The role of RABL-like2 (RABL2) in male fertility and protein transport

2017-02-27T04:03:23Z (GMT) by Lo, Chi Yi
Infertility affects 1 in 20 men in reproductive age [1]. In an effort to identify key pathways and proteins involved in male fertility, a random mouse mutagenesis screen was carried out. In doing so, the O’Bryan lab identified a male sterile mouse line called Mot1. Mot1 homozygous mutants carry an A to G substitution point mutation in the Rab-like 2 (Rabl2) gene which affects two RABL2 isoforms (hereafter called Rabl2Mot/Mot) [2]. Mot1 homozygous male sterility was a result of reduced sperm production, shorter sperm tails and compromised sperm motility. This project aimed to characterise the biochemical mechanism of RABL2 function. Functional studies using unicellular organism have shown the role of RAB-like proteins in flagellum formation but the role of these proteins in mammals remain unknown [3,4]. Phylogenetic analyses conducted within our lab identified the RABL2 orthologues in all ciliated organisms and an absence of the orthologues in non-ciliated species suggesting it is associated with cilia development. RAB-like proteins are small GTPases within an uncharacterised clade of the RAS superfamily. Based on this we predicted they are able to switch between inactive and active states and are likely involved in protein delivery. We predicted in the active GTP-bound RABL2 would bind to a specific set of effector proteins and deliver them to the growing sperm tail that is the affected organelle in the Rabl2Mot/Mot animals. The Mot1 mutation disrupted effector proteins delivery and as such the Rabl2Mot/Mot males were sterile due to reduced levels of effector proteins in sperms. Further, RABL2 binds to intra-flagella transport (IFT) complex B component proteins which are important in cilia/flagella formation. In addition to the fertility phenotype, the Rabl2Mot/Mot animals develop juvenile onset steatosis and adult onset obesity. The weight gain in the Rabl2Mot/Mot animals is a result of increase in adiposity. Although Rabl2Mot/Mot animals have a normal food intake and are sensitive to exogenous leptin, they have insulin resistant, glucose intolerant and are less active than Rabl2WT/WT. Further investigations suggest that metabolic dysfunction is the consequence of primary hepatic dysfunction; specifically Rabl2Mot/Mot animals develop juvenile liver steatosis. They show an increase in pro-lipogenic genes expression and a reduced ability for fatty acid β oxidation which subsequently lead to lipid accumulation. The identified RABL2 putative effector proteins in liver showed enrichment in mitochondrial and peroxisomal proteins indicating RABL2 association in fatty acid metabolism. I hypothesise that RABL2 is involved in the organelle movement associated with microtubules within hepatocytes or it delivers effector proteins required for metabolism to organelles. Furthermore, Rabl2Mot/Mot animals develop age-dependent lung pathology from seven months. Rabl2Mot/Mot lungs were characterised with eosinophilic exudate lining the distal alveolar regions and emphysema. With increasing age, immune infiltrates became prominent. The progressive nature of this phenotype resembles the clinical features in primary ciliary dyskinesia. The data presented in this thesis demonstrates the role of RABL2 in male fertility, fatty acids metabolism and lung function. The co-pathologies presentation in the Rabl2Mot/Mot animals suggested RABL2 is essential for post-natal physiological development. The similar fertility and obesity phenotype in the Rabl2Mot/KO animals have confirmed the Mot1 phenotype is caused by the D73G mutation in Rabl2. 1. McLachlan RI, Mallidis C, Ma K, Bhasin S, de Kretser DM (1998) Genetic disorders and spermatogenesis. Reprod Fertil Dev 10: 97-104. 2. Kennedy CL, O'Connor AE, Sanchez-Partida LG, Holland MK, Goodnow CC, et al. (2005) A repository of ENU mutant mouse lines and their potential for male fertility research. Mol Hum Reprod 11: 871-880. 3. Qin H, Wang Z, Diener D, Rosenbaum J (2007) Intraflagellar transport protein 27 is a small G protein involved in cell-cycle control. Curr Biol 17: 193-202. 4. Silva DA, Huang X, Behal RH, Cole DG, Qin H (2011) The RABL5 homolog IFT22 regulates the cellular pool size and the amount of IFT particles partitioned to the flagellar compartment in Chlamydomonas reinhardtii. Cytoskeleton (Hoboken).