Mechanism of Structured RNA Degradation by the Human Exonuclease Dis3L2

Meze, Katarina (July 2021) Mechanism of Structured RNA Degradation by the Human Exonuclease Dis3L2. PhD thesis, Cold Spring Harbor Laboratory.

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Abstract

RNA degradation pathways are integral to cellular function, with their loss or dysregulation being linked to many human diseases including cancers, developmental disorders and autoimmunity. The autosomal recessive disorder Perlman syndrome, for instance, is caused by the loss of Dis3L2 – an RNase R/II family 3’ exoribonuclease. Dis3L2 targets a wide range of 3’-uridylated coding and non-coding RNAs, including the let-7 microRNAs which have important roles in development. Dis3L2 is particularly noteworthy as it can independently degrade structured substrates, which only some members of the RNase R/II family can do. Although the molecular nature of its U specificity is well characterized, little is known about the mechanism of double-strand (ds) RNA degradation. In this work I present cryoEM structures of human Dis3L2 bound to dsRNA substrates, representing snapshots along the degradation pathway. These structures reveal how the protein accommodates the shortening RNA leading up to double-strand unwinding. Notably, one structure reveals a dramatic conformational change in which two domains (CSD1 and CSD2) reposition themselves by roughly 70 Å. The movement of the CSDs allows the base of the RNA duplex to access Dis3L2's trihelix-linker region, implicating this protein module in strand separation. To assess the functional characteristics of Dis3L2 during degradation, we analyzed nuclease activity of the wild type enzyme and a panel of mutants through pre-steady state kinetic analysis at single nucleotide resolution. The determined kinetic profiles not only defined the contribution of individual domains, but also further confirmed the necessity of the trihelix-linker for dsRNA processing. Together, the structural and kinetic data have allowed us to construct a sequential model of structured RNA degradation by Dis3L2. These findings provide an important foundation for understanding nuclease-coupled dsRNA unwinding as well as a step towards unraveling how the different functionalities of the RNase R/II family evolved.

Item Type:	Thesis (PhD)
Subjects:	bioinformatics bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification bioinformatics > genomics and proteomics > genetics & nucleic acid processing bioinformatics > genomics and proteomics bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > miRNA bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > miRNA
CSHL Authors:	Meze, Katarina
Communities:	CSHL labs > Joshua-Tor lab School of Biological Sciences > Theses
Depositing User:	Kathleen McGuire
Date:	9 July 2021
Date Deposited:	07 May 2024 15:42
Last Modified:	07 May 2024 15:42
URI:	https://repository.cshl.edu/id/eprint/41536

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