Alternative pre-mRNA splicing greatly contributes to the mammalian proteomic diversity. The novel splice variants often emerge through splicing regulation at/nearby the splice sites (SS). A large group of 3′SS in human genes contain REPA (regulatory elements between the Py and 3′AG) G tracts that mostly appear in mammals as splicing silencers. However, the underlying molecular mechanisms and functional consequences remain unknown. We have identified a novel class of REPA G tracts (G)5-8 in a group of human genes including PRMT5 (protein arginine methyl transferase 5) that are significantly enriched in functional clusters of cell growth and proliferation. The PRMT5 G tracts emerged evolutionarily in mammals and repress splicing through recruitment of mainly hnRNP H that interferes with early spliceosome assembly. The splicing repression creates a shorter PRMT5 isoform (PRMT5S) that inhibits cell cycle progression contrary to the role of the full length protein (PRMT5L). Moreover, the expression of a group of genes involved in cell cycle arrest at interphase is preferentially regulated by PRMT5S. We further showed that PRMT5S is differentially expressed among cell and tissue types suggesting tissue-specific regulation. It exhibits distinct subcellular localization pattern from that of PRMT5L and opposite effects on cell cycle-specific structural dynamics of the Golgi apparatus. Moreover, these splice variants are differentially expressed during cell differentiation and PRMT5S promotes the differentiation of dendritic cells whereas PRMT5L shows the opposite effect. The expression of a large number of genes including those involved in crucial cellular processes such as differentiation and apoptosis is regulated by these splice variants of PRMT5. This study provides a direct link between the evolutionary emergence of a novel splicing regulatory G tract element and the generation of a functionally distinct protein isoform. The molecular mechanism underlying the splicing regulation by this G tract is likely common to many mammalian genes and the generation of their protein diversity. / October 2015

No description available.

Protein arginine methyltransferases (PRMTs)mediate the transfer of methyl groups to arginine residues in histone and non-histone proteins. PRMT5 is an important member of PRMTs which symmetrically dimethylates arginine 8 in histone H3 (H3R8) and arginine 3 in histone H4 (H4R3). PRMT5 was reported to inhibit some tumor suppressors in leukemia and lymphoma cells and regulate p53 gene, through affecting the promoter of p53. Through methylation of H4R3, PRMT5 can recruit DNA-methyltransferase 3A (DNMT3A) which regulates gene transcription. All the above suggest that PRMT5 has an important function of suppressing cell apoptosis and is a potential anticancer target. Currently, the enzymatic activities of PRMT5 are not clearly understood. In our study, we improved the protein expression methodology and greatly enhanced the yield and quality of the recombinant PRMT5. In addition, mutagenesis and enzymatic studies implicate an interesting mechanism of PRMT5 activity regulation.

Prostate cancer is one of the most frequently diagnosed cancers and the second leading cause of cancer-related deaths in male population. While localized prostate cancer can be successfully treated with surgery or radiation therapy, the metastatic disease has no curable options. Metastasis can be developed as a result of failed therapy of localized cancer or present at initial diagnosis. As metastasis is the most common cause of prostate cancer-related death, developing novel approaches and improving the efficiency of existing therapies for the metastatic prostate cancer treatment will significantly improve patients’ survival. <div><br><div>The first-line treatment option for metastatic prostate cancer and localized prostate cancer with high risk of recurrence is androgen deprivation therapy (ADT) that decreases androgen receptor (AR) signaling. However, targeting AR signaling inevitably leads to AR reactivation and cancer progression to the castration-resistant prostate cancer (CRPC) that has no curable treatment options. Moreover, about 30% of CRPC cases progress to neuroendocrine prostate cancer (NEPC), highly aggressive and lethal type of prostate cancer. </div><div><br></div><div>Recently my group has shown that protein arginine methyltransferase 5 (PRMT5) functions as an activator of AR expression in hormone-naïve prostate cancer (HNPC). In this dissertation, I demonstrate that PRMT5 also functions as an epigenetic activator of AR transcription in CRPC via symmetric dimethylation of H4R3 at the AR promoter. This epigenetic activation is dependent on pICln, a PRMT5 interaction partner involved in spliceosome assembly, and independent of MEP50, the canonical cofactor of PRMT5. PRMT5 and pICln, but not MEP50, were required for the expression of AR signaling pathway genes. In clinical samples of both HNPC and CRPC, nuclear PRMT5 and pICln protein expressions were highly positively correlated with nuclear AR protein expression. In xenograft tumors, targeting PRMT5 or pICln significantly decreased tumor growth and AR expression. </div><div><br></div><div>Overall, this work identifies PRMT5/pICln as a therapeutic target for HNPC and CRPC treatment that needs to be further evaluated in clinical setting. </div></div>

Indiana University-Purdue University Indianapolis (IUPUI) / Pancreatic ductal adenocarcinoma (PDAC) and colorectal cancer (CRC) are among the most commonly diagnosed forms of cancer in the United States. Due to their widespread prevalence and high mortality rate, it is vital to develop effective therapeutic drugs to combat these deadly diseases. In both PDAC and CRC, the multifunctional factor nuclear factor kappa B (NF-kB), a central coordinator of cellular immune responses, is activated abnormally, leading to tumorigenesis and cancer progression. Therefore, controlling NF-kB activity is critical in the treatment of these cancers. In a previous study, we identified a new mechanism by which NF-kB activity is regulated by an epigenetic enzyme known as protein arginine methyltransferase 5 (PRMT5). We showed that overexpression of PRMT5 not only activated NF-kB, but also significantly promoted several characteristics associated with cancer, including increased cell proliferation, migration, and anchorage-independent growth in both PDAC and CRC cells. Moreover, in order to examine the therapeutic potential of PRMT5 in these cancers, we adapted the state-of-the-art AlphaLISA technique into a high throughput screen (HTS) platform to screen for PRMT5 inhibitors. As a result, we successfully identified the small molecule PR5-LL-CM01 as our lead hit. Further validation experiments confirmed that PR5-LL-CM01 is a potent and specific PRMT5 inhibitor that exhibits significant anti-tumor efficacy in both in vitro and in vivo models of PDAC and CRC. Additionally, in a second screen, we discovered two natural compounds, P1608K04 and P1618J22, that can also function as the PRMT5 inhibitors. These findings further highlight the robustness of the PRMT5- specific AlphaLISA HTS technique. To conclude, we describe here for the first time a novel role of PRMT5 as a tumor-promoting factor in PDAC and CRC through NF-kB activation. By successfully developing and applying an innovative AlphaLISA HTS technique, we discovered PR5-LL-CM01, P1608K04, and P1618J22 as novel PRMT5 inhibitors, with PR5-LL-CM01 showing the strongest potency in both PDAC and CRC models. Therefore, we demonstrated that PRMT5 is a promising therapeutic target in PDAC and CRC, and the novel PRMT5 inhibitor PR5-LL-CM01 could serve as a promising basis for new drug development in PDAC and CRC.

Neste projeto caracterizamos a interação da nucleoproteína viral (N) do Vírus Respiratório Sincicial Humano (HRSV) com as proteínas PRMT5 e WDR77, que constituem o metilosomo celular. Confirmamos que essa interação ocorre através de co-imunoprecipitação em células humanas, e de interação in vitro dessas proteínas purificadas. Demonstramos a co-localização dessas proteínas na célula através de microscopias de imunofluorescência e confocal. Inibindo ou aumentando a expressão de PRMT5 não observamos impacto na replicação viral. Verificamos que ocorre metilação em N tanto em resíduos de argininas como de lisinas, com anticorpos específicos para essas modificações, e por espectrometria de massas, indicando significado funcional. Com essa evidência testamos o efeito de inibidores de metilação e demetilação, em argininas e lisinas. Obtivemos efeito inibitório significativo da replicação do HRSV com um inibidor de metilação de lisina, UNC0646, indicando que a interação N-metilossomo tem potencial como alvo terapêutico contra HRSV. / In this project we had as objective to characterize the interaction observed previously in the laboratory of viral nucleoprotein (N) with PRMT5 and WDR77 proteins that constitute the cell metilosome. We confirmed that this interaction occurs through co-imunoprecipitation in human cells and in vitro interaction of these purified proteins. We also demonstrated the co-localization of these proteins in inclusion bodies, by immunofluorescence and confocal microscopy. Inhibiting or enhancing PRMT5 expression we didnt see effect on viral replication. Our results show that methylation occurs in both arginine and lysine residues, through reactivity with antibodies specific to these modifications, and analysis by mass spectrometry. We tested the effect of arginine and lysine methylation and de-methylation inhibitors in viral replication. We obtained significant inhibitory effect on HRSV replication with a lysine methylation inhibitor, UNC0646, indicating that N-metilossome interaction has the potential to be exploited as a therapeutic target in developing antiviral drugs.

La protéine arginine méthyltransférase 5 est la majeure arginine méthyltransférase de type II chez les mammifères, responsable de la génération de la majorité des arginines protéiques symétriquement diméthylées. Elle est impliquée dans divers processus oncogéniques tel que la progression tumorale et la croissance indépendante de l'ancrage. PRMT5 est surexprimée dans plusieurs cancers comme le cancer de l'ovaire, des poumons et du colon. Cependant, son expression dans le cancer du sein n'est pas assez étudiée. Dans ce projet de thèse, nous avons analysé l'expression de PRMT5 dans une cohorte de 440 tumeurs mammaires. Nos résultats montrent que son expression nucléaire est un facteur de bon pronostic, notamment dans les tumeurs ERa-positives. Nous avons aussi mis en évidence une corrélation entre PRMT5 et la sérine/thréonine kinase LKB1, suggérant un lien entre ces deux protéines. Plusieurs approches in vitro et in cellulo nous ont permis de démontrer que PRMT5 et LKB1 interagissent dans le cytoplasme des cellules mammaires épithéliales. Bien que PRMT5 soit incapable de méthyler LKB1, nous avons montré pour la première fois que PRMT5 est un substrat de cette kinase. Nous avons par la suite identifié les Thr132, 139 et 144 comme cibles de la phosphorylation, au niveau du tonneau TIM en N-terminal de PRMT5. La mutation des thréonines T139/144 en alanine diminue significativement l'activité de PRMT5, probablement suite à une perte de son interaction avec des protéines régulatrices comme MEP50, pICLn et RiOK1. De plus, la modulation de l'expression de LKB1 altère l'activité de PRMT5, témoignant d'un nouveau mécanisme de régulation médié par la phosphorylation identifiée / Protein arginine methyltrasferase 5 is the major type II arginine methyltransferase in humans. It symmetrically dimethylates arginine residues on target proteins in both the cytoplasm and the nucleus. PRMT5 was reported to be an oncoprotein implicated in anchorage independent growth and tumor progression. So far, it has been involved in various cancers such as ovarian cancer, lung cancer and colon cancer, but its expression pattern in breast cancer has not been deeply studied. In this thesis project, we analyzed PRMT5 expression in a cohort of 440 breast tumor samples and we found that its nuclear expression is a good prognosis factor, mainly in ERa-positive tumors. Interestingly, our clinical results analysis showed that PRMT5 expression is correlated with the serine/threonine kinase LKB1, suggesting a relationship between both proteins. Several in vitro and in cellulo approaches gave evidence that PRMT5 and LKB1 interact directly in the cytoplasm of mammary epithelial cells. Moreover, although PRMT5 is not able to methylate LKB1, we found that PRMT5 is a bona fade substrate for LKB1. We next identified Thr132, 139 and 144 residues as target sites for phosphorylation, located in the TIM barrel domain of PRMT5. Interestingly, the Thr139/144 mutation to alanine decreased drastically PRMT5 methyltransferase activity, probably due to the loss of PRMT5 interaction with regulatory proteins such as MEP50, pICLn and RiOK1. In addition, the modulation of LKB1 expression modifies PRMT5 enzymatic activity, highlighting a new regulatory mechanism mediated by the discovered posttranslational modification of this arginine methyltransferase

Indiana University-Purdue University Indianapolis (IUPUI) / The overexpression of protein arginine methyltransferase 5 (PRMT5) is strongly correlated to poor clinical outcomes for colorectal cancer (CRC) patients. Previously, we demonstrated that PRMT5 overexpression could substantially augment activation of NF-κB via methylation of arginine 30 (R30) on its p65 subunit, while knockdown of PRMT5 showed the opposite effect on the transcriptional competence of p65. However, the precise mechanisms governing this PRMT5/NF-κB axis are still largely unknown. We report a novel finding that PRMT5 is phosphorylated on serine 15 (S15) in response to interleukin-1β (IL-1β) stimulation. Overexpression of the serine-to-alanine mutant of PRMT5 (S15A-PRMT5), in either HEK293 cells or HT29, DLD1 and HCT116 CRC cells attenuated NF-κB activation compared to wild type (WT)-PRMT5, confirming that S15 phosphorylation is critical for the activation of NF-κB by PRMT5. Furthermore, we found that overexpression of S15A-PRMT5 mutant attenuated the expression of a subset of NF-κB target genes through decreased p65 occupancy at their respective promoters. Importantly, the S15A-PRMT5 mutant also reduced IL-1β-induced methyltransferase activity of PRMT5 as well as its ability to form a complex with p65. Finally, we observed that the S15A-PRMT5 mutant diminished the growth, migratory and colony-forming abilities of CRC cells compared to the WT-PRMT5. Collectively, our findings provide strong evidence that novel phosphorylation of PRMT5 at S15 is critical to its regulation of NF-κB and plays an essential role in promoting the cancer-associated functions exerted by the PRMT5/NF-κB axis. Therefore, development of inhibitors to block phosphorylation of PRMT5 at S15 could become a potential novel therapeutic approach to treat CRC. / 2020-10-15

No description available.

No description available.