3′ RNA Uridylation in Epitranscriptomics, Gene Regulation, and Disease

Emerging evidence implicates a wide range of post-transcriptional RNA modifications that play crucial roles in fundamental biological processes including regulating gene expression. Collectively, they are known as epitranscriptomics. Recent studies implicate 3′ RNA uridylation, the non-templated addition of uridine(s) to the terminal end of RNA, as a key player in epitranscriptomics. In this review, we describe the functional roles and significance of 3′ terminal RNA uridylation that has diverse functions in regulating both mRNAs and non-coding RNAs. In mammals, three Terminal Uridylyl Transferases (TUTases) are primarily responsible for 3′ RNA uridylation. These enzymes are also referred to as polyU polymerases. TUTase 1 (TUT1) is implicated in U6 snRNA maturation via uridylation. The TUTases TUT4 and/or TUT7 are the predominant mediators of all other cellular uridylation. Terminal uridylation promotes turnover for many polyadenylated mRNAs, replication-dependent histone mRNAs that lack polyA-tails, and aberrant structured noncoding RNAs. In addition, uridylation regulates biogenesis of a subset of microRNAs and generates isomiRs, sequent variant microRNAs that have altered function in specific cases. For example, the RNA binding protein and proto-oncogene LIN28A and TUT4 work together to polyuridylate pre-let-7, thereby blocking biogenesis and function of the tumor suppressor let-7 microRNA family. In contrast, monouridylation of Group II pre-miRNAs creates an optimal 3′ overhang that promotes recognition and subsequent cleavage by the Dicer-TRBP complex that then yields the mature microRNA. Also, uridylation may play a role in non-canonical microRNA biogenesis. The overall significance of 3′ RNA uridylation is discussed with an emphasis on mammalian development, gene regulation, and disease, including cancer and Perlman syndrome. We also introduce recent changes to the HUGO-approved gene names for multiple terminal nucleotidyl transferases that affects in part TUTase nomenclature (TUT1/TENT1, TENT2/PAPD4/GLD2, TUT4/ZCCHC11/TENT3A, TUT7/ZCCHC6/TENT3B, TENT4A/PAPD7, TENT4B/PAPD5, TENT5A/FAM46A, TENT5B/FAM46B, TENT5C/FAM46C, TENT5D/FAM46D, MTPAP/TENT6/PAPD1)

The Intracranial Aneurysm Gene THSD1 Connects Endosome Dynamics to Nascent Focal Adhesion Assembly

Background/aims: We recently discovered that harmful variants in THSD1 (Thrombospondin type-1 domain-containing protein 1) likely cause intracranial aneurysm and subarachnoid hemorrhage in a subset of both familial and sporadic patients with supporting evidence from two vertebrate models. The current study seeks to elucidate how THSD1 and patient-identified variants function molecularly in focal adhesions. Methods: Co-immunostaining and co-immunoprecipitation were performed to define THSD1 subcellular localization and interacting partners. Transient expression of patient-identified THSD1 protein variants and siRNA-mediated loss-of-function THSD1 were used to interrogate gene function in focal adhesion and cell attachment to collagen I in comparison to controls. Results: THSD1 is a novel nascent adhesion protein that co-localizes with several known markers such as FAK, talin, and vinculin, but not with mature adhesion marker zyxin. Furthermore, THSD1 forms a multimeric protein complex with FAK/talin/vinculin, wherein THSD1 promotes talin binding to FAK but not to vinculin, a key step in nascent adhesion assembly. Accordingly, THSD1 promotes mature adhesion formation and cell attachment, while its rare variants identified in aneurysm patients show compromised ability. Interestingly, THSD1 also localizes at different stages of endosomes. Clathrin-mediated but not caveolae-mediated endocytosis pathway is involved in THSD1 intracellular trafficking, which positively regulates THSD1-induced focal adhesion assembly, in contrast to the traditional role of endosomes in termination of integrin signals. Conclusions: The data suggest that THSD1 functions at the interface between endosome dynamics and nascent focal adhesion assembly that is impaired by THSD1 rare variants identified from intracranial aneurysm patients

Utilisation de peptides dérivés des filaments intermédiaires pour leurs propriétés antitumorales et de ciblage des cellules de gliome

Works of our laboratory demonstrated that intermediate filaments, which are one of the three cytoskeleton elements, can bind tubulin dimers in specific sites named TBS (Tubulin-Binding Site). Some of these peptides corresponding to TBS sequences can inhibit in vitro tubulin polymerization in microtubules (MT). Works in this thesis consist of continuing the structural and functional characterization of these peptides. Thus, it has been possible to show that one of these peptides from vimentin protein, Vim-TBS.58-81, is able to enter in T98G human glioblastoma cells and to localize in the nucleus of the cells. When coupled to a pro-apoptotic peptide acting in the nuclear compartment, it is able to inhibit cell proliferation. Another peptide from the light neurofilament subunit, NFL-TBS.40-63, is able to enter in many glioma cell lines, to destabilize MT network and to inhibit cell proliferation and migration without affecting healthy cells of the brain (astrocytes and neurons). Injected by stereotaxy in the tumour of rat bearing F98 glioma, this peptide reduces glioma growth and stays localized in tumour tissue. A structural/functional analyze of this peptide highlights some secondary structures, β-sheet and α-helix. After grafting on lipid nanocapsules (LNC) surface, this peptide enhances their entrance in glioma cells in vitro and in vivo. Finally, LNC containing Paclitaxel or Ferrociphenol and grafted with NFL-TBS.40-63 peptide appeared to be more efficient to inhibit tumour growth in mice bearing GL261 glioma and in rat bearing 9L glioma respectively. All of this work presents new functions of targeting and cellular penetration for peptides from intermediate filaments.Les travaux du laboratoire ont montré que les filaments intermédiaires, qui forment un des trois éléments essentiels du cytosquelette, peuvent lier la tubuline sur des sites spécifiques appelés TBS (Tubulin-Binding Site). Certains peptides correspondant à ces séquences sont capables d'inhiber in vitro la polymérisation des microtubules (MT). Les travaux présentés dans cette thèse consistent à poursuivre la caractérisation structurale et fonctionnelle de ces peptides. Ainsi, il a été possible de montrer qu'un peptide issu de la vimentine, Vim-TBS.58-81, est capable de rentrer dans les cellules de glioblastome humain T98G et de se localiser au niveau nucléaire. Une fois couplé à un peptide proapoptotique agissant au niveau nucléaire, il est capable d'inhiber la prolifération de ces cellules. Un autre peptide issu de la sous-unité légère des neurofilaments, NFL-TBS.40-63, est capable de rentrer dans toutes les lignées de gliome testées jusque-là, de déstabiliser leur réseau de MT et d'inhiber leur prolifération et leur migration sans affecter les cellules saines du cerveau (astrocytes et neurones). Injecté par stéréotaxie en intra tumoral chez des rats porteurs d'un gliome F98, ce peptide ralentit la croissance de la tumeur et reste localisé dans le tissu tumoral. Une analyse structure/fonction de ce peptide a mis en évidence des structures secondaires de type feuillet β et hélice α. Après greffage à la surface de nanocapsules lipidiques (NCL), ce peptide permet également l'amélioration de leur entrée dans les cellules de gliome in vitro et in vivo. Enfin, des NCL contenant du Paclitaxel ou du Ferrociphénol et recouvertes de peptide NFL-TBS.40-63 se sont révélées plus efficaces dans l'inhibition de la croissance tumorale dans un modèle de souris porteuse d'un gliome GL261 et dans un modèle de rat porteur d'un gliome 9L respectivement. L'ensemble de ces travaux révèle de nouvelles fonctions de ciblage et de pénétration cellulaire pour des peptides issus de filaments intermédiaires

Utilisation de peptides dérivés des filaments intermédiaires pour leurs propriétés antitumorales et de ciblage des cellules de gliome

Les travaux du laboratoire ont montré que les filaments intermédiaires, qui forment un des trois éléments essentiels du cytosquelette, peuvent lier la tubuline sur des sites spécifiques appelés TBS (Tubulin-Binding Site). Certains peptides correspondant à ces séquences sont capables d'inhiber in vitro la polymérisation des microtubules (MT). Les travaux présentés dans cette thèse consistent à poursuivre la caractérisation structurale et fonctionnelle de ces peptides. Ainsi, il a été possible de montrer qu'un peptide issu de la vimentine, Vim-TBS.58-81, est capable de rentrer dans les cellules de glioblastome humain T98G et de se localiser au niveau nucléaire. Une fois couplé à un peptide proapoptotique agissant au niveau nucléaire, il est capable d'inhiber la prolifération de ces cellules. Un autre peptide issu de la sous-unité légère des neurofilaments, NFL-TBS.40-63, est capable de rentrer dans toutes les lignées de gliome testées jusque-là, de déstabiliser leur réseau de MT et d'inhiber leur prolifération et leur migration sans affecter les cellules saines du cerveau (astrocytes et neurones). Injecté par stéréotaxie en intra tumoral chez des rats porteurs d'un gliome F98, ce peptide ralentit la croissance de la tumeur et reste localisé dans le tissu tumoral. Une analyse structure/fonction de ce peptide a mis en évidence des structures secondaires de type feuillet b et hélice a. Après greffage à la surface de nanocapsules lipidiques (NCL), ce peptide permet également l'amélioration de leur entrée dans les cellules de gliome in vitro et in vivo. Enfin, des NCL contenant du Paclitaxel ou du Ferrociphénol et recouvertes de peptide NFL-TBS.40-63 se sont révélées plus efficaces dans l'inhibition de la croissance tumorale dans un modèle de souris porteuse d'un gliome GL261 et dans un modèle de rat porteur d'un gliome 9L respectivement. L'ensemble de ces travaux révèle de nouvelles fonctions de ciblage et de pénétration cellulaire pour des peptides issus de filaments intermédiaires.Works of our laboratory demonstrated that intermediate filaments, which are one of the three cytoskeleton elements, can bind tubulin dimers in specific sites named TBS (Tubulin-Binding Site). Some of these peptides corresponding to TBS sequences can inhibit in vitro tubulin polymerization in microtubules (MT). Works in this thesis consist of continuing the structural and functional characterization of these peptides. Thus, it has been possible to show that one of these peptides from vimentin protein, Vim-TBS.58-81, is able to enter in T98G human glioblastoma cells and to localize in the nucleus of the cells. When coupled to a pro-apoptotic peptide acting in the nuclear compartment, it is able to inhibit cell proliferation. Another peptide from the light neurofilament subunit, NFL-TBS.40-63, is able to enter in many glioma cell lines, to destabilize MT network and to inhibit cell proliferation and migration without affecting healthy cells of the brain (astrocytes and neurons). Injected by stereotaxy in the tumour of rat bearing F98 glioma, this peptide reduces glioma growth and stays localized in tumour tissue. A structural/functional analyze of this peptide highlights some secondary structures, b-sheet and a-helix. After grafting on lipid nanocapsules (LNC) surface, this peptide enhances their entrance in glioma cells in vitro and in vivo. Finally, LNC containing Paclitaxel or Ferrociphenol and grafted with NFL-TBS.40-63 peptide appeared to be more efficient to inhibit tumour growth in mice bearing GL261 glioma and in rat bearing 9L glioma respectively. All of this work presents new functions of targeting and cellular penetration for peptides from intermediate filaments.ANGERS-BU Médecine-Pharmacie (490072105) / SudocSudocFranceF

A Tubulin Binding Peptide Targets Glioma Cells Disrupting Their Microtubules, Blocking Migration, and Inducing Apoptosis

International audienceDespite aggressive treatment regimes, glioma remains a largely fatal disease. Current treatment limitations are attributed to the precarious locations within the brain where such tumors grow, their highly infiltrative nature precluding complete resection and lack of specificity among agents capable of attenuating their growth. Here, we show that in vitro, glioma cells of diverse origins internalize a peptide encompassing a tubulin-binding site (TBS) on the neurofilament light protein. The internalized peptide disrupts the microtubule network, inhibits migration and proliferation, and leads to apoptosis. Using an intracerebral transplant model, we show that most, if not all, of these responses to peptide exposure also occur in vivo. Notably, a single intratumor injection significantly attenuates tumor growth, while neither peptide uptake nor downstream consequences are observed elsewhere in the host nervous system. Such preferential uptake suggests that the peptide may have potential as a primary or supplementary glioblastoma treatment modality by exploiting its autonomous microtubule-disrupting activity or engaging its capacity to selectively target glioma cells with other cell-disrupting cargos.</p

Structure-function analysis of the glioma targeting NFL-TBS.40-63 peptide corresponding to the tubulin-binding site on the light neurofilament subunit.

We previously reported that a 24 amino acid peptide (NFL-TBS.40-63) corresponding to the tubulin-binding site located on the light neurofilament subunit, selectively enters in glioblastoma cells where it disrupts their microtubule network and inhibits their proliferation. Here, we analyzed the structure-function relationships using an alanine-scanning strategy, in order to identify residues essential for these biological activities. We showed that the majority of modified peptides present a decreased or total loss to penetrate in these cells, or to alter microtubules. Correspondingly, circular dichroism measurements showed that this peptide forms either β-sheet or α-helix structures according to the solvent and that alanine substitution modified or destabilized the structure, in relation with changes in the biological activities. Moreover, substitution of serine residues by phosphoserine or aspartic acid concomitantly decreased the cell penetrating activity and the structure stability. These results indicate the importance of structure for the activities, including selectivity to glioblastoma cells of this peptide, and its regulation by phosphorylation

Replacement of AA residues with functionally equivalent residues restores the properties of NFL-TBS.40-63.

<p>Using a similar experimental approach to that described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049436#pone-0049436-g001" target="_blank">Figure 1</a>, the amino-acids at position 1, 12, 14 and 16 were replaced by amino-acids with similar properties. While substitution of these AAs by alanine abolished their properties, replacement of these AAs by functionally equivalent residues restore the capacity to penetrate in cells and to alter the MT cytoskeleton. As in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049436#pone-0049436-g001" target="_blank">Figure 1A</a>, experiments were triplicated and a minimum of 200 cells was analyzed in each experiment.</p