siRNA / microRNA epigenetics stem cells

RNA interference (RNAi) • RNAi is a cellular process whereby double-stranded RNA (dsRNA) induces silencing of complementary target genes • RNAi is central to mechanisms of post-transcriptional gene silencing (PTGS), but can also effect transcriptional gene silencing (TGS) • RNAi is highly conserved among all eukaryotic organisms (fungi, protozoans, plants, nematodes, invertebrates, mammals)

Overview of RNAi • Double-stranded RNA (dsRNA) is processed by Dicer, an RNase III family member, to produce 21-23nt small interfering RNAs (siRNAs) • siRNAs are manipulated by a multi-component nuclease called the RNA-induced silencing complex (RISC). • RISC specifically cleaves mRNAs that have perfect complementarity to an siRNA strand

A brief history of RNAi • RNAi was initially discovered and characterized in the C. elegans • It was observed that antisense or sense RNA could equally effect silencing (knock-down) of target gene expression • A later study demonstrated that antisense and sense RNA combined (dsRNA) is 10-times more effective in silencing target gene expression • Genetic studies in C. elegans have helped to identify genes encoding RNAi machinery

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Biological roles of small RNAs • Cellular immune response to viruses (siRNAs) • Genetic stability and heterochromatin maintenance (siRNAs) • Development and cell differentiation (microRNAs)

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Genetic stability • siRNAs repress the mobility of transposable genetic elements in C. elegans and S. pombe • Disruption of Dicer, argonaute, or RdRP increases the relative abundance of transposon RNA and increases transposon mobility • Transposable elements comprise a substantial fraction of the highly repetitive DNA in eukaryotic genomes (another being centromeres) • Highly repetitive DNA is often associated with heterochromatin which is transcriptionally silent

Genetic stability • siRNAs are required to establish and maintain heterochromatin formation and gene silencing at mating type loci and centromeres in yeast (S. pombe) • Disruption of Dicer, argonaute, and RdRP eliminate silencing, decrease histone and DNA methylation, and cause aberrant chromosome segregation • To this point, siRNAs isolated from eukaryotic organisms correspond to centromeric repeats and transposable elements

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siRNAs and transcriptional gene silencing • The role of siRNAs in transcriptional gene silencing appears to be important among all eukaryotic organisms • However, the precise mechanism(s) of siRNA-mediated transcriptional gene silencing are poorly understood

Small RNAs in development • Small RNAs play a critical role in development through the action of a specific class of small dsRNAs termed microRNAs (miRNAs) • miRNAs are the products of endogenous genes! They are processed in part by Dicer but function to effect post-transcriptional gene silencing in a manner distinct from that of siRNAs • The importance of miRNAs in regulating cellular processes through effects on target gene expression is reflected in the fact that many miRNA genes are highly conserved among eukaryotic organisms

A brief history of miRNAs • C. elegans was discovered to possess small noncoding RNAs (let-7 and lin-4) that negatively regulate expression of target genes (lin-41 and lin-28) which direct developmental progress • At that time, the so-called small temporal RNAs (stRNAs) were found to repress translational of the target mRNAs by interacting with complementary sites in their 3’ untranslated regions (UTRs) • It was later appreciated that the stRNAs are processed by Dicer and thus function through related pathway • Disruption of the stRNAs, Dicer, or argonaute genes result in similar developmental abnormalities • With the subsequent discovery that there are many such small RNAs that function through the RNAi pathway, the entire class was renamed microRNAs

Small but plenty • To date, over 10,000 miRNA genes have been identified among over 80 eukaryotic organisms (plants and animals) • There are, for example 174 C. elegans, 157 Drosophila, 579 mouse, and 721 human miRNA genes • About 10 viruses are known to express their own miRNAs • Approximately one third of miRNA genes are intronic with respect to protein coding genes • Approximately two thirds of miRNA genes are intergenic • miRNA genes by their earliest definition should be conserved among species, but there are a growing number of species specific miRNAs

Conservation of miRNA sequence and structure • Certain miRNAs are highly conserved and thus evolutionarily ancient (e.g. let-7) • Sequence conservation must fulfill the require to form a dsRNA hairpin from which the identicle miRNA is processed

miRNA gene transcription • miRNA genes are typically transcribed by RNA pol II • miRNA genes can be arrayed (i.e. clustered and coexpressed)

miRNA biogenesis

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Quantitative real time RT-PCR 22

Real time quantitative RT-PCR From Wikipedia: “amplify and simultaneously quantify a targeted DNA molecule. It enables both detection and quantification (as absolute number of copies or relative amount when normalized to DNA input or additional normalizing genes) of a specific sequence in a DNA sample.” “amplified DNA is quantified as it accumulates in the reaction in real time after each amplification cycle. Two common methods of quantification are the use of fluorescent dyes that intercalate with double-stranded DNA, and modified DNA oligonucleotide probes that fluoresce when hybridized with a complementary DNA.” “Frequently, real-time PCR is combined with reverse transcription polymerase chain reaction to quantify low abundance messenger RNA (mRNA), enabling a researcher to quantify relative gene expression at a particular time, or in a particular cell or tissue type.”

Real time quantitative RT-PCR

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