[影片] 簡介「RNA干擾」現象之短片

lavande 譯

When long double-stranded RNAs were injected into a worms’ gonad, a standard way of introducing transgenes into worms, they blocked the expression of endogenous genes in a sequence-specific manner.

In eukaryotes, most protein-coding genes are transcribed by RNA polymerase II, which generates pre-mRNAs that are then processed to form mature mRNAs. These mRNAs are then transported from the nucleus to the cytoplasm, where they are translated. 

RNAi is a recently discovered process that can regulate endogenous gene expression. In plants, RNAi can be set in motion by genomically encoded short regulatory RNAs known as micro-RNAs. In algae, worms, and flies, RNAi can be activated by endogenous transposition. In plants and cultured insect cells, RNAi also has a role in anti-viral defense, in which viral double-stranded RNAs are targeted for destruction by the RNAi machinery.

When long double-stranded RNAs enter a cell, they are recognized and cleaved by Dicer, which is a member of the RNase III family of double-stranded RNA-specific endonucleases. Cleavage by Dicer creates short double-stranded RNAs that are characterized by two nucleotide long, 3’ overhangs. These are called small-interfering, or siRNAs. In worms, flies and mammals, siRNAs can form a ribonucleic protein complex called RISC, or RNAi silencing complex. This complex includes an unidentified nuclease that has been nicknamed Slicer.

A single-stranded siRNA that is coupled to RISC then binds to a target mRNA in a sequence-specific manner. This binding mediates target mRNA cleavage by Slicer. The site of cleavage falls in the middle of the region of siRNA complementarity. The cleaved mRNA can be recognized by the cell as being aberrant, and then destroyed.  This prevents translation from occurring, silencing the expression of the gene from which the mRNA was transcribed.

In plants, the aberrant RNA that result from RISC-mediated cleavage can also serve as a template for RNA-dependent RNA polymerase, or RdRp, to make a new double-stranded RNA molecule. This process relies on unprimed RNA synthesis, in which the aberrant RNA is used as a template. The resulting double-stranded RNA is a substrate for Dicer activity, which generates more siRNAs.

In some organisms with endogenous RNAi mechanisms, for example, fungi, plants, worms, and mammals, RNAi also involves another amplification step. In this step, single-stranded siRNAs not associated with RISC bind to their target mRNAs in a sequence-specific way, and serve as a primer for RdRp to polymerize the antisense RNA strand. Such specificity is intrinsically sensitive to natural sequence variation. The double-stranded RNA molecule that is created serves as a substrate for Dicer, which cuts it into siRNAs.

In turn, these siRNAs can either unwind and prime RNA-dependent RNA polymerization, or together with RISC mediates the cleavage of target mRNAs. This amplification, coupled with RNAi spreading between cells through an unknown mechanism, is thought to underlie germ line transmission of RNAi in worms.











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