Publication Date

2016-04-10

Availability

Embargoed

Embargo Period

2018-04-10

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Biochemistry and Molecular Biology (Medicine)

Date of Defense

2016-03-11

First Committee Member

Antoni Barrientos

Second Committee Member

Yanbin Zhang

Third Committee Member

Murray Paul Deutscher

Fourth Committee Member

Carlos T. Moraes

Fifth Committee Member

Eric Shoubridge

Abstract

Mammalian mitochondrial ribosomes (mitoribosomes) are macromolecular complexes consisting of nucleus-encoded ribosomal proteins and mitochondrion-encoded rRNAs. They synthesize 13 membrane proteins, all subunits of oxidative phosphorylation system (OXPHOS). The mitoribosome assembly pathway and factors involved are poorly characterized. Here, we have identified a DEAD-box RNA helicase, termed DDX28, as an RNA granule component essential for the biogenesis of the mitoribosome large subunit (mt-LSU). DDX28 interacts with the 16S rRNA and presumably participates in the arrangements of its secondary structures. Silencing of DDX28 in HEK293T cells decreased the steady-state level of both LSU ribosomal proteins and 16S rRNA, which led to a significant mitochondrial protein synthesis defect and attenuated OXPHOS native complex formation. TALENs (Transcription activator-like effector nucleases)-mediated generation of a DDX28-KO model in HEK293T cells further proved the essential role of DDX28 in mitoribosome mt-LSU biogenesis and mitochondrial protein synthesis. In order to study the role of DDX28 ATPase and RNA helicase activities or ATP- and RNA-binding activities in mitoribosome assembly, we reconstituted DDX28-KO cells with DDX28 variants harboring mutations in different conserved motifs. So far, no constituted cells express the mitochondrial translation defect, except one mutant engineered to disrupt mitochondrial localization (ΔMTS+NLS) displayed the same mitochondrial protein synthesis deficiency as DDX28-KO cells, indicating that a correct localization is required for the protein to function. DDX28 locates to RNA granules and was found to associate with a group of proteins involved in all kinds of RNA transactions and with a series of mitoribosome proteins, suggesting the assembly of mitoribosomes occurs in the RNA granules. Analysis of the DDX28 proteome using mitochondrial extracts prepared under different salt stringencies further indicated that the membrane-less RNA granule seems to have a hierarchical interactome, at least surrounding DDX28, with proteins in the central core mostly required for post-transcriptional RNA processing, whereas proteins in the peripheral region are responsible for mitoribosome assembly and translation. In conclusion, our data presents DDX28 as the first DEAD box protein to be involved in mitoribosome assembly, although its precise mechanism of action remains to be fully understood. We have discovered that DDX28 resides in the RNA granule, the submitochondrial matrix compartment where mitoribosome assembly largely occurs.

Keywords

Mitoribosome; RNA helicase; DEAD-box; RNA granule

Available for download on Tuesday, April 10, 2018

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