MCB 252 Topic 15 Regulation of Splicing Polyadenylation and Non coding RNA Biology Prof David Rivier MCB 252 Spring 2015 Lecture Outline Alternative Splicing Alternative Polyadenylation Non coding RNAs siRNAs small inhibitory RNAs miRNAs micro RNAs lncRNAs long non coding RNAs piRNAs PIWI Interacting RNAs Blue Exons Yellow Introns Dscam protein in Drosophila Immunity and Neuronal Wiring Drosophila has 14 000 genes Regulated Alternative Splicing of Doublesex Dsx Sxl only transcribed in females early in development Sxl transcribed in both males and females later in development Sex Determination in Drosophila is dependent on a cascade of alternative splicing events Regulated Alternative Splicing of Doublesex Dsx Rbp1 is an SR protein This is a regulated ESE Based on older technology More recent results 95 of human genes spliced Lecture Outline Alternative Splicing Alternative Polyadenylation Non coding RNAs siRNAs small inhibitory RNAs miRNAs micro RNAs lncRNAs long non coding RNAs piRNAs PIWI RNAs Lecture Outline Regulation of Translation in Some Animal Eggs Alternative Splicing Alternative Polyadenylation Non coding RNAs siRNAs small inhibitory RNAs miRNAs micro RNAs lncRNAs long non coding RNAs piRNAs piwi interacting RNAs An Old Idea to Inhibit Translation of Specific mRNAs A Potentially Powerful Experimental Tool Inhibit the transcription of one type of mRNA by injecting a cell with Anti sense RNA YF Cellular mRNA Injected Antisense RNA dsRNA inhibit translation of YF mRNA less of YFP Unexpected Surprising Result of Control Experiment Sense strand inhibited better than anti sense strand in a control experiment note this sense strand sample was likely contaminated with some dsRNA based on later work Discovery of siRNA RNAi Fire and Mello initially figured out that dsRNA is the inhibiting entity that small dsRNA are involved 21 23bp that the small RNAs likely acted catalytically to inhibit many copies of an mRNA Then did a bunch more stuff Players Involved in Non coding RNA Biology Drosha Dicer RISC RNA Induced Silencing Complex Argonaute family PIWI sub family within the Argonaute family siRNA Pathway Dicer dsRNA 2123 nt segments RISC a sequence specific RNAase Argonaute is the RNA binding nuclease subunit of RISC Argonaute Subunit of RISC Guide RNA Guide RNA makes RISC a sequencespecific RNAase Complementary base pairing of Guide RNA with mRNA Specificity siRNA RNAi For now think of this RNA as perfect dsRNA not loops and hairpins Transposons and Viruses Versions of both produce dsRNA RNAi siRNA Technology is a Major Experimental Tool Start with Sequence of YFG Produce dsRNA complementary to YFG Introduce into Cells or Organisms Determine Phenotype LOF siRNA injected into Worm Embryo mex3 expression eliminated in injected Embryo Dark blue indicates that mex3 mRNA is present Generating dsRNA in the Lab in vitro transcription Chemical Synthesis companies E coli siRNA Mechanisms Some plants and animals distribute siRNAs throughout the organism Some animals can be fed bacteria producing siRNA and see a result in the animal siRNAs often decrease the level of a specific mRNA but don t always eliminate it all together RNAi knockdown Lecture Outline Alternative Splicing Alternative Polyadenylation Non coding RNAs siRNAs miRNAs lncRNAs piRNAs C Elegans a Nematode a Round Worm C elegans as Experimental Organism Brenner Developed C elegans as Model Organism Shared 2002 Nobel with Sulston and Horvitz Who Sidney Brenner Worked Out Cell Lineage and Apoptosis John Sulston Robert Horvitz C elegans Development follows an Invariant Lineage A Genetic Screen for Lineage Defects Screen for Defects in Development of a Vulva Can Produce Fertile Eggs but Cannot Lay the Eggs A Gross Genetic Screen for Lineage Defects Bag of Worms Phenotype lin mutants defects in cell lineage Lin 14 lineage vs Wild type lineage Larval Stage L1 L2 L3 L4 Extra cells formed from P8p Several divisions 1 larval stage earlier than wild type 2008 Victor Ambros lin4 Gary Ruvkun lin14 2015 Connection Between LIN4 and LIN14 Revealed by Cloning LIN4 very small gene that produces a non coding RNA LIN14 2 mutations localized to the 3 UTR of LIN14 mRNA Sequence elements in LIN4 were complementary to sequence elements in the 3 UTR of LIN14 suggesting an RNA RNA interaction between the two gene products Results from Genetics and Molecular Biology LIN4 regulates LIN14 in the absence of LIN4 excess LIN14 protein is made and is made at an earlier time than in wild type cells How does LIN4 RNA activity result in inhibition of LIN14 production How Does LIN4 Regulate LIN14 Production Discovery of the miRNA Pathway LIN4 very small gene that produces a non coding RNA Title miRNA and siRNA have a shared pathway Imperfect pairing of guide RNA inhibit translation Perfect pairing of guide RNA mRNA cleavage Title Lin 4 pre miRNA processed into several miRNAs each of which is partially complementary to distinct elements in the lin 14 3 UTR miRNA Processing Primary transcript priRNA Drosha cleaves pri miRNA to pre miRNA in nucleus Exportin 5 takes pre miRNA to cytoplasm Dicer cleaves pre miRNA into 21 23 bp segments Logic of Regulation One miRNA can inhibit the transcription of mRNAs produced from 100 s of different genes as long as those genes produce mRNA that contain the target sequence usually in the 3 UTR Small genes and small targets doesn t cost much May be easier to add a small target to a UTR than add additional regulatory elements enhancers repressors to gene regulatory regions miRNA Genes in Humans Estimate that there are 2 000 miRNA genes in humans genes that produce miRNAs Estimate that 30 of Human Genes Regulated by miRNAs Lecture Outline Regulation of Translation in Some Animal Eggs Alternative Splicing Alternative Polyadenylation Non coding RNAs siRNAs miRNAs lncRNAs piRNAs Long Non Coding RNAs lncRNAs lncRNAs regulate transcription Often via epigenetic repression histone modification lncRNAs act as a scaffold to recruit histone code writers and readers Some lncRNAs also bp with DNA or RNA to recruit proteins to a specific locus lncRNA and X chromosome Inactivation XIST gene is transcribed only on the inactive Xchromosome 20 000 bp lncRNA XIST RNA in Red XIST is a scaffold that binds PRC2 histone methyltransferase HMT and DNA methyltransferase DNMT XIST in X chromosome Inactivation Spreading of inactive chromatin along the X chromosome Other lncRNAs Regulate Txn at Specific Loci Lecture Outline Regulation of Translation in Some