Transcript Cytoplasm
MBP1007/1008 - Nucleic Acids A functional mRNA: The cytoplasmic story Objectives (1) To discuss the iNUTS and iBOLTS of how mRNAs function in the cytoplasm (2) To review older and introduce newer methods of studying mRNA biology in the cytoplasm (3) Understand the basics of RNAi-induced posttranscriptional gene silencing Breakthrough of the Year in 2002 Nobel prize in 2006 RNAi: RNA Interference Mechanism of post-transcriptional gene silencing mediated by double-stranded RNA molecules Small RNA molecules target mRNAs for degradation or translational repression based upon sequence specificity A classic paper A classic paper In 1998, Fire and Mello reported that dsRNA was a potent trigger for gene silencing in the nematode worm C. elegans (Nature 1998, 391:806) Effect was specific and not generalized Revelation because dsRNA is inherently very stable and incapable of further WatsonCrick base-pairing Passed to neighbouring and daughter cells: mitosis and meiosis RNAi in C. elegans Wild-type RNAi-deficient Wild type and RNAi-deficient worms fed on bacteria expressing GFP dsRNA DNA versus RNA! Structure of RNA versus DNA Life of dsRNA Life of ssRNA Sense and antisense genes Antisense approaches to inhibit gene expression RNA - G:U RNA: Primary, secondary and tertiary structure RNAi: Dicer and RISC Trigger dsRNA length of base pairing segment can be long or short PKR as an anti-viral response Two strands can be derived from two genes (intermolecular interaction), which can be in cis or trans Two strands can be derived from one gene, via an intramolecular interaction Trigger:exogenous dsRNA diced up by Dicer into small interfering RNAs (siRNAs) characteristic structure of 21 to 23 nt complementary RNA hybrid: 5’-phosphate and two nt 3’-overhang 5’ 5’ Dicer Highly evolutionarily conserved Disruption of the Dicer gene in many species leads to lethality or severe developmental phenotypes – Related to lack of miRNA processing Dicer knockout – cell specific Trigger:endogenous dsRNA derived from endogenous primary miRNA transcript processed by Drosha (nuclear) and then Dicer (cytoplasmic) into micro RNAs (miRNAs) same characteristic structure of 21 to 23 nt complementary RNA hybrid: 5’-phosphate and two nt 3’-overhang yet not always perfectly complementary 5’ 5’ MicroRNAs Naturally encoded small (~22nt) noncoding RNA molecules found in eukaryotes Participate in gene regulation, especially translation Found to regulate a wide array of cellular activities in eukaryotes Many are evolutionarily conserved across distantly related species Effector: PTGS RNA-induced silencing complex (RISC) targets one of the two strands in the siRNA and induces RNA degradation cytoplasmic function RNA degradation: the endonuclease is an argonaute protein (Ago 2 in humans) Translational repression: basis for repression not clear Rules of the road: effective Rx siRNAs versus DNA vectors that express shRNAs siRNAs: efficient transfections, transient, dicer-independent shRNAs: plasmids with RNA pol II or pol III promoters, viral gene vectors, stable, dicerdependent Target multiple regions of RNA: mixtures of siRNA targets Focus on function rather than RNA levels Rules of the road Get the right strand into the RISC complex Controls: scrambled oligos and control for transfection method Lowest amount of RNAi Rescue the targeted gene with either RNAisensitive or RNAi-insensitive expression vectors Ensure the processing of dsRNA templates is equal if there is a control and treatment group Grimm et al Nature 2006 Off target effects of RNAi -How? Effector: TGS Thought to only be in lower species: wrong RNA-induced transcriptional silencing (RITS) targets the DNA helix and induces heterochromatic silencing nuclear effect components not clear role of miRNA role of long intergenic small RNAs (lincRNAs) Regulation of Dicer Expression Regulation of Dicer will have downstream effects on miRNA production and, in turn, on gene expression Potentially involved in RNA silencing-mediated antiviral response in both plants and animals MBP1007/1008 - Nucleic Acids A functional mRNA: The cytoplasmic story Objectives (1) To discuss the iNUTS and iBOLTS of how mRNAs function in the cytoplasm (2) To review older and introduce newer methods of studying mRNA biology in the cytoplasm (3) Understand the basics of RNAi-induced posttranscriptional gene silencing