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Background & Aims: The Hippo pathway controls organ size through a negative regulation of the transcription co-activator Yap1. The overexpression of hyperactive mutant Yap1 or deletion of key components in the Hippo pathway leads to increased organ size in different species. Analysis of interactions of this pathway with other cellular signals corroborating organ size control is limited in part due to the difficulties associated with development of rodent models., Methods: Here, we develop a new model of reversible induction of the liver size in mice using siRNA-nanoparticles targeting two kinases of the Hippo pathway, namely, mammalian Ste20 family kinases 1 and 2 (Mst1 and Mst2), and an upstream regulator, neurofibromatosis type II (Nf2)., Results: The triple siRNAs nanoparticle-induced hepatomegaly in mice phenocopies one observed with Mst1(-/-)Mst2(-/-) liver-specific depletion, as shown by extensive proliferation of hepatocytes and activation of Yap1. The simultaneous co-treatment with a fourth siRNA nanoparticle against Yap1 fully blocked the liver growth. Hippo pathway-induced liver enlargement is associated with p53 activation, evidenced by its accumulation in the nuclei and upregulation of its target genes. Moreover, injections of the triple siRNAs nanoparticle in p53(LSL/LSL) mice shows that livers lacking p53 expression grow faster and exceed the size of livers in p53 wild-type animals, indicating a role of p53 in controlling Yap1-induced liver growth., Conclusion: Our data show that siRNA-nanoparticulate manipulation of gene expression can provide the reversible control of organ size in adult animals, which presents a new avenue for the investigation of complex regulatory networks in liver., (Copyright © 2015 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Dysfunctional endothelium contributes to more diseases than any other tissue in the body. Small interfering RNAs (siRNAs) can help in the study and treatment of endothelial cells in vivo by durably silencing multiple genes simultaneously, but efficient siRNA delivery has so far remained challenging. Here, we show that polymeric nanoparticles made of low-molecular-weight polyamines and lipids can deliver siRNA to endothelial cells with high efficiency, thereby facilitating the simultaneous silencing of multiple endothelial genes in vivo. Unlike lipid or lipid-like nanoparticles, this formulation does not significantly reduce gene expression in hepatocytes or immune cells even at the dosage necessary for endothelial gene silencing. These nanoparticles mediate the most durable non-liver silencing reported so far and facilitate the delivery of siRNAs that modify endothelial function in mouse models of vascular permeability, emphysema, primary tumour growth and metastasis.

Argonaute 2 (Ago2) is the only mammalian Ago protein capable of mRNA cleavage. It has been reported that the activity of the short interfering RNA targeting coding sequence (CDS), but not 3′ untranslated region (3′UTR) of an mRNA, is solely dependent on Ago2 in vitro. These studies utilized extremely high doses of siRNAs and overexpressed Ago proteins, as well as were directed at various highly expressed reporter transgenes. Here we report the effect of Ago2 in vivo on targeted knockdown of several endogenous genes by siRNAs, targeting both CDS and 3′UTR. We show that siRNAs targeting CDS lose their activity in the absence of Ago2, whereas both Ago1 and Ago3 proteins contribute to residual 3′UTR-targeted siRNA-mediated knockdown observed in the absence of Ago2 in mouse liver. Our results provide mechanistic insight into two components mediating RNAi under physiological conditions: mRNA cleavage dependent and independent. In addition our results contribute a novel consideration for designing most efficacious siRNA molecules with the preference given to 3′UTR targeting as to harness the activity of several Ago proteins. [ABSTRACT FROM AUTHOR]

Mitochondrial calcium uptake is present in nearly all vertebrate tissues and is believed to be critical in shaping calcium signaling, regulating ATP synthesis and controlling cell death. Calcium uptake occurs through a channel called the uniporter that resides in the inner mitochondrial membrane. Recently, we used comparative genomics to identify MICU1 and MCU as the key regulatory and putative pore-forming subunits of this channel, respectively. Using bioinformatics, we now report that the human genome encodes two additional paralogs of MICU1, which we call MICU2 and MICU3, each of which likely arose by gene duplication and exhibits distinct patterns of organ expression. We demonstrate that MICU1 and MICU2 are expressed in HeLa and HEK293T cells, and provide multiple lines of biochemical evidence that MCU, MICU1 and MICU2 reside within a complex and cross-stabilize each other's protein expression in a cell-type dependent manner. Using in vivo RNAi technology to silence MICU1, MICU2 or both proteins in mouse liver, we observe an additive impairment in calcium handling without adversely impacting mitochondrial respiration or membrane potential. The results identify MICU2 as a new component of the uniporter complex that may contribute to the tissue-specific regulation of this channel. [ABSTRACT FROM AUTHOR]

An outstanding question is how cells control the number and size of membrane organelles. The small GTPase Rab5 has been proposed to be a master regulator of endosome biogenesis. Here, to test this hypothesis, we developed a mathematical model of endosome dependency on Rab5 and validated it by titrating down all three Rab5 isoforms in adult mouse liver using state-of-the-art RNA interference technology. Unexpectedly, the endocytic system was resilient to depletion of Rab5 and collapsed only when Rab5 decreased to a critical level. Loss of Rab5 below this threshold caused a marked reduction in the number of early endosomes, late endosomes and lysosomes, associated with a block of low-density lipoprotein endocytosis. Loss of endosomes caused failure to deliver apical proteins to the bile canaliculi, suggesting a requirement for polarized cargo sorting. Our results demonstrate for the first time, to our knowledge, the role of Rab5 as an endosome organizer in vivo and reveal the resilience mechanisms of the endocytic system. [ABSTRACT FROM AUTHOR]

Argonaute 2 (Ago2) is the only mammalian Ago protein capable of mRNA cleavage. It has been reported that the activity of the short interfering RNA targeting coding sequence (CDS), but not 3′ untranslated region (3′UTR) of an mRNA, is solely dependent on Ago2 in vitro. These studies utilized extremely high doses of siRNAs and overexpressed Ago proteins, as well as were directed at various highly expressed reporter transgenes. Here we report the effect of Ago2 in vivo on targeted knockdown of several endogenous genes by siRNAs, targeting both CDS and 3′UTR. We show that siRNAs targeting CDS lose their activity in the absence of Ago2, whereas both Ago1 and Ago3 proteins contribute to residual 3′UTR-targeted siRNA-mediated knockdown observed in the absence of Ago2 in mouse liver. Our results provide mechanistic insight into two components mediating RNAi under physiological conditions: mRNA cleavage dependent and independent. In addition our results contribute a novel consideration for designing most efficacious siRNA molecules with the preference given to 3′UTR targeting as to harness the activity of several Ago proteins. [ABSTRACT FROM AUTHOR]