Replication of HIV-1 requires nuclear export and translation of the incompletely spliced 4 and 9 kb classes of HIV-1 mRNA, which encode the structural and enzymatic proteins of the virus. HIV-1 Rev binds to the Rev-responsive element (RRE) contained in the introns of incompletely spliced HIV-1 mRNAs and mediates their nuclear export via the Crm1 pathway. Sam68C, is a C-terminal deletion mutant of the endogenous human protein Sam68, and has been shown to be a potent inhibitor of Rev-dependent reporters. In this study we have performed deletion analysis of Sam68C, and determined the minimal mutant required for inhibition of Rev-dependent expression is Sam6814(45-54)-300. Sam68C inhibition is specific to RRE/Rev/Crm1 transported mRNAs: the Rev/Crm1 exported reporter construct GagRRE is inhibited while the Tap/p15 transported GagCTE reporter construct is not. Previous work from our lab showed that Sam68C co-localized with the Rev-exported mRNAs in perinuclear bundles. Further investigation has shown that Sam68C inhibition of incompletely spliced HIV-1 mRNAs is independent of the perinuclear bundling of the viral mRNA. We go on to show that Sam68C specifically inhibits the translation of the incompletely spliced HIV-1 mRNAs. Translational inhibition by Sam68C is correlated with a loss of PABP1 binding with no attendant change in abundance, polyadenylation or polyadenosine tail length of the affected mRNAs. The selective inhibition of Crm1 exported HIV-1 mRNAs by Sam68C suggests that it is able to recognize unique characteristics of these viral mRNPs. We show that Rev and the RRE are required, but individually neither is sufficient for complete Sam68C inhibition. Study of the incompletely spliced HIV-1 mRNP revealed that the nuclear cap binding complex, CBP20/80, is not exchanged for eIF4E. Additionally, in cells expressing the HIV-1 provirus, CBP80 relocalizes to the cytoplasm and co-sediments with polysomes. This supports the hypothesis that incompletely spliced HIV-1 mRNAs are translated in an eIF4E-independent, CBP20/80-dependent fashion. This property of the 9kb and 4kb HIV-1 mRNAs could be utilized to develop new therapeutic approaches to controlling HIV-1 infection.

Replication of HIV-1 requires nuclear export and translation of the incompletely spliced 4 and 9 kb classes of HIV-1 mRNA, which encode the structural and enzymatic proteins of the virus. HIV-1 Rev binds to the Rev-responsive element (RRE) contained in the introns of incompletely spliced HIV-1 mRNAs and mediates their nuclear export via the Crm1 pathway. Sam68C, is a C-terminal deletion mutant of the endogenous human protein Sam68, and has been shown to be a potent inhibitor of Rev-dependent reporters. In this study we have performed deletion analysis of Sam68C, and determined the minimal mutant required for inhibition of Rev-dependent expression is Sam6814(45-54)-300. Sam68C inhibition is specific to RRE/Rev/Crm1 transported mRNAs: the Rev/Crm1 exported reporter construct GagRRE is inhibited while the Tap/p15 transported GagCTE reporter construct is not. Previous work from our lab showed that Sam68C co-localized with the Rev-exported mRNAs in perinuclear bundles. Further investigation has shown that Sam68C inhibition of incompletely spliced HIV-1 mRNAs is independent of the perinuclear bundling of the viral mRNA. We go on to show that Sam68C specifically inhibits the translation of the incompletely spliced HIV-1 mRNAs. Translational inhibition by Sam68C is correlated with a loss of PABP1 binding with no attendant change in abundance, polyadenylation or polyadenosine tail length of the affected mRNAs. The selective inhibition of Crm1 exported HIV-1 mRNAs by Sam68C suggests that it is able to recognize unique characteristics of these viral mRNPs. We show that Rev and the RRE are required, but individually neither is sufficient for complete Sam68C inhibition. Study of the incompletely spliced HIV-1 mRNP revealed that the nuclear cap binding complex, CBP20/80, is not exchanged for eIF4E. Additionally, in cells expressing the HIV-1 provirus, CBP80 relocalizes to the cytoplasm and co-sediments with polysomes. This supports the hypothesis that incompletely spliced HIV-1 mRNAs are translated in an eIF4E-independent, CBP20/80-dependent fashion. This property of the 9kb and 4kb HIV-1 mRNAs could be utilized to develop new therapeutic approaches to controlling HIV-1 infection.

The unspliced 9kb HIV-1 RNA (encoding Gag and GagPol) can undergo multiple splicing events to produce members of the 4kb (encoding Env, Vif, Vpr, and Vpu) or 2kb (encoding Tat, Rev and Nef), respectively. The incompletely spliced 9 and 4kb viral RNAs are exported by HIV-1 Rev which interacts with the RRE (Rev responsive element) in these RNAs as well as the nuclear export receptor Crm1. Several proteins can modulate Rev function and/or HIV-1 gene expression, including the nuclear phosphorprotein Sam68. We have found that overexpression of Sam68 stimulates HIV-1 structural gene expression and increases the proportion of unspliced, 3’ end processed viral RNA. This activity requires the RNA binding activity of Sam68. Surprisingly, Sam68 overexpression does not increase the proportion of unspliced, cleaved RNA found in the cytoplasm, suggesting that Sam68 alters the viral RNP to increase its translation. The Sam68 related proteins Slm1 and Slm2 also stimulate 3’ end cleavage and expression of unspliced HIV-1 RNAs. Sam68 and Slm2 were expressed in Hela cells, whereas Slm1 was not. Therefore, we reduced Sam68 expression alone or in combination with Slm2 to determine if these proteins were required for HIV-1 RNA processing or expression. Knockdown of Sam68 and/or Slm2 had little to no effect on viral RNA cleavage or structural gene expression from transiently transfected reporters. Furthermore, depletion of Sam68 only slightly reduced Gag expression from a stably expressed proviral reporter. These results suggest that additional redundant proteins may be present that functionally replace Sam68 and Slm2. We defined a region encompassing the N-terminal GSG (GRP33, Sam68, Gld1) and KH RNA binding motif as the minimal region of Sam68 required to stimulate HIV-1 gene expression in 293 and 293T cells. The minimal mutant enhanced unspliced RNA cleavage in 293T, but not in 293 cells suggesting that Sam68 may act at other stage of the viral lifecycle to increase gene expression.

The unspliced 9kb HIV-1 RNA (encoding Gag and GagPol) can undergo multiple splicing events to produce members of the 4kb (encoding Env, Vif, Vpr, and Vpu) or 2kb (encoding Tat, Rev and Nef), respectively. The incompletely spliced 9 and 4kb viral RNAs are exported by HIV-1 Rev which interacts with the RRE (Rev responsive element) in these RNAs as well as the nuclear export receptor Crm1. Several proteins can modulate Rev function and/or HIV-1 gene expression, including the nuclear phosphorprotein Sam68. We have found that overexpression of Sam68 stimulates HIV-1 structural gene expression and increases the proportion of unspliced, 3’ end processed viral RNA. This activity requires the RNA binding activity of Sam68. Surprisingly, Sam68 overexpression does not increase the proportion of unspliced, cleaved RNA found in the cytoplasm, suggesting that Sam68 alters the viral RNP to increase its translation. The Sam68 related proteins Slm1 and Slm2 also stimulate 3’ end cleavage and expression of unspliced HIV-1 RNAs. Sam68 and Slm2 were expressed in Hela cells, whereas Slm1 was not. Therefore, we reduced Sam68 expression alone or in combination with Slm2 to determine if these proteins were required for HIV-1 RNA processing or expression. Knockdown of Sam68 and/or Slm2 had little to no effect on viral RNA cleavage or structural gene expression from transiently transfected reporters. Furthermore, depletion of Sam68 only slightly reduced Gag expression from a stably expressed proviral reporter. These results suggest that additional redundant proteins may be present that functionally replace Sam68 and Slm2. We defined a region encompassing the N-terminal GSG (GRP33, Sam68, Gld1) and KH RNA binding motif as the minimal region of Sam68 required to stimulate HIV-1 gene expression in 293 and 293T cells. The minimal mutant enhanced unspliced RNA cleavage in 293T, but not in 293 cells suggesting that Sam68 may act at other stage of the viral lifecycle to increase gene expression.

Background: Conventional chemotherapeutics target bulk tumour cells and generally leave cancer stem cell (CSC) populations unaffected. Recent literature characterized the presence and the role of CSC in several types of solid tumors, including colorectal cancer. Colorectal CSCs (CCSCs) display enhanced WNT/β-catenin pathway activity, sustaining self-renewal and tumor-initiating capacity. Thus, CCSCs are crucial for tumour recurrence and metastasis. As one of the main contributors to sustained self-renewal activity in CCSCs, enhanced formation of β-catenin/CBP complex is fostering transactivation of canonical WNT target genes such as c-myc. However, maintenance of healthy intestinal stem cells also dependents on the canonical WNT pathway. Thus, selective targeting CCSCs while sparring normal intestinal cells is still a significant challenge. Interestingly, Sam68 is a key mediator of the interaction between β-catenin and CBP. It has been reported as a “druggable” target to selectively disrupt β-catenin/CBP in CSCs. Indeed, CWP232228 successfully targets CSCs in AML by facilitating Sam68/CBP complex formation, and consequently lowering the abundance of β-catenin/CBP complexes. CWP232228 was clinically tested on multiple human cancers. Unfortunately, such clinical trials were halted due to unknown causes, and limited information was released on clinical safety and benefits. Consequently, developing more potent pharmacological modulators of Sam68/CBP complex formation is still highly relevant to eradicate CCSCs. Here we describe the discovery and characterization of a new CWP analog, known as YB-0158, which displays enhanced potency and neoplastic selectivity against CCSC. Methods and Results: Following the confirmation that ICG/CWP class of compounds bind to Sam68 in CSCs, I used in silico docking methods to screen for CWP analogs having high predicted affinity for Sam68 Cterminal proline-rich domain. Using high content imaging techniques, I confirmed our top candidate (YB-0158) as more potent vs. CWP parent molecule to compromise cell growth, to induce loss of pluripotency, and to increase Sam68 nuclear localization in a surrogate model of human CSCs. YB-0158 also displayed enhanced selective toxicity in colorectal cancer models vs. normal intestinal epithelium progenitor cells. Moreover, I confirmed that YB-0158 exert negative impact on cancer cell growth by inducing apoptosis and reducing proliferation. Lentiviral-based knockdowns explicitly displayed decrease in drug effectivity in the absence of Sam68, reinforcing the essential role of Sam68 mediating ICG-001/CWP response in CSCs. I demonstrated that Sam68 expression is enriched in tumor-initiating cell fractions derived from primary colorectal tumor tissues vs. bulk heterogeneous tumor organoids. Therefore, YB-0158 showed striking efficacy at supressing tumor-initiation activity in a patient-based serial organoid formation assay. Finally, YB-0158 eradicated CSCs activity in vivo as demonstrated by a syngeneic mouse-to-mouse serial transplantation assay. Conclusion: Overall, YB-0158 is a novel analog of CWP232228 with superior potency to target CCSCs activity through facilitation of Sam68 nuclear localization, thus reducing the interaction frequency between CBP and β-catenin.

Les épines dendritiques sont des petites protubérances remarquablement dynamiques à la surface des dendrites, correspondant à la partie postsynaptique des synapses excitatrices. En réponse à l'activité neuronale, leur géométrie et leur composition biochimique changent, modulant ainsi la force synaptique. Entres autres, la synthèse locale de nouvelles protéines et une modification de la composition protéique membranaire assurent ces changements. Nous avons montré que la ribonucléoprotéine Sam68 régule la traduction de l'ARNm du facteur d'élongation de la traduction eEF1A ; et que TLS, d'ordinaire nucléaire, est localisée dans les dendrites, près des synapses. Nous avons montré que les mutants de CHMP2B (membre du complexe endosomial ESCRT III et mutée dans une maladie neurodégénérative) affectent la morphologie des épines dendritiques, leur taille est réduite, et diminuent la proportion des courants synaptiques de grande amplitude.

Indiana University-Purdue University Indianapolis (IUPUI) / More than 20 million people have died of AIDS since the early eighties, while nearly 34 millions are currently infected with the HIV. Anti-retroviral therapy (ART) directed at key viral enzymes has changed AIDS from uniformly fatal to a manageable chronic disease. However, ART-associated drug resistance and toxicity have posed a great challenge for long-term management of the disease and have called for development of new therapeutics. In this study, we focused on the viral factor Nef and the host factor Sam68. Nef is a major pathogenic viral determinant for HIV-1, and no therapeutics have been targeted to this factor. Sam68 is indispensible for HIV-1 propagation. We revealed that Sam68 variants were very potent in preventing Nef expression. We found that these effects were associated with their ability to form a macromolecular structure called stress granules (SG). In addition, we demonstrated that these variants bound to nef mRNA in a sequence-specific manner. Furthermore, we showed that these variants co-localized with nef mRNA in SG. Importantly, we validated these findings in the context of HIV-1 infection of its natural target cells and found significant loss of Nef function in these cells. Taken together, these results demonstrate that SG induction and nef mRNA sequestration account for translational suppression of Nef expression and offer a new strategy for development of anti-HIV therapeutics. Sam68 is implicated in a variety of other important cellular processes. Our findings that Sam68 variants were able to induce SG formation prompted us to investigate whether wild-type Sam68 was also recruited to SG. We found that Sam68 was increasingly recruited into SG under oxidative stress, and that its specific domains were involved. However, Sam68 knockdown had no effects on SG assembly, suggesting that Sam68 is not a constitutive component of SG assembly. Lastly, we demonstrated that Sam68 complexed with TIA-1, an essential SG component. Taken together, these results provide direct evidence for the first time that Sam68 is recruited into SG through complexing with TIA-1, and suggest that SG recruitment of Sam68 and ensuing changes in Sam68 physiological functions are part of the host response to external stressful conditions.