Because it can expand the range of a protein’s interactions or modulate its activity, post-translational methylation of arginine residues in proteins must be duly coordinated and ‘decoded’ to ensure appropriate cellular interpretation of this biological cue. This can be achieved through modulation of the enzymatic activity/specificity of the protein arginine methyltransferases (PRMTs) and proper recognition of the methylation ‘mark’ by a subset of proteins containing ‘methyl-sensing’ protein modules known as ‘Tudor’ domains. In order to gain a better understanding of these regulatory mechanisms, we undertook a detailed biochemical characterization of the predominant member of the PRMT family, PRMT1, and of the novel Tudor domain-containing protein 3 (TDRD3). First, we found that PRMT1 function can be modulated by 1) the expression of up to seven PRMT1 isoforms (v1-7), each with a unique N-terminal region that confers distinct substrate specificity, and by 2) differential subcellular localization, as revealed by the presence of a nuclear export sequence unique to PRMT1v2. Second, our findings suggest that TDRD3 is recruited to cytoplasmic stress granules (SGs) in response to environmental stress potentially by engaging in methyl-dependent protein-protein interactions with proteins involved in the control of gene expression. We also found that arginine methylation may serve as a general regulator of overall SG dynamics. Finally, we uncovered that alteration of PRMT1, TDRD3, and global arginine methylation levels in breast cancer cells may be closely associated with disease progression and poor prognosis. Therefore, further studies into the pathophysiological consequences ensuing from misregulation of arginine methylation will likely lead to the development of novel strategies for the prevention and treatment of breast cancer.

Because it can expand the range of a protein’s interactions or modulate its activity, post-translational methylation of arginine residues in proteins must be duly coordinated and ‘decoded’ to ensure appropriate cellular interpretation of this biological cue. This can be achieved through modulation of the enzymatic activity/specificity of the protein arginine methyltransferases (PRMTs) and proper recognition of the methylation ‘mark’ by a subset of proteins containing ‘methyl-sensing’ protein modules known as ‘Tudor’ domains. In order to gain a better understanding of these regulatory mechanisms, we undertook a detailed biochemical characterization of the predominant member of the PRMT family, PRMT1, and of the novel Tudor domain-containing protein 3 (TDRD3). First, we found that PRMT1 function can be modulated by 1) the expression of up to seven PRMT1 isoforms (v1-7), each with a unique N-terminal region that confers distinct substrate specificity, and by 2) differential subcellular localization, as revealed by the presence of a nuclear export sequence unique to PRMT1v2. Second, our findings suggest that TDRD3 is recruited to cytoplasmic stress granules (SGs) in response to environmental stress potentially by engaging in methyl-dependent protein-protein interactions with proteins involved in the control of gene expression. We also found that arginine methylation may serve as a general regulator of overall SG dynamics. Finally, we uncovered that alteration of PRMT1, TDRD3, and global arginine methylation levels in breast cancer cells may be closely associated with disease progression and poor prognosis. Therefore, further studies into the pathophysiological consequences ensuing from misregulation of arginine methylation will likely lead to the development of novel strategies for the prevention and treatment of breast cancer.

Because it can expand the range of a protein’s interactions or modulate its activity, post-translational methylation of arginine residues in proteins must be duly coordinated and ‘decoded’ to ensure appropriate cellular interpretation of this biological cue. This can be achieved through modulation of the enzymatic activity/specificity of the protein arginine methyltransferases (PRMTs) and proper recognition of the methylation ‘mark’ by a subset of proteins containing ‘methyl-sensing’ protein modules known as ‘Tudor’ domains. In order to gain a better understanding of these regulatory mechanisms, we undertook a detailed biochemical characterization of the predominant member of the PRMT family, PRMT1, and of the novel Tudor domain-containing protein 3 (TDRD3). First, we found that PRMT1 function can be modulated by 1) the expression of up to seven PRMT1 isoforms (v1-7), each with a unique N-terminal region that confers distinct substrate specificity, and by 2) differential subcellular localization, as revealed by the presence of a nuclear export sequence unique to PRMT1v2. Second, our findings suggest that TDRD3 is recruited to cytoplasmic stress granules (SGs) in response to environmental stress potentially by engaging in methyl-dependent protein-protein interactions with proteins involved in the control of gene expression. We also found that arginine methylation may serve as a general regulator of overall SG dynamics. Finally, we uncovered that alteration of PRMT1, TDRD3, and global arginine methylation levels in breast cancer cells may be closely associated with disease progression and poor prognosis. Therefore, further studies into the pathophysiological consequences ensuing from misregulation of arginine methylation will likely lead to the development of novel strategies for the prevention and treatment of breast cancer.

Because it can expand the range of a protein’s interactions or modulate its activity, post-translational methylation of arginine residues in proteins must be duly coordinated and ‘decoded’ to ensure appropriate cellular interpretation of this biological cue. This can be achieved through modulation of the enzymatic activity/specificity of the protein arginine methyltransferases (PRMTs) and proper recognition of the methylation ‘mark’ by a subset of proteins containing ‘methyl-sensing’ protein modules known as ‘Tudor’ domains. In order to gain a better understanding of these regulatory mechanisms, we undertook a detailed biochemical characterization of the predominant member of the PRMT family, PRMT1, and of the novel Tudor domain-containing protein 3 (TDRD3). First, we found that PRMT1 function can be modulated by 1) the expression of up to seven PRMT1 isoforms (v1-7), each with a unique N-terminal region that confers distinct substrate specificity, and by 2) differential subcellular localization, as revealed by the presence of a nuclear export sequence unique to PRMT1v2. Second, our findings suggest that TDRD3 is recruited to cytoplasmic stress granules (SGs) in response to environmental stress potentially by engaging in methyl-dependent protein-protein interactions with proteins involved in the control of gene expression. We also found that arginine methylation may serve as a general regulator of overall SG dynamics. Finally, we uncovered that alteration of PRMT1, TDRD3, and global arginine methylation levels in breast cancer cells may be closely associated with disease progression and poor prognosis. Therefore, further studies into the pathophysiological consequences ensuing from misregulation of arginine methylation will likely lead to the development of novel strategies for the prevention and treatment of breast cancer.

The dysfunction of autophagy pathways has been linked to the development and progression of numerous human diseases, in particular neurological disorders and cancer. Investigating these pathological autophagy mechanisms is essential to gain insights into the underlying disease mechanisms, identify novel biomarkers, and develop targeted therapies. In this thesis, I present three manuscripts that investigate the regulatory mechanisms of autophagy machinery in human diseases. In the first manuscript (Chitiprolu et al., 2018), we investigated the mechanism of p62-mediated selective autophagic clearance of RNA stress granules implicated in Amyotrophic Lateral Sclerosis (ALS). Repeat expansions in C9ORF72, the major cause of ALS, reduce C9ORF72 levels but how this impacts stress granules is uncertain. By employing mass spectrometry, high resolution imaging and biochemical assays, we demonstrated that the autophagy receptor p62 associates with C9ORF72 to eliminate stress granules by autophagy. This requires p62 to associate with proteins that are symmetrically methylated on arginines. Patients with C9ORF72 repeat expansions accumulate symmetric arginine dimethylated proteins which co-localize with p62. This suggests that C9ORF72 initiates a cascade of ALS-linked proteins (C9ORF72, p62, SMN, FUS) to recognize stress granules for degradation by autophagy and hallmarks of a defect in this process are observable in ALS patients. The second manuscript (Guo, Chitiprolu et al., 2014) describes the mechanism by which autophagy degrades retrotransposon RNA from both long and short interspersed elements, thereby preventing new retrotransposon insertions into the genome. By employing quantitative imaging tools, we demonstrated that retrotransposon RNA localizes to RNA granules that are selectively degraded by the autophagy receptors NDP52 and p62. Mice lacking a copy of Atg6/Beclin1, a gene critical for autophagy, also accumulate both retrotransposon RNA and genomic insertions. This suggests a mechanism for the increased tumorigenesis upon autophagy inhibition and therefore a role for autophagy in tempering evolutionary change. Finally, the third manuscript (Guo, Chitiprolu et al., 2017) examines the intersection of autophagy machinery with exosome release and function in cancer metastasis. By employing dynamic light scattering, Nanosight particle tracking, electron microscopy, super-resolution imaging and Western blotting, we robustly quantified exosome identity and purity in multiple cell lines. We demonstrated that exosome production is strongly reduced in cells lacking Atg5 and Atg16L1, but this is independent of Atg7 and canonical autophagy. The effect of Atg5 on exosome production promotes the migration and in vivo metastasis of orthotopic breast cancer cells. These findings delineate autophagy-independent pathways by which autophagy-related genes can contribute to metastasis. Taken together, data presented in the three manuscripts highlight the molecular mechanisms of autophagy core machinery proteins and selective receptors such as Atg5, p62 and NDP52, in the pathogenesis of cancer and neurodegeneration. In these diseases characterized by mutations in autophagy pathways, the mechanisms we uncover provide insights into their causes and serve as potential therapeutic targets.

Les ARN interagissants avec Piwi (ARNpi) sont des petits ARN non-codants qui sont exprimes dans la ligne grrminale des animaux. Ils interagissent avec les proteines de la branche Piwi de la famille des Argonautes en formant des complexes des ribonucleproteines impliques dans le maintien de l'intégrité du génome. La region N-terminale des quelques proteines Piwi contiennent symetriquement des arginines diméthylées. Il est considere que ce status symmetrique de la dimethylation est responsable du recrutement des proteines possédant des domaines Tudor (TDRDs). Ces domaines peuvent avoir un role comme platforme pour medier les interactions entre les proteines de la voie de l'ARNpi. Nous avons mesure indivindiuellemnt l'affinite de liaison des quatres domaines etendus Tudor (TD) de la proteine murine TDRD1 pour les trois differents peptides de la protein murine Mili qui contiennent de la methyl-arginine. Les resultats montrent une preference des TD2 et TD3 pour les peptides consecutives Mili alors que TD4 et TD1 ont une affinite plus bas et plus faible respectivement pour tous les peptides. Ces observations ont ete confirmees par des experiences pull-down en utilisant des proteines Piwi endogenes et des proteines-interagissent avec Piwi. L'affinite de TD1 pour les peptides qui contiennent de la methyl-arginine peut etre restoree par une seul mutation ponctuelle dans la cage aromatique pour revenir a la sequence consensus. La structure de cristal de la proteine TD3 lie au peptide methyle Mili montre une orientation inattendue de la peptide de liaison et de la chaine latérale de l'arginine methyle dans la cage aromatique. Finalement, le model SAXS des quatres domains tandem Tudor de TDRD1 revele une forme de la proteine flexible et elongee. Globalement, les resultats montrent que la proteine TDRD1 peut accommoder des differents peptides des differentes proteines et ainsi de fonctionner comme une protéine d'échafaudage dans la voie de l'ARNpi. La proteine FKBP6 (FK506 Binding Protein) a ete recemment identifiee comme un nouvel facteur interagissent dans la voie de l'ARNpi. FKBP6 est constituee d'une domaine d'isomerase FK et une domaine de tetratricopeptide (TPR). Une perte de la Fkbp6 conduit a la de –repression des transposons et a la sterilite masculine des souris. Le domaine TPR est implique dans l'interaction avec la proteine chaperone Hsp90 et le domaine FK est une isomerase inactive qui a ete evolue a une module structurale. En effectuant des exepriences biochimiques preliminaires nous avons identifie la region N-terminal du domaine MYND de TDRD1 comme le partenaire d'interaction du domaine FK de la FKBP6. Nous proposons que la proteine TDRD1 est une plateforme moleculaire qui reconnait des marques de methylation de MILI et elle recrute FKB6 pour promouvoir la formation d'un complexe indispensable pour la fonction de la voie de l'ARNpi. / Piwi-interacting RNAs (piRNAs) are small non-coding RNAs expressed in the germ line of animals. They associate with Argonaute proteins of the PIWI subfamily, forming ribonucleoprotein complexes that are involved in maintaining genome integrity. The N-terminal region of some PIWI proteins contains symmetrically dimethylated arginines. This symmetrical dimethylation is thought to be responsible for the recruitment of Tudor domain-containing proteins (TDRDs), which might serve as platforms mediating interactions between various proteins in the piRNA pathway. We measured the binding affinity of the four individual extended Tudor domains (TDs) of murine TDRD1 protein for three different methyl-arginine containing peptides from murine PIWI protein Mili. The results show a preference of TD2 and TD3 for consecutive Mili peptides whereas TD4 and TD1 have respectively lower and very weak affinity for any peptide. These observations were confirmed by pull-down experiments with endogenous PIWI and PIWI-associated proteins. The affinity of TD1 for methyl-arginine peptides can be restored by a single point mutation in the aromatic cage back to the consensus sequence. The crystal structure of TD3 bound to a methylated Mili peptide shows an unexpected, non-canonical orientation of the bound peptide and of the methylated arginine side-chain in the aromatic cage. Finally, the SAXS model of the four tandem Tudor domains of TDRD1 reveals a flexible, elongated shape of the protein. Overall the results show that TDRD1 can accommodate different peptides from different proteins and therefore act as a scaffold protein in the piRNA pathway. FK506-binding protein 6 (FKBP6) was recently identified as a novel piRNA pathway associated factor. It is comprised of an isomerase FK domain and a tetratricopeptide domain (TPR) and loss of Fkbp6 results in transposons de-repression and male sterility in mouse. The TPR domain is implicated in interaction with the chaperone Hsp90 and the FK domain is an inactive isomerase that has evolved into a structural module. We performed preliminary biochemical experiments that identify the N-terminal MYND domain of TDRD1 as the interaction partner of the FK domain of FKBP6. Overall, we propose that TDRD1 protein is a molecular platform that recognizes multiple methylation marks of Mili and recruits FKBP6 for the promotion of a complex formation indispensable for the proper function of the piRNA pathway.