular basis of these diseases often remains unknown. The human genome encodes numerous putative RNA methyltransferases, such as the methyltransferase-like (METTL) proteins and the Nol1/Nop2/SUN domain (NSUN) proteins, however, the molecular targets and functions of several of these enzymes remain unknown. We applied crosslinking and analysis of cDNA (CRAC) to identify the RNA binding sites of METTL16, NSUN6 and NSUN3 in a transcriptome-wide manner and used in vivo and in vitro assays to study their methylation activities. We showed that METTL16 is an N6-methyladenosine (m6A) methyltransferase that targets A43 in the U6 snRNA, which lies within an evolutionarily conserved sequence that base pairs with 5’ splice sites in pre-mRNAs, suggesting that this modification contributes to the regulation of pre-mRNA splicing. Furthermore, our results indicated that while NSUN6 introduces m5C72 on some cytoplasmic tRNAs during a late step of their biogenesis, NSUN3 installs m5C34 on the mitochondrial (mt)-tRNAMet. We have also identified ALKBH1 as the dioxygenase responsible for the oxidation of m5C34 to f534 of mt-tRNAMet and shown that these modifications are important for expanding codon recognition by mt-tRNAMet to enable efficient mitochondrial translation. We studied how these proteins recognize their substrates and showed that the CCA nucleotides at the 3’ end of tRNAs are important for recognition by NSUN6. Moreover, we found that the stability of the anticodon stem loop (ASL) is required for recognition by NSUN3, explaining why pathogenic mutations in mt-tRNAMet that disrupt the ASL impair methylation by NSUN3 and lead to disease. We further explored the link between RNA modification enzymes and disease by analysing effects of an aspartate 86 to glycine exchange (D86G) in the nucleolar ribosomal (r)RNA methyltransferase EMG1, which has been observed in patients with Bowen-Conradi syndrome (BCS). We discovered that EMG1D86G is chaperoned by the IMP/7 heterodimer in the cytoplasm and, upon the disassembly of the import complex in the nucleus, EMG1D86G aggregates and is degraded by the proteasome, leading to ribosome biogenesis defects. Taken together, our studies characterized substrates of novel RNA-modifying enzymes and provided insights into their cellular functions and the link between defects in these enzymes and
dc.contributor.coRefereeStülke, Jörg Prof. Dr.
dc.contributor.thirdRefereeRehling, Peter Prof. Dr.
dc.contributor.thirdRefereeDoenecke, Detlef Prof. Dr.
dc.contributor.thirdRefereeHöbartner, Claudia Prof. Dr.
dc.subject.engRNA biology+RNA modification+RNA and disease+the epitranscriptomede
dc.affiliation.instituteGöttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB)de
dc.subject.gokfullBiologie (PPN619462639)de

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