The tRNA ligase complex in neurogenesis and maintenance of neuronal functions

Our laboratory has recently identified an elusive protein complex in human cells displaying tRNA ligase activity. The human tRNA ligase is a pentamer consisting of the catalytic subunit HSPC117, the DEAD box helicase Ddx1 and three proteins of unknown function - CGI-99, Fam98B and Ashwin. Only cells depleted of the essential subunit HSPC117 fail to complete tRNA splicing. We envision additional roles for the mammalian tRNA ligase in RNA processing and RNA metabolic pathways, particularly given the more extensive functions of tRNA ligases in other organisms. In line with this, our data suggest that the mammalian tRNA ligase participates in Xbp1 mRNA splicing during the Unfolded Protein Response. To elucidate the in vivo function of the mammalian tRNA ligase, we generated an HSPC117 knockout mouse. Since the complete knock-out of HSPC117 leads to early embryonic lethality, I have focused on the depletion of HSPC117 in the brain (HSPC117fl/fl Nestin-Cre) given the crucial role of RNA processing enzymes in neurons and the presence of HSPC117 and other subunits of the complex in neuronal RNA transport granules, structures that are important for neurogenesis and neuronal functions. I will pursue a detailed phenotypical characterization of these mice (Aim 1) using histological and immunohistochemical analyses of brains and spinal cords from HSPC117fl/fl Nestin-Cre mice, to evaluate defects in neuronal development, morphology and survival. I will also use this model to study the mechanism of HSPC117 action in neuronal cells (Aims 2) by investigating neuronal differentiation and function combining biochemical, immunofluorescence and quantitative, real-time imaging techniques. Together we believe that results from this study will provide novel functional insights in both RNA ligation and neurobiology. Mammalian RNA ligation and RNA ligase enzymes are relatively unexplored. This timely proposal and results thereof will reveal fundamental insights into the role of mammalian RNA ligation in diverse cellular functions, and potentially connect the failure of these functions to neuronal pathologies and other diseases.

Activity of the mammalian RNA ligase RTCB within a pentameric complex is known to be important for splicing of intron-containing tRNAs. Within this project we described a second function of this protein. We showed that RTCB together with a stimulatory co- factor called Archease catalyzes unconventional RNA splicing of Xbp1 mRNA that encodes for a key transcription factor during the unfolded protein response (UPR). Apart from tRNA splicing and Xbp1 mRNA ligation, the presence of RTCB has been described in neuronal RNA granules that transport mRNAs to distal axons and dendrites and play essential roles in the regulation of local translation during memory formation and neuronal development. However the importance of RTCB in neurons has never been addressed. Therefore the main goal of the project was to evaluate the role of RTCB in neurogenesis and the maintenance of neuronal functions. To this end we have generated two mouse models, Rtcbfl/fl Nestin-Cre mice, in which Rtcb is deleted during embryogenesis in neuronal progenitors and post-mitotic neurons and Rtcbfl/fl Camk2a-Cre mice, in which Rtcb is deleted after birth in post-mitotic, glutamatergic, excitatory neurons. We found that deletion of Rtcb during neuronal development causes early postnatal death. Although loss of RTCB during development did not affect general brain morphology, it triggered neuronal loss in the olfactory lobes, the cerebral cortex and the cerebellum and caused microcephaly and hydrocephalus. These defects are not caused by the absence of mature tRNAs or severe defects in global translation. Instead, we postulate that lack of RTCB introduces defects in neuronal progenitors that are independent of tRNA maturation and the unfolded protein response and may influence neural stem cell proliferation and the generation of post-mitotic neurons. Using the Rtcbfl/fl Camk2a-Cre mouse model, we furthermore showed that deletion of Rtcb in adult neurons causes increased synaptic signaling that ultimately leads to synaptic dysfunction and neurodegeneration. In summary we could show that the absence of RTCB in neurons has pleiotropic effects that regulate both neurogenesis and neuronal function. Our data suggests that apart from tRNA maturation and the UPR, RTCB mediates multiple essential processes in vivo.