During spliceosome activation, a large structural rearrangement occurs that involves the release of two small nuclear RNAs, U1 and U4, and the addition of a protein complex associated with Prp19p. We show here that the Prp19p-associated complex is required for stable association of U5 and U6 with the spliceosome after U4 is dissociated. Ultraviolet crosslinking analysis revealed the existence of two modes of base pairing between U6 and the 5' splice site, as well as a switch of such base pairing from one to the other that required the Prp19p-associated complex during spliceosome activation. Moreover, a Prp19p-dependent structural change in U6 small nuclear ribonucleoprotein particles was detected that involves destabilization of Sm-like (Lsm) proteins to bring about interactions between the Lsm binding site of U6 and the intron sequence near the 5' splice site, indicating dynamic association of Lsm with U6 and a direct role of Lsm proteins in activation of the spliceosome.
Fragile X syndrome is caused by the absence of functional fragile X mental retardation protein (FMRP), an RNA binding protein. The molecular mechanism of aberrant protein synthesis in fmr1 KO mice is closely associated with the role of FMRP in mRNA transport, delivery, and local protein synthesis. We show that GFP-labeled Fmr1 and CaMKIIα mRNAs undergo decelerated motion at 0-40 min after group I mGluR stimulation, and later recover at 40-60 min. Then we investigate targeting of mRNAs associated with FMRP after neuronal stimulation. We find that FMRP is synthesized closely adjacent to stimulated mGluR5 receptors. Moreover, in WT neurons, CaMKIIα mRNA can be delivered and translated in dendritic spines within 10 min in response to group I mGluR stimulation, whereas KO neurons fail to show this response. These data suggest that FMRP can mediate spatial mRNA delivery for local protein synthesis in response to synaptic stimulation.fragile X syndrome | dendritic mRNA targeting | local translation F ragile X syndrome (FXS) is the most common form of inherited mental retardation and is caused by the loss of function of the FMR1 gene, which encodes fragile X mental retardation protein (FMRP) (1). FXS affects 1 in 4,000 males and 1 in 6,000 females on average and is characterized by hyperactivity, attention deficits, autistic-like behaviors, and seizures (2). Dendritic spine morphology in the cerebral cortex of FXS patients and in the fmr1 KO mouse model shows more immature long thin spines than mature stubby, mushroom-shaped spines (3). Furthermore, group I mGluR-dependent long-term depression in the hippocampus is exaggerated in the fmr1 KO model (4). These findings suggest that FMRP functions in synaptic development and plasticity.Activity-dependent local translation is a fundamental mechanism underlying synaptic plasticity (5, 6). Inhibition of protein synthesis attenuates specific types of long-term plasticity (7,8). Morphological changes in dendritic spines can be blocked by protein synthesis inhibitors (9). In the fmr1 KO model, it has been shown that aberrant synthesis of individual proteins such as CaMKIIα, PSD-95, and MAP1b, upon group I mGluR stimulation, is associated with defective long-term plasticity (10-12). Here we have studied specific molecular mechanisms to elucidate aberrant localized translation in the fmr1 KO model. The molecular basis of FMRP's role in translation-dependent plasticity remains unclear despite extensive study. FMRP is a ribosome-associated RNA binding protein with selective affinity (13,14). Upon neuronal stimulation, FMRP may regulate protein levels by mediating translational regulation and mRNA trafficking (11, 15). FMRP, mRNA, and other RNA binding proteins can form ribonucleoprotein (RNP) or granule structures and couple with motor proteins to be transported in dendrites (16-18). Dendritic transport of FMRP and associated mRNAs, such as Fmr1, CaMKIIα, and MAP1b, are regulated by group I mGluR signaling (15, 19). It is not yet fully understood how and when mRNA is delivered to th...
Diabetes is linked to loss of pancreatic beta-cells. Pluripotent stem cells offer a valuable source of human beta-cells for basic studies of their biology and translational applications. However, the signalling pathways that regulate beta-cell development and functional maturation are not fully understood. Here we report a high content chemical screen, revealing that H1152, a ROCK inhibitor, promotes the robust generation of insulin-expressing cells from multiple hPSC lines. The insulin expressing cells obtained after H1152 treatment show increased expression of mature beta cell markers and improved glucose stimulated insulin secretion. Moreover, the H1152-treated beta-like cells show enhanced glucose stimulated insulin secretion and increased capacity to maintain glucose homeostasis after transplantation. Conditional gene knockdown reveals that inhibition of ROCKII promotes the generation and maturation of glucose-responding cells. This study provides a strategy to promote human beta-cell maturation and identifies an unexpected role for the ROCKII pathway in the development and maturation of beta-like cells.
bThe yeast Sad1 protein was previously identified in a screen for factors involved in the assembly of the U4/U6 di-snRNP particle. Sad1 is required for pre-mRNA splicing both in vivo and in vitro, and its human orthologue has been shown to associate with U4/U6.U5 tri-snRNP. We show here that Sad1 plays a role in maintaining a functional form of the tri-snRNP by promoting the association of U5 snRNP with U4/U6 di-snRNP. In the absence of Sad1, the U4/U6.U5 tri-snRNP dissociates into U5 and U4/U6 upon ATP hydrolysis and cannot bind to the spliceosome. The separated U4/U6 and U5 can reassociate upon incubation more favorably in the absence of ATP and in the presence of Sad1. Brr2 is responsible for mediating ATP-dependent dissociation of the tri-snRNP. Our results demonstrate a role of Sad1 in maintaining the integrity of the tri-snRNP by counteracting Brr2-mediated dissociation of tri-snRNP and provide insights into homeostasis of the tri-snRNP. Splicing of precursor mRNA takes place via two steps of transesterification reactions and is catalyzed by a large ribonucleoprotein complex called the spliceosome. The spliceosome consists of five small nuclear RNAs (snRNAs), U1, U2, U4, U5, and U6, and nearly a hundred proteins. These snRNAs are associated with specific protein components to form small nuclear ribonucleoprotein particles (snRNPs), among which U4 and U6 base pair with each other to form a U4/U6 di-snRNP, which can further associate with U5 snRNP to form a U4/U6.U5 tri-snRNP. The spliceosome is assembled via sequential binding of snRNPs to the pre-mRNA in the order U1, U2, and then the tri-snRNP. U1 binds to the 5= splice site through base pairing of the U1 snRNA with the 5= splice site sequence, and U2 recognizes the branch site through base pairing of U2 snRNA with the branch site sequence (1-5). A subsequent conformational rearrangement results in the release of U1 snRNP and U4 snRNP, accompanied by new base pairings between U6 and U2 and between U6 and the 5= splice site (6-8). A protein complex associated with Prp19, called NTC (for nineteen complex), is then added to the spliceosome to stabilize specific interactions of U5 and U6 with pre-mRNA to form the activated spliceosome (9-11), which can catalyze transesterification reactions.The release of U1 and U4 from the spliceosome involves destabilization of base pair interactions between U1 and the 5= splice site and the unwinding of the U4/U6 duplex, which are mediated by DEXD/H-box RNA helicases Prp28 and Brr2, respectively. Genetic studies have revealed a requirement for Prp28 to destabilize U1-5= splice site base pairing, and this requirement can be eliminated by mutations in U1 snRNA or U1-C protein that destabilize the base pair interaction of U1 and the 5= splice site (12, 13). Prp28 may act by directly unwinding the U1-5= splice site duplex or by displacing U1C to destabilize U1-5= splice site base pairing (12,14,15). Brr2 is an intrinsic component of the U5 snRNP and is associated with the spliceosome, along with the binding of the trisnRNP. Br...
The Prp19-associated complex, consisting of at least eight protein components, is involved in spliceosome activation by specifying the interaction of U5 and U6 with pre-mRNA for their stable association with the spliceosome after U4 dissociation. We show here that yeast cells depleted of one or two of the Prp19-associated components, accumulate the free form of U4. In NTC25-deleted cells, the level of U6 was also reduced. Extracts prepared from NTC25-deleted cells contained neither free U4 nor U6 and were ineffective in spliceosome recycling in the in vitro splicing reaction. Overexpression of U6 partially rescued the temperature-sensitive growth defect and decreased the relative amount of free U4 in NTC25-deleted cells, indicating that the accumulation of free U4 was ac onsequence of insufficient amounts of U6 snRNA. Extracts prepared from U6-overproducing NTC25-deleted cells containing free-form U6 were capable of spliceosome recycling, suggesting ar ole of free U6 RNP in spliceosome recycling. Our results demonstrate that in addition to direct participation in spliceosome activation, the Prp19-associated complex has an indirect role in spliceosome recycling through affecting the biogenesis of U4/U6 snRNP in the in vivo splicing reaction.Keywords: Prp19; U4; U6; spliceosome recycling INTRODUCTIONSplicing of pre-mRNAt akes place on al arge dynamic ribonucleoprotein complex, the spliceosome, which assembles through ordered interactions of four small nuclear ribonucleoproteinparticles (snRNPs),U1, U2,U4/U6,and U5,and numerous proteinf actors (Willa nd Lü hrmann 1997;S taley andG uthrie 1998;B urge et al.1 999;Brow 2002;H artmuth et al.2 002;J uricae ta l. 2002;Z houe ta l. 2002).D uring spliceosomea ssembly, U1 firstb inds to the5 9 -splices iteo f thep re-mRNA, followed by bindingo fU 2t ot he branch site. The pre-formed U4/U6.U5t ri-snRNP is then recruited to the spliceosome to form ac omplexc ontaining all five snRNAs. Subsequently, al arge conformational rearrangement in the spliceosome occurs in which U1 and U4 are released, accompanied by formation of new base-pairing betweenU6and U2,and U6 andthe 5 9 -splicesiteofthe premRNA. This leads to the activation of the spliceosome, on which catalytic reactions can then take place (Brow 2002).Activationo ft he spliceosome requires unwinding of base-pairing between U1 and the 5 9 -splice site and between U4 and U6 as the first step,f reeing the 5 9 -splice site sequence and U6 for new base-pair formation. Factors that mediate unwinding of these base-pairings have notb een directly demonstrated, although Brr2 has been implicated in the unwinding of U4/U6 (Raghunathan and Guthrie 1998a), andP rp28 in displacing U1 from the 5 9 -splice site (Staley and Guthrie1999; Chenetal. 2001b). U4 snRNA is presumed to function as the repressor of U6 prior to the activation of the spliceosome and does not participate in catalytic reactions (Yean and Lin 1991). After release from the spliceosome, U4 appears to reassociate with U6 immediately to form the di-snRNPcomplex, an...
Cells with different cohesive properties self-assemble in a spatiotemporal and context-dependent manner. Previous studies on cell self-organization mainly focused on the spontaneous structural development within a short period of time during which the cell numbers remained constant. However the effect of cell proliferation over time on the self-organization of cells is largely unexplored. Here, we studied the spatiotemporal dynamics of self-organization of a co-culture of MDA-MB-231 and MCF10A cells seeded in a well defined space (i.e. non-adherent microfabricated wells). When cell-growth was chemically inhibited, high cohesive MCF10A cells formed a core surrounded by low cohesive MDA-MB-231 cells on the periphery, consistent with the differential adhesion hypothesis (DAH). Interestingly, this aggregate morphology was completely inverted when the cells were free to grow. At an initial seeding ratio of 1 : 1 (MDA-MB-231 : MCF10A), the fast growing MCF10A cells segregated in the periphery while the slow growing MDA-MB-231 cells stayed in the core. Another morphology developed at an inequal seeding ratio (4 : 1), that is, the cell mixtures developed a side-by-side aggregate morphology. We conclude that the cell self-organization depends not only on the cell cohesive properties but also on the cell seeding ratio and proliferation. Furthermore, by taking advantage of the cell self-organization, we purified human embryonic stem cells-derived pancreatic progenitors (hESCs-PPs) from co-cultured feeder cells without using any additional tools or labels.
A simple, robust, and cost-effective method is developed to fabricate nanofibrous micropatterns particularly microposts and microwells of controlled shapes. The key to this method is the use of an easily micropatternable and intrinsically conductive metal alloy as a template to collect electrospun fibers. The micropatterned alloy allows conformal fiber deposition with high fidelity on its topographical features and in situ formation of diverse, free-standing micropatterned nanofibrous membranes. Interestingly, these membranes can serve as structural frames to form robust hydrogel micropatterns that may otherwise be fragile on their own. These hybrid micropatterns represent a new platform for cell encapsulation where the nanofiber frames enhance the mechanical integrity of hydrogel and the micropatterns provide additional surface area for mass transfer and cell loading.
SummaryAlthough endothelial cells have been shown to affect mouse pancreatic development, their precise function in human development remains unclear. Using a coculture system containing human embryonic stem cell (hESC)-derived progenitors and endothelial cells, we found that endothelial cells play a stage-dependent role in pancreatic development, in which they maintain pancreatic progenitor (PP) self-renewal and impair further differentiation into hormone-expressing cells. The mechanistic studies suggest that the endothelial cells act through the secretion of EGFL7. Consistently, endothelial overexpression of EGFL7 in vivo using a transgenic mouse model resulted in an increase of PP proliferation rate and a decrease of differentiation toward endocrine cells. These studies not only identified the role of EGFL7 as the molecular handle involved in the crosstalk between endothelium and pancreatic epithelium, but also provide a paradigm for using hESC stepwise differentiation to dissect the stage-dependent roles of signals controlling organogenesis.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.