BIOLOGY 305 1st Edition Lecture 25 Outline of Last Lecture I. Introduction to EpigeneticsII. 5 Classic Epigenetic PhenomenaIII. Stem CellsIV. Biotechnologies and EthicsOutline of Current Lecture I. RNA InterferenceA. The Mechanism and Function of RNA interferenceB. CRISPR SequencesC. Comparison and Application of RNAiII. TransposonsIII. TGS, miRNA, and lncRNA as RegulatorsIV. Key Vocabulary and Sample QuestionsCurrent LectureI. RNA InterferenceThe important questions are: Why do RNA have so many functions?How does ncRNA regulate or serve as an immune system?Dark matter – something we can’t see but we know does something importantHowever, Non coding-RNA is the opposite: they are things we can see but don’t know if it contributes to anythingRNA interference (RNAi) – first observed in 1990, RNA interference turns gene off instead of contributing to gene productEx: Richard Jorgensen inserted pigment-producing gene under control of an active promoter in order to produce a deeper color, found variegated phenotypes instead:Cosuppression – expression of both introduced by pigment gene and the petunia’s natural pigment genes were suppressed.RNAi is the fundamental mechanism behind this post-transcriptional regulation involves double-stranded RNA. It silences gene expression by either1) Blocking transcription of targeted genes2) Block gene transcription post-transcriptionallyafter small regulatory RNAs bind to mRNA using complementary base pairingA. The Mechanism and Function of RNA interferenceRNA is introduced  RNA is restricted into very small fragments called ‘small RNA’ (sRNA, 21-25bp)  these have the ability to turn of genes  results in the repression of homologous genesRegulatory RNA in RNAi become double stranded (called dsRNA) in two ways:1) They form hairpin loops2) RNA dependent RNA polymerase enzyme produces the second strandThe double stranded RNA is able to bind to a protein called dicer, which chops it into sRNAsRNA  silencing complexes (RISCs) as mediated by Argonaute protein. Argonaut proteins (within RISC) gives sequence specificity to sRNA: siRNA = small interfering RNAThe silencing complexes, called RNA-induced Silencing Complex (RISCs) denatures the dsRNA into either:Guide strand – which is biologically activePassenger strand – which is usually degradedThe silencing complex together with the guide strand can:1) Repress translation as microRNA (miRNA)RISC-guide RNA binds to complementary mRNA to block translation2) Degrade mRNA as siRNA  post-transcriptional gene silencing PTGSGuide strand complements to mRNA, RNAse domain of RISC cuts mRNA3) Repress transcription as siRNA  transcriptional gene silencing TGScomplex directs chromatin modifying enzymes to nucleus, silences transcriptionmiRNA and siRNA are produced in a similar wayPrimary microRNA (pri-miRNA) and pri-siRNA from the nucleus  cut by DicerIn animals, the Drosha enzyme complex cuts pri-miRNA before processed by dicer + RISCBoth miRNA and siRNA are bound by RISC after being producedB. CRISPR sequences Clustered regularly interspaced short palindromic repeats found in bacteria, containing short sequences derived from phages. They give rise to crRNA and target the destruction of cDNAAfter multiple bacterial genomes and bacteriophage genomes are sequenced, people realized they have a lot in common with each other  this is because of convergent evolutionColors are different sequences coming from various sequences in the bacteriophage genomeIn biogenesis, the short fragments of RNA contain important info about phage DNA (this function is similar that of the dicer)The crispr RNA (crRNA) is like Argonaut as it gives the an effector complex sequence specificityStops genes from functioning(this function is similar to that of Argonaut)C. Comparison and Application of RNAiRNAi EvolutionLikely used to protect genome against mutation by transposons and viral infectionMutations in Dicer, Argonaut, RNAi machinery  susceptible to viruses and mutationThe siRNA produced by dicer binds to Argonaut and is then able to bind to viral RNA and destroy itRNAi detects RNA only, double-stranded RNA is recognized by dicer, can also recognize incorrectfunctioning RNA, is a function of virus resistanceFunction in Plants vs. AnimalsPlants: miRNA have more complementarity and degradation of mRNA, less translation Animals: miRNA have less complement of mRNA, more blocking of translationRNAi ApplicationResearch: Use RNAi to knock out expression of genesBiomedical: Use RNAi to control over-expression or erroneous transcripts in diseaseSuggestion: Make a table of all the different functions of the crRNA, RNAi, etc…Assign practical applications to RNAi (which are eukaryote only) or CRISPR (works in bacteria, but can be transplanted into eukaryotes):II. Transposons Transposable ElementsMust be turned off or genome integrity may be compromisedProduces abnormal mRNA:Are sometimes present as tandem repeats (if the same RNA is produced more than once, may lead to the production of dsRNA)Inverted repeats  hairpins (which are dsRNA)Recognized by RNA dependent RNA-PolymeraseAre targeted and silenced by TGSHeterochromatin formation  TE turned offPlants have two extra RNA polymeraseRDRna polymerase recognizes and creates dsRNAdsRNA diced by dicersiRNA is formed and methylated to work with argonauteArgonaut is able to bind to specific locus to establish heterochromatin on this genomic regionHistone proteins, etc., help to work in removal of active histone marks and establishment of repressive histone marksIII. TGS, miRNA, and lncRNA as RegulatorsTGS (transcription gene silencing sRNA)silences transposons and regulates gene expressionChIP-sequence – found that Argonaut protein was strongly enriched on gene promoters microRNAThey are specialized to regulate gene expressionComposed of double stranded RNA produced by short hairpinpri-miRNA  pre-miRNA  processed into miRNA by dicerRepressed protein production by affecting translation or RNA stabilityBinds to argonaute and this binds to ribosome and stops translationTurns off gene expression by turning of protein expression10% of human genes are turned off by miRNA, and they are very conserved1 miRNA controls several genes, so if this is mutated, you can get very sick.Long-noncoding RNA (lncRNA):The Xic (X inactivation center) produces several lncRNAsXist coats the inactivated X and recruits PRC2Once recruited, turns off all the