small-interfering RNA (siRNA): basics and essential publication (1)
siRNA & quantitative real-time RT-PCR (2)
siRNA & quantitative real-time RT-PCR (3)
siRNA & quantitative real-time RT-PCR (4)
RNAinterference (RNAi) microRNA (miRNA) small activating RNA (saRNA)
News and Views Q&A (by Helge Großhans and Witold Filipowicz)
Molecular biology: The expanding world of small RNAs
Molecular cell biology has long been dominated by a protein-centric view. But the emergence of small, non-coding RNAs challenges this perception. These plentiful RNAs regulate gene expression at different levels, and have essential roles in health and disease.
Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, are a class of 20-25 nucleotide-long double-stranded RNA molecules that play a variety of roles in biology. Most notably, it is involved in the RNA interference (RNAi) pathway where the siRNA interferes with the expression of a specific gene. In addition to their role in the RNAi pathway, siRNAs also act in RNAi-related pathways, e.g. as an antiviral mechanism or in shaping the chromatin structure of a genome; the complexity of these pathways is only now being elucidated. SiRNAs were first discovered by David Baulcombe's group in Norwich, England, as part of post-transcriptional gene silencing (PTGS) in plants, and published there findings in Science in a paper titled "A species of small antisense RNA in posttranscriptional gene silencing in plants." Shortly thereafter, in 2001, synthetic siRNAs were then shown to be able to induce RNAi in mammalian cells by Thomas Tuschl and colleagues in a paper, "Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells." published in Nature and Genes & Development. This discovery led to a surge in interest in harnessing RNAi for biomedical research and drug development.
RNAi induction using siRNAs or their biosynthetic precursors
Transfection of an exogenous siRNA can be problematic, since the gene knockdown effect is only transient, particularly in rapidly dividing cells. One way of overcoming this challenge is to modify the siRNA in such a way as to allow it to be expressed by an appropriate vector, e.g. a plasmid. This is done by the introduction of a loop between the two strands, thus producing a single transcript, which can be processed into a functional siRNA. Such transcription cassettes typically use an RNA polymerase III promoter (e.g. U6 or H1), which usually direct the transcription of small nuclear RNAs (snRNAs) (U6 is involved in gene splicing; H1 is the RNase component of human RNase P). It is assumed (although not known for certain) that the resulting siRNA transcript is then processed by Dicer.
Dicer is an RNAse III nuclease that cleaves double-stranded RNA (dsRNA) and pre-microRNA (miRNA) into short double-stranded RNA fragments called small interfering RNA (siRNA) of about 20-25 nucleotides long, usually with a two-base overhang on the 3' ends. Dicer contains two RNase domains and one PAZ domain; the distance between these two regions of the molecule is determined by the length and angle of the connector helix and determines the length of the siRNAs it produces. Dicer catalyzes the first step in the RNA interference pathway and initiates formation of the RNA-induced silencing complex (RISC), whose catalytic component argonaute is an endonuclease capable of degrading messenger RNA (mRNA) whose sequence is complementary to that of the siRNA guide strand.
The enzyme Dicer was given its name by Emily Bernstein, a graduate student in Greg Hannon's group at the Cold Spring Harbor Laboratory, who first demonstrated its dsRNA "dicing" activity.
Mechanism of siRNA silencing http://www.uni-konstanz.de/FuF/chemie/jhartig/