NucleicAcids18 Eukaryotic MicroRNAs.pdf

BIOC 3041 Nucleic Acids Biochemistry Regulatory RNAs Regulatory RNAs • Early work on gene regulation proposed a role for regulatory RNAs but evidence was lacking until the early 1990s – found small RNAs… • What was formerly believed to be “junk DNA” is now known as template for non-protein coding...

BIOC 3041 Nucleic Acids Biochemistry Regulatory RNAs Regulatory RNAs • Early work on gene regulation proposed a role for regulatory RNAs but evidence was lacking until the early 1990s – found small RNAs… • What was formerly believed to be “junk DNA” is now known as template for non-protein coding RNAs…mostly • This may explain why eukaryotes were originally predicted to have far more protein-coding genes based on genome size but were later found by genome analysis to have far fewer proteins • “non-coding RNAs” (ncRNAs) now known to have regulatory roles • Can now exploit regulatory RNA knowledge to experimentally alter gene transcriptional activity to study gene function, medical purposes RNAs can serve as regulators of gene transcription and translation RNA Interference Is A Major Regulatory Mechanism in Eukaryotes • Eukaryotes have very short RNAs that can repress / silence genes that have homology to these short RNAs • This is RNA interference (RNAi), is a well-used method of choice to suppress gene expression in model organisms to study gene function • RNAi can cause: 1) inhibition of translation of gene (blocks ribosome), 2) dsRNA formation and then destruction of dsRNA, 3) short RNA binding to gene’s promoter and block it • These very short RNAs made from longer dsRNAs by specific enzymes • Worm (C. elegans) and plant (Arabidopsis) research shows RNAi to be important in development and antiviral defence RNAi uses very short RNAs to bind & shut off matching regions in target genes Eukaryotes use different classes of small RNAs • Eukaryotes small RNAs are ~ 20-30 nt long, come from longer transcripts • Eukaryotic sRNAs bind with Argonaute-family proteins, which facilitate interactions with targets • Eukaryotic sRNA are grouped into three main classes: MicroRNAs (miRNAs) –come from primary transcripts -usually downregulate RNAs by translational repression and mRNA decay Small interfering RNAs (siRNAs) -from longer double stranded RNAs -they target RNAs for degradation - a cellular defense mechanism Repeat-associated small interfering RNAs (rasiRNAs) -from repetitive regions of the genome -downregulate transcription from repetitive regions -focus on mammalian subclass (piRNAs) Small RNAs derived from different sources and used in various ways Short RNAs That Silence Genes Are Produced from a Variety of Sources and Direct the Silencing of Genes in 3 Different Ways • Small RNAs made from dsRNA precursors are small interfering RNAs (siRNAs) • RNAs encoded by dedicated genes rather than longer dsRNAs are called micro RNAs (miRNAs) • A RNase enzyme (‘Dicer’) chops up dsRNA precursor to yield 19-25 nucleotide tiny RNAs RISC – RNA Induced Silencing Complex Dicer enzymes process small RNAs into tiny bits that stop gene expression Short RNAs That Silence Genes Are Produced from a Variety of Sources and Direct the Silencing of Genes in Three Different Ways - 2 • Proteins involved in these different means of gene expression suppression are same • RNA-Induced Silencing Complex (RISC) is both protein and siRNA/miRNA • Argonaute-family proteins in RISC complex • RISC complex denatures the RNA and allows it to bind to matching regions so it may interfere • Perfect target matches cause mRNA destruction • Imperfect target matches just inhibits translation RISC protein needed to match interfering RNA with target in mRNA or genome Short RNAs That Silence Genes Are Produced from a Variety of Sources and Direct the Silencing of Genes in Three Different Ways -#3 • Argonaute-family proteins often called “Slicer” and mRNA cleavage called “slicing” • RISC can recruit chromatin remodelling enzymes when targeted to nucleus and modify promoter leading to gene silencing • E.g., yeast centromeric regions use this method to silence themselves by making miRNA • RNAi very potent since RNA-Dependent RNA Polymerase (RDRP) can be recruited by RISC and make even more dsRNA precursor which amplifies the process http://www.youtube.com/watch?v=cK-OGB1_ELE at 0:30 sec RISC can shut down promoters and recruit RDRP to amplify the RNAi signal • RNAi very potent since RNA-Dependent RNA Polymerase (RdRP) can be recruited by RISC and make even more dsRNA precursor which amplifies the process miRNAs Have a Characteristic Structure That Assists in Identifying Them & Their Target Genes • Genetically-encoded micro RNAs (miRNAs) have characteristic, predictable structure • Each pre-miRNA composed of 2 “arms” on either side of the hairpin’s loop within the primary miRNA 5’ 3’ DROSHA • primary miRNA (pri-miRNA) is a precursor made of hairpin RNA plus ssRNA sequence • Pri-mRNA cleaved by ‘DROSHA’ RNase to give hairpin precursor miRNA (pre-miRNA) that are exported to cytosol • Second RNase cleavage reaction by DICER liberates the mature miRNA sequences miRNAs need to undergo post-transcriptional processing to activate them miRNAs Have a Characteristic Structure That Assists in Identifying Them & Their Target Genes - 2 • Pre-miRNA has two arms, each of which may have their own targets (e.g. miR-1 and miR-1*) • Lin4 and let-7 miRNAs discovered by genetics in pre-genome era • miRNAs found after called miR-## using bioinformatic searches of sequenced genomes • “seed region” (bases 2-9) has high complementarity to target RNA and can help identify targets www.youtube.com/watch?v=_-9pROnSD-A Pre-miRNAs can yield 1-2 miRNAs that bind complementary target regions miRNAs Have a Characteristic Structure That Assists in Identifying Them & Their Target Genes - 3 • Pre-miRNA may be found in any part of an RNA molecule • miRNAs can be found in exons, introns, leader sequences • One or more can be made from the same transcript • DICER and miRNA-specific RNase called Drosha/DGCR8 recognize pre-miRNA structures - not their sequences miRNAs may be encoded by any RNA transcript but need DICER & Drosha processing An Active miRNA Is Generated Through a 2-Step Nucelolytic Processing • Drosha cuts the pri-mRNA to liberate pre-miRNA with help of specificity subunit protein (Pasha a.k.a. DGCR8) • Drosha + Pasha/DGCR8 is the microprocessor complex • Drosha/DGCR8 cuts first (yields 65-70 base pre-miRNA), removes the lower stem (few mismatches) to leave upper stem and terminal loop • Drosha/DGCR8 needs single-stranded RNA in basal segments to help guide the cleavage reaction (targets only dsRNA) • Drosha/DGCR8 leaves a 2 nucleotide 3′ overhang required for DICER Drosha cleaves pri-mRNA to yield pre-mRNA containing upper stem + loop Dicer Is the Second RNA-Cleaving Enzyme Involved in miRNA Production • Pre-miRNA cleaved by Drosha in nucleus is exported to cytoplasm where Dicer recognizes the pre-miRNA’s structure, not sequence • Dicer made of two RNase domains and a PAZ domain cupping the pre-miRNA’s 3′ overhang • PAZ domain shared among Piwi$, Argonaute, and Zweille$ proteins to serve as the base of the helical “ruler” • Ruler domain used to measure 22-25 base pairs so that each of the RNase domains will cut one of the two double-stranded RNA molecules Dicer recognizes the Drosha-cleaved pre-miRNA and cleaves off terminal loop Incorporation of a Guide Strand RNA into RISC Makes A Mature Complex ready to Silence Gene Expression • Dicer gives a ~22 bp miRNA that will serve as “guide RNA” that binds a mRNA target using the RISC protein complex (RNA-Induced Silencing Complex) • Double-stranded miRNA bound by RISC and denatured to yield the guide miRNA strand and a ‘passenger strand’ that is discarded and destroyed • Argonaute protein is usually the ‘slicer’ protein that cuts the base paired guide miRNA-target mRNA molecule – but other Argonaute isoforms do lead to simple repression of translation (no cuts) • RISC composed of several proteins including the Argonaute protein that also has a PAZ domain • Argonaute lines up guide & target RNAs to allow RNase domain to cleave target in the middle N- PAZ MID PIWI (RNase) -C Argonaute subunit of RISC pairs miRNA & target mRNA so it can be silenced PIWI RNAs serves as template • made from single-stranded piRNA precursor RNA that are antisense to transposon mRNAs • Zucchini nuclease cleaves initial transcript into fragments that are loaded onto Piwi proteins (Piwi/Aub) • These primary piRNAs direct the generation of additional piRNAs in the ‘ping-pong’ cycle • Aub-loaded piRNA directs transcript cleavage, making another piRNA • piRNA then loaded into an Argonaut protein, which directs cleavage of piRNA precursor to make another piRNA from the antisense PIWI RNAs help to suppress expression of otherwise damaging transposons siRNAs Are Regulatory RNAs Generated from Long Double-Stranded RNAs • miRNAs made as pri-mRNAs with hairpin loops, siRNAs don’t have these • siRNAs are made from longer dsRNAs, thus Drosha is unneeded • siRNAs discovered when scientist tried to make darker flowers by overexpression of pigment enzymes • More enzyme overexpression caused loss of colour!  something was “knocking down” the level of the pigment enzyme transcript dsRNA formation! • dsRNA can be formed several ways in cells; Unidirectional transcription siRNAs differ from miRNAs in that they don’t have hairpins or need Drosha Did RNAi Evolve As an Immune System • RNAi might’ve been ancient immune system recruited for use with miRNAs to regulate gene expression • RNAi machinery perhaps once used to suppress viruses & transposons (45% of our genome is made up of former transposons…) • Transposons often transcriptionally silent and linked to heterochromatin loss of RNAi machinery reactivates transposons causing them to ‘jump’ around and cause mutations • Plants use RNAi to suppress viral genes by spreading siRNAs made from viral genome itself around the plant to stop spread of viral infection • • • • Viruses have retaliated by producing anti- RNAi proteins that 1) stop production of siRNAs, No virus 2) destabilize siRNAs, or 3) stop export of siRNAs Virus-infected RNAi is likely an ancient immune system recently used for gene regulation • • • • • • • • • • • • • • Lecture Recap RNAs can serve as regulators of gene transcription and translation RNAi uses very short RNAs to bind & shut off matching regions in target genes Dicer enzymes process small RNAs into tiny bits that stop gene expression RISC protein needed to match interfering RNA with target in mRNA or genome RISC can shut down promoters and recruit RDRP to amplify the RNAi signal miRNAs need to undergo post-transcriptional processing to activate them Pre-miRNAs can yield 1-2 miRNAs that bind complementary target regions miRNAs may be encoded by any RNA transcript but need DICER & Drosha processing Drosha cleaves pri-mRNA to yield pre-mRNA containing upper stem + loop Dicer recognizes the Drosha-cleaved pre-miRNA and cleaves off terminal loop Argonaute subunit of RISC pairs miRNA & target mRNA so it can be silenced siRNAs differ from miRNAs in that they don’t have hairpins or need Drosha RNAi is likely an ancient immune system recently used for gene regulation Natural RNAi machinery can be exploited to artificially silence target genes www.youtube.com/watch?v=5YsTW5i0Xro Reading – MBPGF pages 528-43, 547-8, MBOG7 pg 711-719, 725-726, MBOG6 pg 633-657