The compendium of RNA-binding proteins (RBPs) has been greatly expanded by the development of RNA-interactome capture (RIC). However, it remained unknown if the complement of RBPs changes in response to environmental perturbations and whether these rearrangements are important. To answer these questions, we developed "comparative RIC" and applied it to cells challenged with an RNA virus called sindbis (SINV). Over 200 RBPs display differential interaction with RNA upon SINV infection. These alterations are mainly driven by the loss of cellular mRNAs and the emergence of viral RNA. RBPs stimulated by the infection redistribute to viral replication factories and regulate the capacity of the virus to infect. For example, ablation of XRN1 causes cells to be refractory to SINV, while GEMIN5 moonlights as a regulator of SINV gene expression. In summary, RNA availability controls RBP localization and function in SINV-infected cells.

Background: Recent evidence has accumulated that MicroRNA (miRNA) dysregulation occurs in the majority of human malignancies including acute myeloid leukemia (AML) and may contribute to onco-/leukemo-genesis.Methods: The expression levels of miR-370 and FoxM1 were assessed in 48 newly diagnosed AML patients, 40 AML patients in 1^stcomplete remission (CR) and 21 healthy controls. Quantitative real-time PCR, western blots, colony formation assay, and β-Galactosidase ( SA-β-Gal) staining were used to characterize the changes induced by overexpression or inhibition of miR-370 or FoxM1.Results: We found that the down-regulation of miR-370 expression was a frequent event in both leukemia cell lines and primary leukemic cells from patients with de novo AML. Lower levels of miR-370 expression were found in 37 of 48 leukemic samples from AML patients compared to those in bone marrow cells derived from healthy adult individuals. Ectopic expression of miR-370 in HL60 and K562 cells led to cell growth arrest and senescence. In contrast, depletion of miR-370 expression using RNA interference enhanced the proliferation of those leukemic cells. Mechanistically, miR-370 targets the transcription factor FoxM1, a well established oncogenic factor promoting cell cycle progression. Moreover, when HL60 and K562 cells were treated with 5-aza-2^′-deoxycytidine, a DNA methylation inhibitor, miR-370 expression was up-regulated, which indicates epigenetic silencing of miR-370 in leukemic cells.Conclusions: Taken together, miR-370 may function as a tumor suppressor by targeting FoxM1, and the epigenetic silence of miR-370 thus leads to derepression of FoxM1 expression and consequently contributes to AML development and progression.

The DNA methylation inhibitor induces telomere dysfunction and apoptosis of leukemia cells that is attenuated by telomerase over-expression The DNA methylation inhibitor induces telomere dysfunction and apoptosis of leukemia cells that is attenuated by telomerase over-expression

The somatic mutation of FLT3 occurs in 30% of acute myeloid leukemia (AML), with the majority of mutations exhibiting internal tandem duplication (ITD). On the other hand, the induction of telomerase reverse transcriptase (hTERT) and the activation of telomerase is a key step in AML development. Here, we sought to determine whether FLT3ITD regulates hTERT expression in AML cells and whether hTERT expression affects FLT3 inhibitors’ therapeutic efficacy on AML. FLT3ITD-harboring AML cell lines and primary cells treated with the FLT3 inhibitor PKC412 displayed a rapid decline in the levels of hTERT mRNA and telomerase activity. Moreover, PKC412 inhibited hTERT gene transcription in a c-MYC-dependent manner. The ectopic expression of hTERT significantly attenuated the apoptotic effect of PKC412 on AML cells. Mechanistically, hTERT enhanced the activity of FLT3 downstream effectors or alternative RTK signaling, thereby enhancing AKT phosphorylation, in AML cells treated with PKC412. Collectively, PKC412 downregulates hTERT expression and telomerase activity in a MYC-dependent manner and this effect is required for its optimal anti-AML efficacy, while hTERT over-expression confers AML cells resistance to a targeted therapeutic agent PKC412. These findings suggest that the functional interplay between FLT3ITD and hTERT contributes to the AML pathogenesis and interferes with the efficacy of FLT3ITD-targeted therapy.

Bortezomib-mediated down-regulation of telomerase and disruption of telomere homeostasis contributes to apoptosis of malignant cells

The induced pluripotent stem cell (iPSC) technology has provided a unique opportunity to develop disease-specific models and personalized treatment for genetic disorders, and is well suitable for the study of Werner syndrome (WS), an autosomal recessive disease with adult onset of premature aging caused by mutations in the RecQ like helicase (WRN) gene. WS-derived fibroblasts were previously shown to be able to generate iPSCs; however, it remains elusive how WS-derived iPSCs behave and whether they are able to mimic the disease-specific phenotype. The present study was designed to address these issues. Unexpectedly, we found that a specific WS fibroblast line of homozygous truncation mutation was difficult to be reprogrammed by using the Yamanaka factors even under hypoxic conditions due to their defect in induction of hTERT, the catalytic unit of telomerase. Ectopic expression of hTERT restores the ability of this WS fibroblast line to form iPSCs, although with a low efficiency. To examine the phenotype of WRN-deficient pluripotent stem cells, we also generated WRN knockout human embryonic stem (ES) cells by using the CRISPR/Cas9 method. The iPSCs derived from WS-hTERT cells and WRN-/- ESCs are fully pluripotent, express pluripotent markers and can differentiate into three germ layer cells; however, WS-iPSCs and WRN-/- ESCs show S phase defect in cell cycle progression. Moreover, WS-iPSCs and WRN-/- ESCs, like WS patient-derived fibroblasts, remain hypersensitive to topoisomerase inhibitors. Collectively, WS-derived iPSCs and WRN-/- ESCs mimic the intrinsic disease phenotype, which may serve as a suitable disease model, whereas not be good for a therapeutic purpose without gene correction.