MicroRNAs (miRNA) are short RNAs averaging 22 nucleotides, complementary to the 3' UTR regions of target mRNAs. They are well-conserved in eukaryotic organisms, and thought to play important roles in gene silencing. Enzymes and proteins involved in microRNA form part of the regulatory RNA pathway.
Gene transcription: RNA polymerase II, and less often RNA polymerase III transcribe from genomics sequence and produce primary miRNA (pri-miRNA), which could be hundreds or thousands of nucleotides long and contain more than one miRNA stem loops. Just like the regular mRNA molecules, the pri-miRNA is capped, polyadenylated and spliced.
Nuclear processing:Some pri-miRNAs undergo RNA editing, usually through adenosine deaminases acting on RNA (see ADAR1). Microprocessor, formed by DiGeorge Syndrome Critical Region 8 (DGCR8, also called Pasha) and enzyme Drosha, cleaves pri-miRNA and generates the pre-miRNA hairpin.
Nuclear export:Nucleocytoplasmic transporter Exportin-5 recognizes the two-nucleotide overhang at the 3' end of pre-miRNA hairpin and transports pre-miRNAs to the cytoplasm.
Cytoplasmic processing:RNase III enzyme Dicer cleaves out the loop of a pre-miRNS hairpin and generates miRNA:miRNA duplex. One strand of miRNA:miRNA duplex is incorporated into the RNA-induced silencing complex.
RNA-induced silencing complex (RISC), also known as microRNA ribonucleoprotein complex (miRNP), contains the mature miRNA and many other proteins, including members of argonaute family (see AGO1, AGO2, AGO3, AGO4, and PIWIL1, PIWIL2, PIWIL3, PIWIL4), human immunodeficiency virus (HIV) transactivating response RNA (TAR) binding protein (TRBP), protein activator of the interferon induced protein kinase (PACT), fragile X mental retardation protein (FMRP), Tudor staphylococcal nuclease-domain-containing protein (Tudor-SN). RISC binds target mRNA through its cognate mature miRNA and degrades mRNA.
quantitative real-time PCR:
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microarray:
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locked nucleic acid oligo:
Locked nucleic acid oligo can be used to detect miRNA in situ.
locked nucleic acid oligo:The 2' oxygen and 4' carbon of the ribose in a locked nucleic acid is connected through a methylene bridge, thus increases thermal stability of oligonucleotides. LNA is the only method that can detect miRNA in situ effectively.
Morpholino oligo: Morpholino oligo mimics the DNA oligo, where the deoxyribose rings are replaced with morpholine rings and phosphorodiamidate groups are used to link based instead of phosphates. They bind to complementary sequences of RNA by standard nucleic acid base-pairing, and block the access of other molecules to that portion of sequences, and thus are used to block miRNA activity and maturation.
2'-O-methyl RNA oligo:2′-O-Methyl modification can protect oligoribonucleotides from degradation in cell extracts or cultured cells, and 2'-O-Methyl oligoribonucleotides can quickly and stably hybridize to single-stranded RNA. 2′-O-methyl oligoribonucleotides antisense to the guide strand positioned in the RNA silencing complex can inhibit effectively RISC and miRNP complexes in cell lysates and when transfected, in cultured cells.
antagomir: Antagomirs are small synthetic RNAs, that bind specific miRNA targets with either mispairing at the cleavage site of Ago2 or other modifications to inhibit Ago2 cleavage. They can be used to silence endogenouse microRNA in vivo.
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Precursor clones for all human, mouse, and rat microRNAs. Samples:
Inhibitor clones for all human, mouse, and rat microRNAs. Samples:
Synthetic inhibitors for all human, mouse, and rat microRNAs. Samples:
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