RCC1L (WBSCR16) isoforms coordinate mitochondrial ribosome assembly through their interaction with GTPases

Abstract

Mitochondrial translation defects can be due to mutations affecting mitochondrial- or nuclear-encoded components. The number of known nuclear genes involved in mitochondrial translation has significantly increased in the past years. RCC1L (WBSCR16), a putative GDP/GTP exchange factor, has recently been described to interact with the mitochondrial large ribosomal subunit. In humans, three different RCC1L isoforms have been identified that originate from alternative splicing but share the same N-terminus, RCC1L^V1, RCC1L^V2 and RCC1L^V3. All three isoforms were exclusively localized to mitochondria, interacted with its inner membrane and could associate with homopolymeric oligos to different extent. Mitochondrial immunoprecipitation experiments showed that RCC1L^V1 and RCC1L^V3 associated with the mitochondrial large and small ribosomal subunit, respectively, while no significant association was observed for RCC1L^V2. Overexpression and silencing of RCC1L^V1 or RCC1L^V3 led to mitoribosome biogenesis defects that resulted in decreased translation. Indeed, significant changes in steady-state levels and distribution on isokinetic sucrose gradients were detected not only for mitoribosome proteins but also for GTPases, (GTPBP10, ERAL1 and C4orf14), and pseudouridylation proteins, (TRUB2, RPUSD3 and RPUSD4). All in all, our data suggest that RCC1L is essential for mitochondrial function and that the coordination of at least two isoforms is essential for proper ribosomal assembly. Mitochondria are essential organelles responsible for the production of most of the energy required by the eukaryotic cells and hence their proper function is essential for cell survival. About 1,500 proteins are estimated to be present in human mitochondria but new proteins are still being reported as mitochondrial. While most proteins present in mitochondria are synthesized in the cytosol and then imported into mitochondria, 13 proteins encoded in the mitochondrial DNA are synthesized inside mitochondria by a dedicated set of proteins. RCC1L (WBSCR16) has recently been described as a protein involved in mitochondrial protein synthesis and more precisely in mitochondrial ribosome biogenesis. Three different RCC1L transcripts resulting in three different isoforms have been described; however, their role in mitochondrial translation has not been fully studied. Here we show that all three isoforms are present in mitochondria and that at least two of them play a role in mitochondrial ribosome biogenesis though the interaction with specific proteins that play a role in the assembly of either the large or the small ribosome subunit. This protein is an example of how biological processes may be more complex than anticipated as different isoforms of the same gene can have different roles in the cell.