Mechanisms for control of mitoribosome assembly by the GTPase ERAL1 - Abstract Mitochondria assemble their own ribosomes in order to translate the membrane-bound components of the electron transport chain. To do this, ribosomal RNA processing and folding and its sequential assembly with more than eighty protein subunits must be finely coordinated. Impairments of mitoribosome assembly through mutation of ribosomal subunits, assembly factors, or mitochondrial ribosomal RNA processing cause diverse mitochondriopathies. The GTPase ERAL1 is conserved as an essential RNA-binding GTPase functioning in assembly of the ribosomal small subunit between E. coli (Era) and human mitochondria. Deletion of ERAL1 is lethal in mice and its mutation causes a form of Perrault syndrome, which is characterized by sensorineural hearing loss and ovarian dysgenesis with variable neurological pathology. The molecular function(s) by which ERAL1 contributes to ribosome assembly, however, have not been described. ERAL1 binds the small subunit rRNA (the 12S RNA) until mid-assembly and is important for its stability, but what transformation of the 12S RNA and small subnit ERAL1 conducts has not been defined. In addition, the mitochondrial AAA+ protease CLPX-CLPP complex (CLPXP) appears to be important to remove ERAL1 from the assembling small subunit, to either terminate the normal function of ERAL1 or to resolve an off-pathway intermediate. Loss of CLPXP causes similar mitoribosomal dysfunction to loss of ERAL1 and mutations in CLPP also cause Perrault syndrome. In preliminary studies, we have reconstituted direct degradation of ERAL1 by CLPXP and discovered several factors that enhance or inhibit degradation, restricting degradation of ERAL1 to a particular state. We also determined that a Perrault mutation in ERAL1 blocks regulation by one of these factors. The goal of this proposal is to establish a molecular framework for the function and regulation of ERAL1 in mitoribosome assembly. Through the strategies we propose in Aim 1, we will determine how CLPXP senses the state of ERAL1 and how this is linked to mitoribosome assembly to terminate ERAL1-small subunit association, using our biochemically reconstituted system to examine degradation complex assembly and function. In Aim 2, we will define how ERAL1 GTPase activity and removal by CLPXP is coupled to maturation of the 12S RNA, using cell culture-based analysis of 12S RNA processing. These studies will provide direct molecular insight into a mechanism for disease in Perrault syndrome as well as identify potential targets for treatment of the broader spectrum of mitochondrial disease resulting from impaired mitoribosome biogenesis.
