MicroRNA-mediated up-regulation of an alternatively polyadenylated variant of the mouse cytoplasmic β-actin gene

Actin is a major cytoskeletal protein in eukaryotes. Recent studies suggest more diverse functional roles for this protein. Actin mRNA is known to be localized to neuronal synapses and undergoes rapid deadenylation during early developmental stages. However, its 3′-untranslated region (UTR) is not characterized and there are no experimentally determined polyadenylation (polyA) sites in actin mRNA. We have found that the cytoplasmic β-actin (Actb) gene generates two alternative transcripts terminated at tandem polyA sites. We used 3′-RACE, EST end analysis and in situ hybridization to unambiguously establish the existence of two 3′-UTRs of varying length in Actb transcript in mouse neuronal cells. Further analyses showed that these two tandem polyA sites are used in a tissue-specific manner. Although the longer 3′-UTR was expressed at a relatively lower level, it conferred higher translational efficiency to the transcript. The longer transcript harbours a conserved mmu-miR-34a/34b-5p target site. Sequence-specific anti-miRNA molecule, mutations of the miRNA target region in the 3′-UTR resulted in reduced expression. The expression was restored by a mutant miRNA complementary to the mutated target region implying that miR-34 binding to Actb 3′-UTR up-regulates target gene expression. Heterogeneity of the Actb 3′-UTR could shed light on the mechanism of miRNA-mediated regulation of messages in neuronal cells

Dendritic effect of ligand-coated nanoparticles: enhanced apoptotic activity of silica−berberine nanoconjugates

We describe the synthesis and biological characterization of a novel prototype, namely, silica nanoconjugates bearing a covalently linked berberine, a plant alkaloid known to have antiproliferative activity. The effect of synthesized nanoconjugates on cell proliferation, the cell cycle profile, and apoptosis in the human cervical carcinoma cell line (HeLa), human hepatocellular liver carcinoma cell line (HepG2), and human embryonic kidney (HEK) 293T cell line has been studied and compared with the results obtained for free berberine. Our results show that all the nanoconjugates display higher antiproliferative activity than free berberine. The ability of these nanoconjugates to inhibit cellular proliferation is mediated by the cell cycle arrest at the G1 phase. Moreover, silica nanoconugates caused selective apoptotic arrest with a higher efficiency than free berberine followed by apoptotic cell death as shown by quantitative fluorescence-activated cell sorting analyses. Efficiency of the nanoconjugates increases upon an increase in the linker chain length, demonstrating the distinct role of the spacer chain that conjugates nanoparticles and ligands. The actual reason to show enhanced efficiency by the nanoconjugates has not been elucidated in the present study; however, we hypothesize that an increase in local concentration due to the confinement of a ligand on the nanosurface (“dendritic” effect) might have led to the observed effect

Identification of Novel Targets for miR-29a Using miRNA Proteomics

<div><p>MicroRNAs (miRNAs) are short regulatory RNA molecules that interfere with the expression of target mRNA by binding to complementary sequences. Currently, the most common method for identification of targets of miRNAs is computational prediction based on free energy change calculations, target site accessibility and conservation. Such algorithms predict hundreds of targets for each miRNA, necessitating tedious experimentation to identify the few functional targets. Here we explore the utility of miRNA-proteomics as an approach to identifying functional miRNA targets. We used Stable Isotope Labeling by amino acids in cell culture (SILAC) based proteomics to detect differences in protein expression induced by the over-expression of miR-34a and miR-29a. Over-expression of miR-29a, a miRNA expressed in the brain and in cells of the blood lineage, resulted in the differential expression of a set of proteins. Gene Ontology based classification showed that a significant sub-set of these targets, including Voltage Dependent Anion Channel 1 and 2 (VDAC1 and VDAC2) and ATP synthetase, were mitochondrial proteins involved in apoptosis. Using reporter assays, we established that miR-29a targets the 3′ Untranslated Regions (3′ UTR) of VDAC1 and VDAC2. However, due to the limited number of proteins identified using this approach and the inability to differentiate between primary and secondary effects we conclude that miRNA-proteomics is of limited utility as a high-throughput alternative for sensitive and unbiased miRNA target identification. However, this approach was valuable for rapid assessment of the impact of the miRNAs on the cellular proteome and its biological role in apoptosis.</p> </div

miR-29a targets 3′ untranslated region of VDAC1, 2 and 3.

<p>Binding pattern of miR-29a with the 3′UTR of the VDAC1(A),VDAC2 (B) AND VDAC3 (C). Free energies of the interactions are mentioned in Kcal/mol. (D,E,F) Luciferase activity for VDAC1, VDAC2 and VDAC3 was determined 24hours after co-transfection with Luciferase-VDAC-3′ UTR fusion and hsa-miR-29a overexpression constructs. Relative luciferase activity is shown after normalization to Firefly Luciferase. Error bars represent standard deviation from three independent experiments.</p

Differential Expression of VDAC1 by miR-29a.

<p>HEK293T cells were transfected with mock LNA, LNA modified anti-miR-29a, Vector or plasmid over-expressing miR-29a. (A) Immunoblotting was performed as mentioned in materials and methods and B) Band intensities quantified and normalized to loading control (GAPDH).</p

Synaptobrevin2 is the v-SNARE required for cytotoxic T-lymphocyte lytic granule fusion.

Cytotoxic T lymphocytes kill virus-infected and tumorigenic target cells through the release of perforin and granzymes via fusion of lytic granules at the contact site, the immunological synapse. It has been postulated that this fusion process is mediated by non-neuronal members of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex protein family. Here, using a synaptobrevin2-monomeric red fluorescence protein knock-in mouse we demonstrate that, surprisingly, the major neuronal v-SNARE synaptobrevin2 is expressed in cytotoxic T lymphocytes and exclusively localized on granzyme B-containing lytic granules. Cleavage of synaptobrevin2 by tetanus toxin or ablation of the synaptobrevin2 gene leads to a complete block of lytic granule exocytosis while leaving upstream events unaffected, identifying synaptobrevin2 as the v-SNARE responsible for the fusion of lytic granules at the immunological synapse

Experimental schema for miRNA-proteomics.

<p>Cells were transfected with either miRNA expressing vector or LNA modified oligonucleotide against miRNA. The lysate mix was analyzed on LTQ-ORBITRAP mass spectrometer. The data was analyzed as mentioned in materials and methods. The quantification data was then compared with other miRNA-target identification methods.</p

Gene Ontology (GO) classification of miR-29a Upregulated and Downregulated Targets.

<p>Gene Ontology (GO) classification of miR-29a Upregulated and Downregulated Targets.</p