MiRNAs

Fig. 2 RNAi inducers used in antiviral strategies. In general, RNAi is induced either by transfection of synthetic siRNAs into cells, or by stable or transient intracellular expression of double-stranded siRNA precursors (shRNA, e-shRNA, IhRNA, or pri-miRNAs). After transcription in the nucleus shRNAs, IhRNAs and e-shRNAs are exported to the cytoplasm and subsequently diced into mature siRNAs. Pri-miRNAs modified to encode antiviral siRNAs first undergo cleavage by Drosha before they are exported to the cytoplasm. Here the antiviral pre-miRNAs (also called shRNA-miRs) are processed by Dicer into the mature miRNAs. After loading of the antisense strand of the siRNAs/miRNAs into RISC, the complex will target and cleave viral transcripts bearing the complementary sequences...

IhRNAs have also been used to induce an anti-HIV-1 RNAi response (Barichievy et al. 2007 Konstantinova et al. 2006, 2007 Liu et al. 2007). These IhRNAs can be processed into multiple effective siRNAs, thus preventing the chance of viral escape. Although IhRNA have been shown to effectively inhibit HIV-1 replication, there is currently no data on their ability to prevent viral escape. The use of multiple siRNAs or IhRNAs should take into account the increased danger of side effects due to interference with cellular miRNA processing and function. 

Biological function of targets (pathway dissection, miRNA/siRNA/ shRNA studies, model building in vitro and in vivo)... 

Comment Application of this method makes the further assumption that miRNA-mediated repression results from a graded reduction in translation of all functional mRNA molecules, rather than a complete translational shutdown of a subpopulation thereof. It also critically depends on reproducible transfection conditions, because the reporter enzyme activity cannot be... 

Bagga, S., Bracht, J., Hunter, S., Massirer, K., Holtz, J., Eachus, R., and Pasquinelli, A. E. (2005). Regulation by let-7 andlin-4 miRNAs results in target mRNA degradation. Cell 122, 553-563. 

Nottrott, S., Simard, M.J., and Richter, J. D. (2006). Human let-7a miRNA blocks protein production on actively translating polyribosomes. Nat. Struct. Mol. Biol. 13, 1108-1114. 

Microreactor technology, application in combinatorial chemistry, 7 401, 422 Micro RNAs (miRNA), 17 620-621 Micro-routing, in waste collection, 25 869 Microsampling methods, in infrared spectroscopy, 14 232-233 Microscale microbial cultures, shaken, 16 406... 

A number of modified nucleotides have been tested and described in siRNA design. These are mostly modifications of the 2 OH group of the ribose. By the incorporation of chemically modified nucleotides into siRNAs, the on-target efficiency of the siRNAs can be increased (42 4). On the other hand, different types of siRNA off-target effects can be reduced by the use of chemically modified nucleotides immunostimulatory effects (reviewed in (20)) as well as sequence-dependent miRNA-like off-target effects (26, 44,45) and passenger strand incorporation (46, 47). In addition, chemical modifications can be used to improve the cellular delivery of siRNAs in living animals and are important tools to enhance the serum stability of siRNAs (48). 

Filipowicz W, Jaskiewicz L, Kolb FA et al (2005) Post-transcriptional gene silencing by siRNAs and miRNAs. Curr Opin Struct Biol 15 331-341... 

Meister G, Landthaler M, Patkaniowska A et al (2004) Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs. Mol Cell 15 185-197... 

Doench JG, Petersen CP, Sharp PA (2003) siRNAs can function as miRNAs. Genes Dev 17 438-442... 

Khvorova A, Reynolds A, Jayasena SD (2003) Functional siRNAs and miRNAs exhibit strand bias. Cell 115 209-216... 

Jordan SD, Kruger M, Willmes DM et al (2011) Obesity-induced overexpression of miRNA-143 inhibits insulin-stimulated AKT activation and impairs glucose metabolism. Nat CeU Biol 13 434-446... 

Due to their functional involvement in specific molecular pathways, miRNAs have become interesting targets for therapeutic intervention. Examples of disease-related mechanisms affected by miRNAs include single-nucleotide polymorphisms (SNPs) in 3 UTR sequences (23, 24) or in miRNA genes (25, 26) that may affect normal protein expression and has been identified in association with heart failure (27) and cancer (25, 28). Chromosomal deletions and epigenetic changes involving miRNA also cause abnormal protein expression that is associated with malfunctions... 

The evaluation of miRNA ISH results requires considerations on the specificity of the signal. Specificity control analysis is an inherent part of molecular histology. For miRNA ISH analyses, there are several ways to address this question (40). Specific for double fluorescence analyses, one key point to address is preventing cross-reaction between the two parallel assays (see Note 7). 

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