Mutations in proteins like FUS which cause Amyotrophic Lateral Sclerosis (ALS) result in the aberrant formation of stress granules while ALS-linked mutations in other proteins impede elimination of ...stress granules. Repeat expansions in C9ORF72, the major cause of ALS, reduce C9ORF72 levels but how this impacts stress granules is uncertain. Here, we demonstrate that C9ORF72 associates with the autophagy receptor p62 and controls elimination of stress granules by autophagy. This requires p62 to associate via the Tudor protein SMN with proteins, including FUS, that are symmetrically methylated on arginines. Mice lacking p62 accumulate arginine-methylated proteins and alterations in FUS-dependent splicing. 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.
Tudor domain containing protein 3 (TDRD3) is a modular protein identified based on its ability to recognize methylated arginine motifs through its Tudor domain. We have previously shown that TDRD3 ...localizes to cytoplasmic stress granules, a structure shown to promote survival upon treatment with chemotherapeutic drugs in cancer cells. Here, we report TDRD3 as a novel regulator of cell proliferation and invasion in breast cancer cells. Our study also demonstrates that TDRD3 depletion inhibits tumor formation and metastasis to the lung in vivo. Furthermore, we show that TDRD3 regulates the expression of a number of key genes associated with promotion of breast cancer tumorigenesis and disease progression. Strikingly, we report that TDRD3 regulates some of these key targets at the level of translation. These findings provide the first experimental demonstration of a functional role for TDRD3 in promoting breast cancer development and progression, and identify TDRD3 as a potential new therapeutic target for breast cancer.
Loss of 'Survival of Motor Neurons' (SMN) leads to spinal muscular atrophy (SMA), a disease characterized by degeneration of spinal cord alpha motor neurons, resulting in muscle weakness, paralysis ...and death during early childhood. SMN is required for assembly of the core splicing machinery, and splicing defects were documented in SMA. We previously uncovered that Coactivator-Associated Methyltransferase-1 (CARM1) is abnormally up-regulated in SMA, leading to mis-regulation of a number of transcriptional and alternative splicing events. We report here that CARM1 can promote decay of a premature terminating codon (PTC)-containing mRNA reporter, suggesting it can act as a mediator of nonsense-mediated mRNA decay (NMD). Interestingly, this pathway, while originally perceived as solely a surveillance mechanism preventing expression of potentially detrimental proteins, is now emerging as a highly regulated RNA decay pathway also acting on a subset of normal mRNAs. We further show that CARM1 associates with major NMD factor UPF1 and promotes its occupancy on PTC-containing transcripts. Finally, we identify a specific subset of NMD targets that are dependent on CARM1 for degradation and that are also misregulated in SMA, potentially adding exacerbated targeting of PTC-containing mRNAs to the already complex array of molecular defects associated with this disease.
Recent evidence points to the protein arginine methyltransferase (PRMT) family of enzymes playing critical roles in cancer. PRMT7 has been identified in several gene expression studies to be ...associated with increased metastasis and decreased survival in breast cancer patients. However, this has not been extensively studied. Here we report that PRMT7 expression is significantly upregulated in both primary breast tumour tissues and in breast cancer lymph node metastases. We have demonstrated that reducing PRMT7 levels in invasive breast cancer cells using RNA interference significantly decreased cell invasion in vitro and metastasis in vivo. Conversely, overexpression of PRMT7 in non-aggressive MCF7 cells enhanced their invasiveness. Furthermore, we show that PRMT7 induces the expression of matrix metalloproteinase 9 (MMP9), a well-known mediator of breast cancer metastasis. Importantly, we significantly rescued invasion of aggressive breast cancer cells depleted of PRMT7 by the exogenous expression of MMP9. Our results demonstrate that upregulation of PRMT7 in breast cancer may have a significant role in promoting cell invasion through the regulation of MMP9. This identifies PRMT7 as a novel and potentially significant biomarker and therapeutic target for breast cancer.
Although recent advances in gene therapy provide hope for spinal muscular atrophy (SMA) patients, the pathology remains the leading genetic cause of infant mortality. SMA is a monogenic pathology ...that originates from the loss of the
gene in most cases or mutations in rare cases. Interestingly, several
mutations occur within the TUDOR methylarginine reader domain of SMN. We hypothesized that in
mutant cases, SMA may emerge from aberrant protein-protein interactions between SMN and key neuronal factors. Using a BioID proteomic approach, we have identified and validated a number of SMN-interacting proteins, including fragile X mental retardation protein (FMRP) family members (FMR
). Importantly, SMA-linked SMN
mutant forms (SMN
) failed to interact with FMR
In agreement with the recent work, we define biochemically that SMN forms droplets in vitro and these droplets are stabilized by RNA, suggesting that SMN could be involved in the formation of membraneless organelles, such as Cajal nuclear bodies. Finally, we found that SMN and FMRP co-fractionate with polysomes, in an RNA-dependent manner, suggesting a potential role in localized translation in motor neurons.
► Hypertonicity increases NKA α and α-ENaC, but sgk1decreased. ► Other CCD sodium transporters (β-NKA, β-ENaC, NHE) were unchanged. ► Hypertonicity also increased PGE2 but EP4 receptor mRNA was ...unaltered. ► PGE2 increased Na+ and cAMP, but PGE2-cAMP was attenuated by hypertonicity. ► COX inhibition and EP4 siRNA did not alter hypertonicity induced NKA.
Cyclooxygenase-2 (COX-2) expression is increased by hypertonicity. Therefore we hypothesized that hypertonicity increased PGE2 can modulate the sodium transporters (Na+/K+-ATPase: NKA, epithelial sodium channel: ENaC, and sodium hydrogen exchanger: NHE) in M1 cortical collecting duct (CCD) cells. We demonstrated by immunoblotting a 2-fold increase in NKA expression and activity following hypertonic treatment. α-ENaC was also increased, however sgk1, an ENaC activator, decreased in response to hypertonicity. Other CCD sodium transporters (β-ENaC, NHE) were unchanged. Hypertonicity also increased PGE2 but EP4 receptor mRNA was unaltered. PGE2 increased intracellular Na+ and cAMP production in M1 cells, but PGE2-stimulated cAMP response was attenuated by hypertonicity. Overall, PGE2 had no effect on sodium transporter levels. Since neither COX inhibition nor EP4 siRNA altered the induction of NKA, we propose that sodium transporter regulation by hypertonicity is independent of PGE2. Altogether, these data indicate that despite a concomitant increase in PGE2 production and sodium transporter expression in hypertonicity, both pathways are acting independently of each other.
Peroxisome proliferator-activated receptor (PPAR)-γ is highly expressed in the collecting duct (CD), yet little is known about the effects of PPAR-γ ligands, thiazolidinediones (TZDs), on CD cell ...structure and function. M1 mouse cortical CD cells were treated with 5 μM troglitazone (TRO) and rosiglitazone (ROSI). First, growth was measured by (3)Hthymidine and (3)Hleucine incorporation, as well as analysis of cyclin D1 and the CDK inhibitor p27 by Western blot. (3)Hthymidine incorporation was reduced by 56 and 24% by TRO and ROSI at 6 h, and (3)Hleucine by 21 and 10%. A similar growth inhibition was also observed after 24 h for thymidine, but leucine was reduced by 48 and 24%, respectively. Likewise, cyclin D1 was diminished 60% by TRO, and p27 was elevated 1.6- and 1.7-fold in response to TRO and ROSI. Next, epithelial cell integrity was assessed by measuring different markers by Western blot analysis. While fibronectin and α-smooth muscle actin levels were unchanged, by 24 h E-cadherin was decreased by 50%, and β-catenin levels were reduced 2- and 1.5-fold in response to TRO and ROSI, respectively. GW9662, a PPAR-γ antagonist, did not reverse any of the TZD responses in M1 cells. Of interest, phosho-p38 levels were also elevated 2-fold in response to TRO and 2.3-fold to ROSI, but MAPK inhibition by PD98059 or SB203580 caused an additive inhibition of cell growth and did not alter E-cadherin or β-catenin in response to TZDs. Finally, apoptotic death was assessed by Western blot, but cleaved caspase-3 levels were unchanged from 15 min to 24 h in response to TZDs, and TRO did not affect cell viability or reactive oxygen species generation. Our data suggest that TZDs cause a disruption of M1 cell integrity that is preceded by an inhibition of cell growth. This response is independent of p38 or PPAR-γ activation.
Ce volume rassemble les communications du colloque tenu à l’École nationale des chartes et à la bibliothèque Sainte-Geneviève les 30 et 31 mars 2009, à l’initiative conjointe du Centre d’études ...supérieures de la Renaissance et de l’École nationale des chartes. L’articulation entre imprimerie et humanisme, trop souvent perçue comme évidente, est le fil conducteur de ces contributions qui interrogent la place singulière des imprimeurs-libraires dans la communauté savante du XVIe siècle. Véritables médiateurs, ces « passeurs de textes » figurent aux côtés des auteurs parmi ceux qui recueillent un héritage autant qu’ils le transmettent. La variété méthodologique des analyses – portant aussi bien sur les catalogues des maisons d’édition que sur des collaborations suivies avec des auteurs, des mises en texte originales, la portée et la réception de certaines éditions, la diffusion en réseaux commerciaux ou culturels – a permis de préciser les notions d’« imprimeur humaniste » et d’« humaniste imprimeur », tout en décrivant des réalités et des stratégies très différentes : du savant qui se fait typographe au marchand-libraire soucieux avant tout de profit, le rapport à l’humanisme peut en effet se déployer sur un large éventail.
Protein arginine methylation is catalyzed by protein arginine methyltransferases (PRMTs) and plays an important role in many cellular processes. Aberrant PRMT expression has been observed in several ...common cancer types; however, their precise contribution to the cell transformation process is not well understood. We previously reported that the PRMT1 gene generates several alternatively spliced isoforms, and our initial biochemical characterization of these isoforms revealed that they exhibit distinct substrate specificity and subcellular localization. We focus here on the PRMT1v2 isoform, which is the only predominantly cytoplasmic isoform, and we have found that its relative expression is increased in breast cancer cell lines and tumors. Specific depletion of PRMT1v2 using RNA interference caused a significant decrease in cancer cell survival due to an induction of apoptosis. Furthermore, depletion of PRMT1v2 in an aggressive cancer cell line significantly decreased cell invasion. We also demonstrate that PRMT1v2 overexpression in a non-aggressive cancer cell line was sufficient to render them more invasive. Importantly, this novel activity is specific to PRMT1v2, as overexpression of other isoforms did not enhance invasion. Moreover, this activity requires both proper subcellular localization and methylase activity. Lastly, PRMT1v2 overexpression altered cell morphology and reduced cell-cell adhesion, a phenomenon that we convincingly linked with reduced β-catenin protein expression. Overall, we demonstrate a specific role for PRMT1v2 in breast cancer cell survival and invasion, underscoring the importance of identifying and characterizing the distinct functional differences between PRMT1 isoforms.
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