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The tiny RNA world.

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Animals↗

RNAi versus small molecules: different mechanisms and specificities can lead to different outcomes.

The methodology of RNA interference (RNAi) arrived on the scene of mammalian systems research in 2001. Since that time, there has been widespread use of this technology in both academic and industrial settings. RNAi and small molecules were initially considered as equivalent methods for inhibiting protein activity within cells, but as our understanding of the mechanism of RNAi has improved, it has become apparent that differences in their cellular mechanisms have unexpected consequences. There can be profound differences in the phenotypic outcomes of treatment with RNAi versus small molecules. This review discusses the similarities, as well as the predicted differences between RNAi and small molecule effects on therapeutic paradigms and drug discovery.

Animals↗

Factors involved in self-renewal and pluripotency of embryonic stem cells.

Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of blastocyst stage embryos. These cells possess two unique characteristics: an indefinite self-renewal capacity and pluripotency, the ability to differentiate to cells from the three germ layers. Both human and mouse ES cells are currently at the center of intensive research. One of the burning issues in this research is the way in which these cells remain undifferentiated and maintain their pluripotency. In the past years, data has accumulated concerning the pathways responsible for the unique phenotype of these cells, in both human and mouse. This paper will review the main extrinsic factors and intrinsic transcriptional pathways currently implicated in the self-renewal and pluripotency of ES cells.

Animals↗

Regulation of gene expression in mast cells: micro-rNA expression and chromatin structural analysis of cytokine genes.

Despite deriving from two different compartments of the immune system (myeloid and lymphoid respectively), Th2 cells and mast cells produce the same panel of cytokines, interleukin (IL)4, IL5 and IL13. We have compared the chromatin structure of the RAD50/IL13/IL4 locus in Th2 cells and mast cells. Th2 and mast cells display strong overlap in their patterns of DNase I hypersensitivity throughout this locus, except that the first intron of the IL13 gene (MCHS) is DNase I hypersensitive only in mast cells and the conserved non-coding sequence (CNS)-1 in the IL4/IL13 intergenic region is DNase I hypersensitive only in Th2 cells (explaining why cytokine expression is impaired in Th2 cells but not in mast cells of CNS-1-deleted mice). We have also examined the role of micro-RNAs (miRNAs) in the development and activation of mast cells and T cells. miRNAs are 21- to 25-nucleotide small RNAs that regulate gene expression posttranscriptionally by targeting protein-coding mRNAs. Using oligonucleotide arrays to analyse miRNA expression in murine T cells and mast cells, we have identified distinctive cell type-specific patterns of miRNA expression as well as changes related to differentiation and cell activation. We are studying the biological functions of selected miRNAs.

Animals↗

[Genome-wide lentivector-based pooled shRNA library optimization].

We have optimized lentiviral vector constructs and cassettes for expression of short hairpin RNAs (shRNAs) in order to create genome-wide library capable of inhibition of full variety of human mRNAs. The vector optimization has resulted in 15-20-fold improvement in virus stock titers. We found that in the context of lentiviral vector the most effective structure for the shRNA is simple hairpin with 21 nucleotide stem. The shRNA-expressing lentiviral constructs contain choice of puro(R), copGFP or H-2K(k) selective markers. The efficiency of the optimized library was evaluated in experiments on screening of shRNAs that reactivate oncosuppressor p53 in HeLa cells. The cells contained reporter construct with p53-dependent expression of a fluorescent protein, which allows cytofluorimetric isolation of cell population with reactivated p53.

Gene Expression Regulation↗

[Trans-acting short interfering RNAs].

RNA is now considered a key factor in the regulation of gene expression. There are several classes of small regulatory RNAs in plants, functioning in posttranscriptional gene silencing (PTGS) or epigenetic DNA modification. Trans-acting short interfering RNAs (tasiRNAs) form a class of small regulatory RNAs which has been distinguished only recently. To date, five genes encoding tasiRNAs have been identified in Arabidopsis thaliana. TasiRNAs derive from non-coding RNA precursors which are initially targeted for cleavage by a miRNA. Cleavage products are then converted into dsRNAs by a RNA dependent RNA polymerase and sequentially cleaved into 21-nt tasiRNAs. Like the majority of plant miRNAs, tasiRNAs regulate gene expression at the posttranscriptional level, guiding cleavage of ARF and PPR transcripts. Here, we briefly present tasiRNAs and speculate whether they form a homogeneous class of siRNAs.

Gene Expression Profiling↗