Analysis of multifunctional RNA helicase regulation by protein cofactors in different gene expression processes
by Nidhi Kanwal
Date of Examination:2023-08-28
Date of issue:2023-09-11
Advisor:Prof. Dr. Markus Bohnsack
Referee:Prof. Dr. Markus Bohnsack
Referee:Prof. Dr. Henning Urlaub
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EnglishThe biogenesis of ribonucleoprotein (RNP) complexes is a fundamental process in the gene expression network. Assembly of eukaryotic ribosomes and spliceosomes is highly orchestrated and entails the formation of numerous RNA-RNA and RNA-protein interactions. Ensuring correct and timely formation of such interactions often require the action of trans acting assembly factors, including RNA helicases that can chaperone RNA folding as well as drive structural and compositional remodelling of RNPs. RNA helicases are NTP-dependent enzymes required in several aspects of RNA metabolism, such as pre-mRNA splicing, ribosome biogenesis and translation. DHX15 is a multifunctional RNA helicase involved in pre-mRNA splicing, ribosome biogenesis and the innate immune response to viral infection. The regulation of DHX15 is achieved via association with G-patch cofactor proteins that modulate its helicase activity and provide substrate specificity. While 10 out of 22 members of human G-patch protein family bind DHX15 via the conserved glycine-rich G-patch domain, other members of the G-patch protein family remain poorly characterised. The present study focuses on the characterisation of GPATCH4. We identify DHX15 as the cognate RNA helicase of GPATCH4 and show that the G-patch protein functions as a cofactor, increasing the ATPase activity of the helicase. In vivo crosslinking approaches reveal that GPATCH4 associates with rDNA and binds nascent pre-rRNA. By employing small RNA RIP-seq, we find that GPATCH4 interacts with snoRNAs and scaRNAs implicated in guiding the modifications of rRNAs and snRNAs, respectively. In line with this, using RiboMeth-seq analysis to analyse global rRNA and snRNA 2’-O-methylation, revealed that knockout of GPATCH4 impairs methylation at various sites. In addition, our data show that the regulation of 2’-O- methylation by GPATCH4 is both dependent on, and independent of, its interaction with DHX15. RNase H-based cleavage assays indicate that the ATPase activity of DHX15 is required for efficient methylation at DHX15-dependent sites in the rRNA. This suggests that GPATCH4- mediated stimulation of DHX15 catalytic activity contributes to the regulation of snoRNA-guided 2’-O-methylation of rRNA. Taken together, these findings expand our understanding of the function and regulation of the multifunctional helicase DHX15 by its network of G-patch protein cofactors.
Keywords: Multifunctional RNA helicase; RNA modification; ribosome; ribosome biogenesis; small nucleolar RNA; 2’-O- methylation