Discussion paper on the role of government in the construction and building materials industry Latvian construction and building materials industrial sector study

Available from Latvian Academic Library / LAL - Latvian Academic LibrarySIGLEThe European Union PHARE Programme, Brussels (Belgium)LVLatvi

MicroRNA-9: Functional evolution of a conserved small regulatory RNA

The functional significance of microRNA-9 (miR-9) during evolution is evidenced by its conservation at the nucleotide level from flies to humans but not its diverse expression patterns. Recent studies in several model systems reveal that miR-9 can regulate neurogenesis through its actions in neural or non-neural cell lineages. In vertebrates, miR-9 exerts diverse cell-autonomous effects on the proliferation, migration and differentiation of neural progenitor cells by modulating different mRNA targets. In some developmental contexts, miR-9 suppresses apoptosis and is misregulated in several types of cancer cells, influencing proliferation or metastasis formation. Moreover, downregulation of miR-9 in postmitotic neurons is also implicated in some neurodegenerative diseases. Thus, miR-9 is emerging as an important regulator in development and disease through its ability to modulate different targets in a manner dependent on the developmental stage and the cellular context

miR-92a and miR-92b target their host gene <i>jigr1</i>.

<p>(A) Predicted miR-92a and miR-92b binding sites in the 3’UTR of <i>jigr1</i>. The seed sequences of miR-92a and miR-92b are the same and shown in blue and mutated nucleotides are shown in red. (B) Dual luciferase assay of HEK 293T cell lysates cotransfected with miR-92a, miR-92b or miR-124 expression vectors together with dual luciferase construct containing the wild type or mutant 3’UTR of <i>jigr1</i> at 3’ of Renilla luciferase. Bar graph shows normalized mean luciferase activity of cells transfected with miR-92 expression plasmid to that of cells transfected with control miRNA from three independent experiments. Statistical significance was determined by two-way ANOVA. (C) Predicted miR-92a and miR-92b binding sites at the 5’UTR of <i>jigr1</i>. Mutated nucleotides are shown in red. (D) Dual luciferase assay of lysates of HEK 293T cells co-transfected with miR-92a, miR-92b, or control miRNA (miR-124) expression vectors together with a firefly luciferase construct containing the wild type or mutant 5’UTR of <i>jigr1</i>. Bar graph shows normalized mean values of the luciferase activity of cells transfected with a miR-92 expression plasmid relative to that of cells transfected with control miRNA from three independent experiments. Statistical significance was determined by two-way ANOVA. Values are mean ± s.e.m. in all graphs. *: p < 0.05, **: p < 0.005, ***: p < 0.001, ****: p < 0.0001.</p

Expression profile of Jigr1 in third instar larval brain.

<p>(A) Jigr1 expression (green) in wild type third instar larval brain. Z-projection of optical sections is shown. Scale bar: 100 μm. CB: Central Brain, OL: Optic lobe, VNC: Ventral Nerve Cord. (B) Jigr1 expression (green) in post mitotic neurons labeled with Elav (red). Scale bar: 20 μm (C) Jigr1 expression (green) in glial cells labeled with glial specific transcription factor, Repo, (red) (D) High-magnification images of boxed area in A. Neuroblasts are labeled with Miranda (Mira, red), and ganglion mother cells (GMCs) and neurons are immunostained with Prospero (Pros, blue). Single optical section is shown. Arrows indicate neuroblasts. Scale bar: 20 μm. (E) Horizontal sections of third instar larval brains are labeled with miR-92a (red) and Jigr1 (green). Dashed lines outlines neuroepthelial cells (NE). Scale Bar: 20 μm. (F) Magnified view of dashed box in 2E. Arrows indicate miR-92a positive cells. Scale Bar: 5 μm.</p

<i>Jigr1</i> upregulation is responsible for premature neuroblast differentiation in <i>miR-92</i>^{<i>–/–</i>} flies.

<p>(A) Neuroblast number in third instar larval brains of the indicated genotypes at 120 hr ALH. <i>w</i>^<i>1118</i> (n = 17); <i>miR-92</i>^<i>4/4</i> (n = 15); <i>miR-92</i>^<i>1/4</i> (n = 20); <i>Def(3R)BSC321/miR-92</i>^<i>4</i> (n = 17); <i>Insc-GAL4/UAS-Jigr1</i>^<i>RNAi</i><i>; miR-92</i>^<i>1/4</i> (n = 14). (B) The size of Adult brains of indicated genotypes. <i>w</i>^<i>1118</i> (n = 10); <i>miR-92</i>^<i>4/4</i> (n = 10); <i>miR-92</i>^<i>1/4</i> (n = 8); <i>Def(3R)BSC321/miR-92</i>^<i>4</i> (n = 12); <i>Insc-GAL4/UAS-Jigr1</i>^<i>RNAi</i><i>; miR-92</i>^<i>1/4</i> (n = 8). (C) Immunostaining of third instar larval brains of wild type (left panel), <i>Insc-GAL4/UAS-jigr1</i> (middle panel) and <i>Insc-GAL4/UAS-jigr1-RNAi</i> (right panel) flies. Green: Jigr1. Red: Miranda. Scale Bar: 20 m. (D) Percentage of BrdU^<i>+</i> neuroblasts in wild type control (n = 12), <i>Insc-GAL4/UAS-jigr1</i> (n = 15) and <i>Insc-GAL4/UAS-jigr1-RNAi</i> (n = 9) larval brains. (E) Immunostaining of <i>Insc-GAL4/UAS-jigr1</i>brains with Prospero (green) and Dpn (red) at 120 hr ALH. (F) Adult brain volume of wild type control (n = 10) and <i>Insc-GAL4/UAS-jigr1</i> (n = 9) flies. (G) Working model summarizing the expression and the role of miR-92 in larval neuroblasts. Statistical significance was determined by One-Way ANOVA. Values are mean ± s.e.m. in all graphs. *: p < 0.05, **: p < 0.005, ***: p < 0.001, ****: p < 0.0001.</p

Generation of <i>miR-92a</i>^{<i>–/–</i>} and <i>miR-92b</i>^{<i>–/–</i>} flies by ends-out gene targeting.

<p>(A) Schematic of ends-out gene targeting strategy (modified from Huang et al., 2009). (B) DNA gel electrophoresis of PCR-amplified miR-92a (left) and miR-92b (right) locus from genomic DNA of wild type and <i>miR-92a</i>^{<i>–/–</i>} and <i>miR-92b</i>^{<i>–/–</i>} single knockout (KO) flies. (C) Northern blot analysis of miR-92a, miR-92b, and U6 expression in wild type, <i>miR-92a</i>^{<i>–/–</i>}, <i>miR-92b</i>^{<i>–/–</i>}, and <i>miR-92</i>^{<i>–/–</i>} mutant third instar larvae.</p

miR-92a and miR-92b regulate neuroblast self-renewal by inhibiting premature differentiation.

<p>(A) Number of brain neuroblasts at 120 hr ALH. 1: <i>w</i>^<i>1118</i> (n = 17); 2: <i>miR-92a</i>^<i>-/-</i> (n = 13); 3: <i>miR-92b</i>^<i>-/-</i> (n = 12); 4: <i>miR-92</i>^<i>4/4</i> (n = 15); 5: <i>miR-92</i>^<i>1/4</i> (n = 20); 6: <i>Insc-GAL4/UAS-miR-92a; miR-92</i>^<i>4/4</i> (n = 8); 7: <i>Insc-GAL4/UAS-miR-92b; miR-92</i>^<i>4/4</i> (n = 8); 8: <i>Insc-GAL4/UAS-miR-92a; miR-92</i>^<i>1/4</i> (n = 16); 9: <i>Insc-GAL4/UAS-miR-92b; miR-92</i>^<i>1/4</i> (n = 15). Statistical significance was determined by one-way ANOVA. (B) Number of neuroblasts in wildtype (red) and <i>miR-92</i>^<i>4/4</i> (green) mutants from 24 to 120 hr ALH. Statistical significance was determined by Student’s <i>t</i> test. Error bars show S.D. (C) Percentage of neuroblasts with diameter > 10 μm (black bar) and < 10 μm (grey bar) in wild type (n = 60) and <i>miR-92</i>^{<i>–/–</i>} (n = 60) mutant brains at 96 hr ALH. Fisher’s exact test was used for statistical analysis. (D) Percentage of BrdU^<i>+</i> wild type control (n = 15) and <i>miR-92</i>^{<i>–/–</i>} mutant (n = 16) larval neuroblasts. Statistical significance was determined by Student’s <i>t</i> test. (E) Neuroblast clones of wild type control and <i>miR-92</i>^<i>4</i> mutants. Clones are marked with CD8::GFP (green). Yellow arrows mark Dpn^<i>+</i> neuroblasts. White dashed lines indicate the position of clones and yellow dashed line encircles the nucleus of neuroblast cell. Single focal planes are shown. (F) Comparison of wild type control (type I; n = 57, type II; n = 27) and <i>miR-92</i>^<i>-/-</i> mutant (type I; n = 67, type II; n = 33) clone sizes. (G) Whole-mount image of wild type and <i>miR-92</i>^{<i>–/–</i>} mutant male adult brains immunostained with the anti-HRP (green) to label axonal membranes and anti-Repo (red) to label glial cell nuclei. Scale bar: 100 μm. (H) Adult brain volume measured using ImageJ. 1: <i>w</i>^<i>1118</i> (n = 10); 2: <i>miR-92</i>^<i>4/4</i> (n = 10); 3: <i>miR-92</i>^<i>1/4</i> (n = 8); 4: <i>Insc-GAL4/UAS-miR-92a; miR-92</i>^<i>4/4</i> (n = 7); 5: <i>Insc-GAL4/UAS-miR-92a; miR-92</i>^<i>1/4</i> (n = 9); 6: <i>Insc-GAL4/UAS-miR-92b; miR-92</i>^<i>1/4</i> (n = 10). Statistical significance was determined by one-way ANOVA. Values are mean ± s.e.m. in all graphs unless otherwise stated. *: p < 0.05, **: p < 0.005, ***: p < 0.001, ****: p < 0.0001.</p

<i>miR-92a</i> and <i>miR-92b</i> are transcribed in the same transcriptional unit.

<p>(A) Schematic representation of the genomic arrangement of <i>miR-92a</i>, <i>miR-92b</i> and <i>jigr1</i> based on results from this study. Red lines indicate deleted regions. (B) Northern blot analysis of total RNA extracted from third instar larval heads of the indicated genotypes. RNA was probed for miR-92a, miR-92b, U6, Jigr1 (exon 6 probe), Jigr1 (long 3’UTR probe) and rp49. Deletion lines, <i>Del #4</i> and <i>Del #7</i>, are homozygous lethal but in trans to the bigger deficiency covering the locus, <i>Def(3R)BSC321</i>, they are viable and used like this for the analysis. (C) Northern blot analysis of RNA from wild type whole third instar larvae (lane 1), wild type larval heads (lane 2) and <i>Del #1</i> (lane 3) third instar larval heads. Probes specific for all the isoforms of <i>jigr1</i> (exon 6 probe, first panel), for the long 3’UTR (second panel), and for alternatively spliced noncoding exon 2 (third panel), and rp49 (bottom panel) were used. (D) Schematic representation of <i>jigr1</i> constructs. (E) Northern blot analysis of total RNA from HEK 293T cells transfected with UAST-<i>jigr1</i>-long-3’UTR or UAST-<i>jigr1</i>-short-3’UTR plasmids together with the Actin-Gal4 plasmid. Cells transfected with UAST-<i>jigr1</i>-long-3’UTR alone served as negative controls. miR-92b, miR-92a, and control U6 probes were used. The miR-92b probe recognizes endogenous miR-92b in HEK 293T cells.</p