Biological function of SLIMP, a mitochondrial seryl-tRNA synthetase paralog

Abstract

Our research group focuses on protein translation and more specifically on the mechanism of transfer RNA (tRNA) aminoacylation by a family of essential and universal enzymes called aminoacyl-tRNA synthetases (aaRSs). aaRS are essential and universal components of the genetic code and their long evolutionary history explains the growing number of functions being discovered for aaRS and for aaRS homologues, beyond their canonical role in gene translation. During the process of constructing a model for human disorders caused by mitochondrial tRNA aminoacylation deficiencies in Drosophila melanogaster in the laboratory has been identified a previously uncharacterized paralog of a seryl-tRNA synthetase (SerRS) named SLIMP. Given the conservation of SLIMP in all available insect genomes, a characterization of the phenotype upon SLIMP depletion was conducted in Drosophila melanogaster. We analyzed the specific effect of SLIMP knockdown in muscles and glia, and we observed a dramatic reduction of the lifespan in both mutants. Interestingly, SLIMP knockdown in the glia causes neurodegeneration visualized as vacuolization in the brain of mutant flies. The sequence identity that SLIMP shares with SerRS allowed to predict that this protein has an identical fold of a seryl-tRNA synthetase. It was previously shown that SLIMP does not posses tRNA aminoacylation activity, but it retains the property to bind mitochondrial tRNASer isoacceptors as a possible reflection of the evolutionary origin of the protein. We characterized the RNA-and DNA-binding property of SLIMP through in vitro and in vivo methodologies. We found that SLIMP binds in vitro all RNA sequences that are encoded in the mitochondrial genome. We suggest that the composition of these sequences (AT enrichment) and the respective folding energy (low .G) are the main determinants for SLIMP binding to RNA. We performed ribonucleoprotein immunoprecipitation assay (RIP) in cells to study SLIMP-RNA interaction in vivo and we showed that SLIMP interact with almost all mitochondrial transcripts. Pull-down experiments combined to mass spectrometry analysis revealed at least two SLIMP protein interactors: SRS2 and LON protease. We demonstrated that SLIMP and SRS2 are interdependent, as the knockdown or the overexpression of one protein reduced or increase respectively the level of the other. We suggest that SLIMP and SRS2 might form a functional complex that is maintained at a given SLIMP:SRS2 ratio. LON was the other identified SLIMP interactor. Our data shows that LON and SRS2 do not interact, but SRS2 might be a substrate of LON proteolytic activity. We also found OPA1 as a potential interactor of LON. OPA1 is a dynamin-like GTPase that is responsible for inner membrane fusion. Our results indicated that the overexpression of LON protease results in an increase of the smaller OPA1 isoforms that correlates with mitochondrial stress. Given the proposed role of SLIMP in RNA binding, we aimed to determine whether SLIMP has an effect on mitochondrial transcription. The results obtained by Northern blot screening of some mitochondrial mRNAs revealed that knockdown of SLIMP significantly reduced the steady-state levels of COX2 and COX3 mRNA, and 12S and 16S rRNAs whereas tRNA levels were found constant. Our results suggest that the defect in transcription upon SLIMP depletion, might be limited to mature mRNAs and may reveal a specific function for SLIMP in the binding and stabilization of processed mitochondrial mRNAs rather than a role in the control of their transcriptional rate or processing. We showed that knockdown of SLIMP affects also cellular growth and cell cycle progression, in fact, upon SLIMP depletion, we observed a dramatic increase of cells in G2/M phase. This result suggests that the mitochondrial role of SLIMP, or a consequence of its function, may be acting as a crosstalk between mitochondria and nuclear transcription factors that regulate cell proliferation. Collectively, the work described in this thesis has contributed to the characterization of a mitochondrial seryl-tRNA synthetase paralog that has acquired an essential function in insects despite a relatively modest divergence from a canonical SerRS structure.

El nostre grup de recerca es centra en la traducció de proteïnes i més específicament en el mecanisme d’aminoacilació dels àcids ribonucleics (ARNs) de transferència (ARNt) per una família d’enzims essencials i universals anomenats aminoacil-ARNt sintetases (aaRSs). Al laboratori s’han analitzat el paper de les aaRSs en la traducció proteica, les seves funcions no canòniques, la seva evolució, així com la seva implicació en malalties humanes. Les aaRSs són components universals i essencials del codi genètic. La seva llarga historia evolutiva explica el creixent número de funcions que s’estan descobrint, tant per a elles com per a proteïnes paràlogues, més enllà del seu paper canònic en traducció genètica. Al laboratori, durant el procés d’obtenció d’un model a Drosophila melanogaster per a l’estudi de malalties humanes degudes a deficiències en l’aminoacilació d’ARNt, es va identificar un nou gen, paràlog de la seril-ARNt sintetasa (SeRS) mitocondrial, anomenat SLIMP. La proteïna SLIMP representa un nou tipus de proteïna similar a aaRS que ha adquirit una funció essencial a insectes, tot i la relativament baixa divergència respecta a una estructura d’SeRS canònica. Tot i amb això, són necessaris estudis addicionals per a identificar el paper biològic de SLIMP. Per aconseguir aquesta fita, s’ha portat a terme el projecte descrit en aquest manuscrit, el qual consisteix en anàlisis addicionals del fenotip resultant de la depleció de SLIMP in vivo, seguits d’estudis detallats de les interaccions moleculars amb àcids nucleics i proteïnes, per acabar amb un estudi dels efectes de SLIMP en la fisiologia cel•lular. En conjunt, els nostres resultats demostren que SLIMP s’uneix específicament a ARNs mitocondrials in vivo i in vitro. SLIMP interacciona amb SerRS2 i les dues són interdependents a nivell d’estabilitat proteica. La depleció de SLIMP o de SerRS2 redueix els nivells basals d’alguns ARNm mitocondrials, però la transcripció d’ARNt és manté inalterada. Es proposa un rol en la regulació post-transcripcional o en l’estabilitat dels ARNm madurs. Hem observat també que la depleció de SLIMP indueix l’aturada del cicle cel•lular en la transició G2/M. Aquests resultats suggereixen que SLIMP, o una conseqüència de la seva funció, podria tenir un paper d’enllaç entre els mitocondris i els factors de transcripció nuclears que regulen la proliferació cel•lular.