[Show abstract][Hide abstract] ABSTRACT: 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.
Proceedings of the National Academy of Sciences 01/2015; 112(3). DOI:10.1073/pnas.1424217112 · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The interactions between the nephrogenic mesenchyme and the ureteric bud during kidney development are well documented. While recent studies have shed some light on the importance of the stroma during renal development, many of the signals generated in the stroma, the genetic pathways and interaction networks involving the stroma are yet to be identified. Our previous studies demonstrate that retinoids are crucial for branching of the ureteric bud and for patterning of the cortical stroma. In the present study we demonstrate that autocrine retinoic acid (RA) signaling in stromal cells is critical for their survival and patterning, and show that Extracellular matrix 1, Ecm1, a gene that in humans causes irritable bowel syndrome and lipoid proteinosis, is a novel RA-regulated target in the developing kidney, which is secreted from the cortical stromal cells surrounding the cap mesenchyme and ureteric bud. Our studies suggest that Ecm1 is required in the ureteric bud for regulating the distribution of Ret which is normally restricted to the tips, as inhibition of Ecm1 results in an expanded domain of Ret expression and reduced numbers of branches. We propose a model in which retinoid signaling in the stroma activates expression of Ecm1, which in turn down-regulates Ret expression in the ureteric bud cleft, where bifurcation normally occurs and normal branching progresses.
PLoS ONE 12/2013; 8(12):e84155. DOI:10.1371/journal.pone.0084155 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Pre-mRNA splicing and polyadenylation are critical steps in the maturation of eukaryotic mRNA. U1 snRNP is an essential
component of the splicing machinery and participates in splice-site selection and spliceosome assembly by base-pairing to the 5′ splice site. U1 snRNP also plays an additional, nonsplicing global function in 3′ end mRNA processing; it actively suppresses the polyadenylation machinery from using early, mostly intronic polyadenylation signals which would lead to aberrant, truncated mRNAs. Thus, U1 snRNP safeguards pre-mRNA transcripts against premature polyadenylation and contributes to the regulation of alternative polyadenylation. Here, we review the role of U1 snRNP in 3′ end mRNA processing, outline the evidence that led to the recognition of its physiological, general role in inhibiting polyadenylation, and finally highlight the possibility of manipulating this U1 snRNP function for therapeutic purposes in cancer.
International Journal of Cell Biology 10/2013; 2013:846510. DOI:10.1155/2013/846510
[Show abstract][Hide abstract] ABSTRACT: Next-generation antisense technologies are re-emerging as viable and powerful approaches to the treatment of several genetic diseases. Similar strategies are also being applied to cancer therapy. Reprogramming of the expression of endogenous oncogenic products to replace them with functional antagonists, by interfering with alternative splicing (AS) or polyadenylation, provides a promising novel approach to address acquired drug resistance and previously undruggable targets.
Drug Discovery Today Therapeutic Strategies 09/2013; 10(3). DOI:10.1016/j.ddstr.2013.06.002
[Show abstract][Hide abstract] ABSTRACT: The microRNA (miRNA)-induced silencing complex (miRISC) controls gene expression
by a posttranscriptional mechanism involving translational repression and/or
promoting messenger RNA (mRNA) deadenylation and degradation. The GW182/TNRC6
(GW) family proteins are core components of the miRISC and are essential for
miRNA function. We show that mammalian GW proteins have distinctive functions in
the miRNA pathway, with GW220/TNGW1 being essential for the formation of GW/P
bodies containing the miRISC. miRISC aggregation and formation of GW/P bodies
sequestered and stabilized translationally repressed target mRNA. Depletion of
GW220 led to the loss of GW/P bodies and destabilization of miRNA-targeted mRNA.
These findings support a model in which the cellular localization of the miRISC
regulates the fate of the target mRNA.
The Journal of Cell Biology 08/2012; 198(4):529-44. DOI:10.1083/jcb.201201153 · 9.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In humans and mice, mutations in the Ret gene result in Hirschsprung's disease and renal defects. In the embryonic kidney, binding of Ret to its ligand, Gdnf, induces a program of epithelial cell remodeling that controls primary branch formation and branching morphogenesis within the kidney. Our previous studies showed that transcription factors belonging to the retinoic acid (RA) receptor family are crucial for controlling Ret expression in the ureteric bud; however, the mechanism by which retinoid-signaling acts has remained unclear. In the current study, we show that expression of a dominant-negative RA receptor in mouse ureteric bud cells abolishes Ret expression and Ret-dependent functions including ureteric bud formation and branching morphogenesis, indicating that RA-receptor signaling in ureteric bud cells is crucial for renal development. Conversely, we find that RA-receptor signaling in ureteric bud cells depends mainly on RA generated in nearby stromal cells by retinaldehyde dehydrogenase 2, an enzyme required for most fetal RA synthesis. Together, these studies suggest that renal development depends on paracrine RA signaling between stromal mesenchyme and ureteric bud cells that regulates Ret expression both during ureteric bud formation and within the developing collecting duct system.
Development 01/2010; 137(2):283-92. DOI:10.1242/dev.040287 · 6.27 Impact Factor