Ch7+8 RNA interference

• RNA interference, also known as RNA silencing, co-suppression, and transcriptional gene silencing, TGS, is a process whereby specific gene expression is downregulated through the action of small RNAs.
• Natural RNA interference involves the use of small RNAs.
• Tools for investigating natural small RNAs include small RNA cloning and sequencing.
• Small RNAs can be used as a tool in research and biotechnology.
• TGS can result in long-term stable epigenetic modifications to gene expression that can be passed on to daughter cells during cell division.
• Promoter-targeted small RNAs interact with various proteins to guide TGS, beginning in the first 24 h, with direct interactions with AGO1 and AGO2.
• TGS is mechanistically distinct from the RNA interference (RNAi) gene-silencing pathway.
• The discovery of RNA interference was made by injecting double-stranded RNA (dsRNA) corresponding to the muscle protein coding gene unc-22 in C. elegans gonads, causing a 'twitching' phenotype in the progeny due to the lack of the muscle protein.
• Forward genetics seeks to find the genetic basis of a phenotype or trait, while reverse genetics seeks to find what phenotypes are controlled by particular genetic sequences.
• In 1984, a significant step in the direction of RNA interference was reported by Izant and Weintraub, who transformed tissue culture cells with a DNA construct engineered to express antisense RNA complementary to thymidine kinase mRNA, inhibiting the activity of thymidine kinase protein in these transgenic cells.
• The initial study on antisense inhibition was followed by several more, demonstrating the efficacy of using an antisense strategy to inhibit the activity of specific genes.
• In antisense studies using RNAs synthesized in vitro, sense RNAs have been typically introduced as negative controls for specificity.
• Craig Mello and colleagues coined the phenomenon of RNA interference, or RNAi, to distinguish it from classic antisense inhibition.
• Mello and coworkers observed that silencing spread to cells beyond the site of injection, suggesting that the interfering RNA could be transported from the site of initial delivery to most cells and tissues in the worm, eliciting a systemic response.
• A small RNA duplex generated by Dicer is subsequently loaded onto an AGO protein to form an effector complex called RNA-induced silencing complex (RISC).
• Many C. elegans researchers embraced RNAi, even in the absence of an understanding of how the knockdown in expression was being achieved.
• There were at least three possible explanations for the dsRNA’s potency: it could have been amplified, it could have acted catalytically, or it could have targeted the gene directly.
• Andrew Fire at the Carnegie Institution of Washington’s Department of Embryology first observed that double-stranded RNA (dsRNA), rather than single-stranded antisense RNA, was responsible for triggering the sequence-specific degradation of targeted endogenous mRNAs in C. elegans.
• Early studies focused on predicting and identifying the physiological roles of miRNA and siRNA by identifying their target transcripts in the cell.
• Deadenylation as well as displacement of poly(A)-binding protein (PABP) through GW182 and CCR4–NOT also contribute to the overall miRNA-mediated translational repression.
• Fire reasoned that sense and antisense RNA preps generated by in vitro transcription reactions using plasmid templates might be contaminated with small amounts of RNA of the opposite polarity, owing to the infidelity of viral RNA polymerases used in the in vitro synthesis reactions.
• The dsRBD of Drosha is necessary but not sufficient for substrate interaction.
• The AGO proteins are divided into three subclades: AGO, PIWI and worm-specific AGO proteins (WAGOs).
• In humans, no strict small-RNA-sorting system exists, and the four AGO proteins (AGO1–4) are associated with almost indistinguishable sets of miRNAs.
• The strand with a relatively unstable terminus at the 5′ side is typically selected as the guide strand.
• Following miRNA duplex loading, the pre-RISC (in which AGO proteins associate with RNA duplexes) quickly removes the passenger strand to generate a mature RISC.
• The seed concept is central to the recognition of the target mRNA.
• An additional determinant for strand choice is the first nucleotide sequence: AGO proteins select for guide strands with a U at nucleotide position 1.
• The recognition of the target mRNA is a key step in the miRNA pathway.
• RISC assembly involves two steps: the loading of the RNA duplex and its subsequent unwinding.
• Slicing-competent AGO proteins (namely, AGO2 in flies and humans) can cleave the passenger strand if the duplex is matched at the centre.
• The released passenger strand is degraded quickly, resulting in a strong bias towards the guide strand in the mature miRNA pool.
• All four human AGO proteins can incorporate both siRNA and miRNA duplexes, with a preference for small RNA duplexes with central mismatches (nucleotide positions 8–11).
• The less abundant passenger strand (miRNA * ) is also active in silencing, albeit usually less potently than the more abundant guide strand.
• RISC is very stable and can be more than 3 weeks.
• As strand selection is not completely strict, the strand that is not favoured can also be selected with varying frequency.
• Proteins of the AGO subclade are ubiquitously expressed and associate with miRNAs or siRNAs, whereas PIWI proteins are germ-cell-specific and interact with piRNAs.
• Stanislaw A. Gorski, Jörg Vogel & Jennifer A. Doudna are recognized for their work on the seed concept.
• The guide strand is determined during the AGO loading step, mainly on the basis of the relative thermodynamic stability of the two ends of the small RNA duplex.
• The distance between the 5' monophosphate of the guide RNA and the 5' phosphate-binding pocket at the interface between the MID and PIWI domain is typically 21–25 nucleotides in length and depends on the species and the type of Dicer.