MicroRNAs repress mRNA translation by guiding Argonaute proteins to partially complementary binding sites, primarily within the 3′ untranslated region (UTR) of target mRNAs. In cell lines, Argonaute-bound microRNAs exist mainly in high molecular weight RNA-induced silencing complexes (HMW-RISC) associated with target mRNA. Here we demonstrate that most adult tissues contain reservoirs of microRNAs in low molecular weight RISC (LMW-RISC) not bound to mRNA, suggesting that these microRNAs are not actively engaged in target repression. Consistent with this observation, the majority of individual microRNAs in primary T cells were enriched in LMW-RISC. During T-cell activation, signal transduction through the phosphoinositide-3 kinase–RAC-alpha serine/threonine-protein kinase–mechanistic target of rapamycin pathway increased the assembly of microRNAs into HMW-RISC, enhanced expression of the glycine-tryptophan protein of 182 kDa, an essential component of HMW-RISC, and improved the ability of microRNAs to repress partially complementary reporters, even when expression of targeting microRNAs did not increase. Overall, data presented here demonstrate that microRNA-mediated target repression in nontransformed cells depends not only on abundance of specific microRNAs, but also on regulation of RISC assembly by intracellular signaling.
A robust network of transcription factors and an open chromatin landscape are hallmarks of the naïve pluripotent state. Recently, the acetyllysine reader Brd4 has been implicated in stem cell maintenance, but the relative contribution of Brd4 to pluripotency remains unclear. Here we show that Brd4 is dispensable for self-renewal and pluripotency of embryonic stem cells (ESCs). When maintained in their ground state, ESCs retain transcription factor binding and chromatin accessibility independent of Brd4 function or expression. In metastable ESCs, Brd4 independence can be achieved by increased expression of pluripotency transcription factors including STAT3, Nanog or Klf4 so long as the DNA methylcytosine oxidases, Tet1 and Tet2, are present. These data reveal that Brd4 is not essential for ESC self-renewal. Rather, the levels of pluripotency transcription factor abundance and Tet1/2 function determine the extent to which bromodomain recognition of protein acetylation contributes to the maintenance of gene expression and cell identity.
Septins (SEPTs) are filamentous guanosine-5′-triphosphate (GTP)-binding proteins, which affect microtubule (MT)-dependent functions including membrane trafficking and cell division, but their precise role in MT dynamics is poorly understood. Here, in vitro reconstitution of MT dynamics with SEPT2/6/7, the minimal subunits of septin heteromers, shows that SEPT2/6/7 has a biphasic concentration-dependent effect on MT growth. Lower concentrations of SEPT2/6/7 enhance MT plus-end growth and elongation, while higher and intermediate concentrations inhibit and pause plus-end growth, respectively. We show that SEPT2/6/7 has a modest preference for GTP- over guanosine diphosphate (GDP)-bound MT lattice and competes with end-binding protein 1 (EB1) for binding to guanosine 5′- O-[γ-thio]triphosphate (GTPγS)-stabilized MTs, which mimic the EB1-preferred GDP-Pi state of polymerized tubulin. Strikingly, SEPT2/6/7 triggers EB1 dissociation from plus-end tips in cis by binding to the MT lattice and in trans when MT plus ends collide with SEPT2/6/7 filaments. At these intersections, SEPT2/6/7 filaments were more potent barriers than actin filaments in pausing MT growth and dissociating EB1 in vitro and in live cells. These data demonstrate that SEPT2/6/7 complexes and filaments can directly impact MT plus-end growth and the tracking of plus end–binding proteins and thereby may facilitate the capture of MT plus ends at intracellular sites of septin enrichment. [Media: see text]
Cell growth and proliferation require the coordinated activation of many cellular processes, including cap-dependent mRNA translation. MicroRNAs oppose cap-dependent translation and set thresholds for expression of target proteins. Emerging data suggest that microRNA function is enhanced by cellular activation due in part to induction of the RNA-induced silencing complex (RISC) scaffold protein GW182. In the current study, we demonstrate that increased expression of GW182 in activated or transformed immune cells results from effects of phosphoinositol 3-kinase-Akt-mechanistic target of rapamycin (PI3K-AktmTOR) and Jak-Stat-Pim signaling on the translation of GW182 mRNA. Both signaling pathways enhanced polysome occupancy and eukaryotic initiation factor 4E (eIF4E) binding to the 5= 7mG cap of GW182 mRNA. The effect of Jak-Stat-Pim signaling on polysome occupancy and expression of GW182 protein was greater than that of PI3K-Akt-mTOR signaling, likely resulting from enhanced eIF4A-dependent unwinding of G-quadruplexes in the 5= untranslated region of GW182 mRNA. Consistent with this, GW182 expression and microRNA function were reduced by inhibition of mTOR or Pim kinases, translation initiation complex assembly, or eIF4A function. Taken together, these data provide a mechanistic link between microRNA function and cap-dependent translation that allows activated immune cells to maintain microRNA-mediated repression of targets despite enhanced rates of protein synthesis.
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