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5/20/05 Biol/chem 473

More stem cell cloningnews from the SouthKoreans:See articles posted onweb site

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From previous lecture:Lack of homology between two sequence is often more apparent whencomparisons are made at the amino acid level (Why**)

DNA sequence: identities in red; non-identities in blueThese sequences are 76% identifical. Are they sequenceshomologous?

when this sequence is translated into amino acids, the identity falls to28% (identities in brown; non-identities in green)

Bottom line: the comparison between amino acidsequences suggests that these genes are not homologousand that the similarity at the nucleotide level wasfortuitous

**Think of the possible # of readouts with a digital codewith 20 possibilities at each position in the code versus 4possibilites

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6/6/03 Biol/Chem 473 RNAihttp://www.orbigen.com/RNAi_Orbigen.html

RNAi 2002 Scientific Breakthrough of the YearScience Dec 20, 2002

RNAi at the movies:http://www.nature.com/focus/rnai/animations/index.html

Science 308: 480 4/22/05 Human RNA slows down a primateretrovirus

Nature 402: 128 11/11/99 Policing Rogue Genes

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Scientific discovery byaccident:The plant thread of the story beginswith the search for a more purpleflower

The quest for purplerpetunias

• Pant biotechnologistsstrategy was to try to boostthe activity of an enzymeinvolved in the production ofanthrocyanin pigments

• The researchers hooked upthe gene to a powerfulpromoter sequence andintroduced this artificialconstruct into their petunias

• The investigators expecteddeep purple flowers from ahigh level of transcription ofthe transgene

• Instead of being deep purple,many of the flowers grew upvirgin white or variegated(above)

• In the white or variegatedflowers, not only was thetransgene not activated, butthe endogenous anthrocyaningenes had been inactivated

• the white phenotype could bepassed onto the nextgeneration -- but someflowers reverted to purple

• Was this phenonmenoncontrolled by some sortof unstable nucleic acid?

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The worm thread of the story: when controls don’tbehave properly

Older naïve idea:: antisense technology

Unexpected results from controls suggested that “antisense” techniquesweren’t functioning via the expected mechanism:• sense RNA also worked to abrogate gene function• double-stranded RNA worked 10 times better than sense or

antisense RNA

Various studies concluded that ds RNA was the “active agent” and thatprevious results showing effects of single-stranded antisense (or sense)RNA were due to double-stranded RNA that contaminated the preps

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These and other investigations (with funnyoutcomes) in other organisms converged on anancient RNA silencing system

This system consists of an RNA silencingmechanism that is conserved among speciesfrom different kingdoms:fungi, animals and plant(what’s true for brewer’s yeast is true for theelephant…..)

HUH? WHAT?

How does it work? What triggers it?How have molecular biologists made use of it?

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RNAi has roles in:

• normal developmental events that arecontrolled by micro RNAs (miRNAs)

• an ancient “immune system” that protectscells from foreign and/or aberrant nucleicacids

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CH3, modified DNA or chromatin; AAAA, poly-adenosine tail; TGA, translationtermination codon copied directly from legend in the paper!

Figure legend on next page

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Legend to figure on previous page; Model depicting distinctroles for dsRNA in a network of interacting silencing pathways.• In some cases dsRNA functions as the initial stimulus (or

trigger), for example when foreign dsRNA is introducedexperimentally.

• In other cases dsRNA acts as an intermediate, for examplewhen 'aberrant' mRNAs are copied by cellular RdRP.

• Transcription can produce dsRNA by readthrough fromadjacent transcripts, as may occur for repetitive genefamilies or high-copy arrays (blue dashed arrows).Alternatively, transcription may be triggeredexperimentally or developmentally, for example in theexpression of short hairpin (shRNA) genes and endogenoushairpin (miRNA) genes.

siRNAs, the small (~23 nt) RNA products of the Dicer-mediated dsRNA processing reaction guide distinct proteincomplexes to their targets.These silencing complexes include:

1. the RNA-induced silencing complex (RISC), which isimplicated in mRNA destruction and translationalrepression, and

2. the RNA-induced transcriptional silencing complex(RITS), which is implicated in chromatin silencing.

Sequence mismatches between a miRNA and its target mRNAlead to translational repression (black solid arrow), whereas nearperfect complementarity results in mRNA destruction (blackdashed arrow). Feedback cycles permit an amplification andlongterm maintenance of silencing.

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Micro RNAs: Be sure to read pg. 570 in Watson• hundreds of microRNAs (miRNAs) have been

discovered in the genomes of eukaryotes• miRNAS appear to be involved in many critical

cellular and developmental functions• miRNAs are involved in regulation of gene

expression at the level of translation of a messenger -- important in development in worms and plants andwho knows who else

• there is some evidence that miRNAs in humans maytarget the genetic material of viruses, not humangenes

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All genomes of complex organisms are thepotential targets of rogue nucleic acids:

Categories of rogue nucleic acids?

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Transposable elements

Viruses

• 45% of the human genome consists ofremnants of previous transposon/virusinvasions and elements that are still activetoday

• 8% of our genome is retrovirus likesequence

A priori, one would expect that organisms needto fight off such invasionsto prevent the genomefrom being completely taken over by molecularinvaders

The two problems faced by the organism inprotecting the genome are similr to those facedby the vertebrate immune system:

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How to recognize self from non-self?

How amplify an initial response in a specificfashion?

Vertebrate immune system solution:solved the specificity issue by generating a moreor less random repertoire which is then limitedby a filtering process

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How does this genomic “immune system”work?

Specificity:recognize self-vs. non-selfVersatilityMemory/Amplification

• RNA silencing uses common mechanisms inall organisms studied thus far

• There is a high degree of homology betweengenes required for this process

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The NON-self Silencing trigger:

ONE KNOWN TRIGGER:Double-stranded RNA triggers RNAi or RNAsilencing or PTGS

Based on what you know about “normal”cellular ribonucleic acids does this make sense?

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Gagging order; using dsRNA, specific genescan be silenced

dicer

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RNA silencing pathways in the usual suspects• depends on a common trigger: DS RNA• can result in silencing in three (maybe

more?) different ways

Figure 1 RNA silencing pathways in different organisms. Long dsRNA and miRNA precursorsare processed to siRNA/miRNA duplexes by the RNase enzyme called Dicer. The short dsRNAsare subsequently unwound and assembled into effector complexes: RISC, RITS (RNA-inducedtranscriptional silencing) or miRNP. RISC mediates mRNA-target degradation, miRNPs guidetranslational repression of target mRNAs, and the RITS complex guides the condensation ofheterochromatin. In animals, siRNAs guide cleavage of complementary target RNAs, whereasmiRNAs mediate translational repression of mRNA targets. rasiRNAs guide chromatinmodification. S. pombe, C. elegans and mammals carry only one Dicer gene. In D. melanogasterand A. thaliana, specialized Dicer or DLC proteins preferentially process long dsRNA ormiRNA precursors. 7mG, 7-methyl guanine; AAAA, poly-adenosine tail; Me, methyl group; P,5' phosphate.

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Science 296: 1263 A model for the molecular steps in RNA silencingRNAi animation featuring species differences in the RNAi specifics:http://imgenex.com/rnai_anim.php

More acronyms: RdRP =

What makes a ssRNA aberrant? Nobody knows yet for sure.How does it work? Although mechanims of gene silencing are far from completely understood,the working hypothesis goes like this: the initial trigger is the presence in the host's cells of anaberrant RNA. This could be a double-stranded RNA, a shortened RNA that lacks its 'cap' or'tail', or a conventional RNA that is present in unusually large quantities. The host organism'sresponse is to call on enzymes that slice and dice the offending RNA into pieces around 25nucleotides long. At some stage — either before or after the formation of these fragments — therogue RNA is copied many times over, to amplify the alarm signal. The fragments then spreadthroughout the host. Antisense strands, complementary to the target mRNA, bind to the targetand prompt other enzymes to disable it.

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Amplifying the RNAi signal

Science 296: 1271

Figure 2. (A) Degradative and synthetic pathways linking dsRNA, siRNAs, and target mRNAin RNA silencing. Black arrows denote classical RNA silencing degradative pathways. Yellowarrows denote RdRp-dependent synthetic pathways leading to generation or amplification ofdsRNA. RdRp may act on siRNA-primed dsRNA (1), siRNA-primed mRNA (2), or asRNA-primed mRNA (3) to generate or amplify some or all of the inducing dsRNA sequences

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RNA silencing involves molecular machines

Nature 418: 244Figure 2 Dicer and RISC (RNA-induced silencing complex). a, RNAi is initiated by the Dicerenzyme (two Dicer molecules with five domains each are shown), which processes double-stranded RNA into 22-nucleotide small interfering RNAs36. Based upon the known mechanismsfor the RNase III family of enzymes, Dicer is thought to work as a dimeric enzyme. Cleavageinto precisely sized fragments is determined by the fact that one of the active sites in each Dicerprotein is defective (indicated by an asterisk), shifting the periodicity of cleavage from 9–11nucleotides for bacterial RNase III to 22 nucleotides for Dicer family members40. The siRNAsare incorporated into a multicomponent nuclease, RISC (green). Recent reports suggest thatRISC must be activated from a latent form, containing a double-stranded siRNA to an activeform, RISC*, by unwinding of siRNAs41. RISC* then uses the unwound siRNA as a guide tosubstrate selection31. b, Diagrammatic representation of Dicer binding and cleaving dsRNA (forclarity, not all the Dicer domains are shown, and the two separate Dicer molecules are coloureddifferently). Deviations from the consensus RNase III active site in the second RNase III domaininactivate the central catalytic sites, resulting in cleavage at 22-nucleotide intervals

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Direct Evidence that RNA silencing suppressestransposon activity in C. elegans:• there are 30 mutant C. elegans lines that have a

higher than normal activity of a specifictransposable element

• in 22 of these mutant strains, RNAi (RNAsilencing system) is compromised

• one of the mutated genes codes for an RNase

What about RNAi mediated Defense againstviruses?

See abstract

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Where do DS RNAs come “naturally”?

How double-stranded RNA can be generated

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AHA -- YET another way for molecularbiologists to abrogate gene function

As usual molecular biologists have appropriated this naturallyoccuring genomic immune system for their own purposes:

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» » » Dicer siRNA Generation KitHome Catalog Gene Delivery

Dicer siRNA Generation Kit

The Dicer siRNA Generation Kit allows easy and cost-effective generation of a large number of small interfering RNAs (siRNAs) from full-length target genes (1). siRNAs are 21- to 23-nucleotide RNA molecules that can cause targeted gene silencing in mammalian cells through a process known as RNA interference (2,3,4). In nature, siRNAs are generated by ribonuclease III cleavage of longer double stranded RNAs (dsRNAs). When dsRNAs are transfected directly into mammalian cells, they activate the interferon system and provoke non-specific gene suppression and cytotoxic response (2). siRNAs have proven to be effective at specifically silencing gene expression without causing any interferon response.

The Dicer siRNA Generation Kit mimics the natural RNA interference process by using recombinant human dicer enzyme, a double-stranded RNA-specific endonuclease, to cleave in vitro transcribed dsRNA templates into a pool of 22 bp siRNAs (Figure 1, 2). Compared to conventional siRNA construction methods such as chemical synthesis and hairpin siRNA expression vectors, the Dicer siRNA Generation Kit offers the following advantages:

Description

No guesswork – A mixture of siRNAs has a better chance of success than a single siRNA design.No wasted time and money due to failed siRNA designsMore regions of the genes can be screened for silencingProvided with the proven GeneSilencer™ siRNA Transfection Reagent

Search:

Diced GFP siRNA Effectively Suppresses GFP Expression in HeLa Cells (Click to enlarge)

Files

Examples of Highly Specific Gene Silencing Using the Dicer Kit (pdf - 172 KB)More Dicer Product Data (pdf - 133 KB)

Manuals

Dicer siRNA Generation Kit Manual (pdf - 498 KB)Recombinant Dicer Enzyme Kit Manual (pdf - 164 KB)TurboScript T7 Transcription Kit Manual (pdf - 168 KB)RNA Purification Column 1 Manual (pdf - 134 KB)RNA Purification Column 2 Manual (pdf - 136 KB)

6/5/03 1:10 PMGene Therapy Systems - Catalog - Gene Delivery - Dicer siRNA Generation Kit

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For maximum convenience, the Dicer siRNA Generation Kit contains everything that is required for preparing double stranded RNA, RNA cleavage, siRNA cleanup and transfection. Both the Recombinant Dicer Enzyme and the RNA Purification Columns are also available separately.

Sufficient material is provided for transcribing, cleaving, and transfecting siRNAs for 50 transfection experiments in 24-well plates and with up to 5 different genes.

Contents

The Dicer siRNA Generation Kit is shipped frozen. Both the TurboScript™ T7 Transcription Kit and the recombinant human dicer enzyme should be stored at –20°C upon receipt. If stored properly, all reagents are stable for 6 months.

Storage

1. Myers, J.W. et al. (2003) Nature Biotechnology 21: 324-328.2. Elbashir, S.M. et al. (2001) Nature 411: 494-498.3. Caplen, N.J. et al. (2001) Proc Natl Acad Sci USA 98: 9742-9747.4. Sharp, P.A. (2001) Genes and Development 15: 485-490.

References:

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GeneSilencer siRNA Transfection ReagentGeneSilencer 96 siRNA Transfection Reagent Plates

For maximum convenience, the Dicer siRNA Generation Kit contains everything that is required for preparing double stranded RNA, RNA cleavage, siRNA cleanup and transfection. Both the Recombinant Dicer Enzyme and the RNA Purification Columns are also available separately.

Sufficient material is provided for transcribing, cleaving, and transfecting siRNAs for 50 transfection experiments in 24-well plates and with up to 5 different genes.

The Dicer siRNA Generation Kit is shipped frozen. Both the TurboScript™ T7 Transcription Kit and the recombinant human dicer enzyme should be stored at –20°C upon receipt. If stored properly, all reagents are stable for 6 months.

1. Myers, J.W. et al. (2003) Nature Biotechnology 21: 324-328.2. Elbashir, S.M. et al. (2001) Nature 411: 494-498.3. Caplen, N.J. et al. (2001) Proc Natl Acad Sci USA 98: 9742-9747.4. Sharp, P.A. (2001) Genes and Development 15: 485-490.

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6/5/03 1:10 PMGene Therapy Systems - Catalog - Gene Delivery - Dicer siRNA Generation Kit

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Knockout (knockdown) of gene function via RNAior PTGS (Post-transcriptional gene silencing)

Does is work?

d. the white gene of Drosophila inactivated byRNAi: left panel

Next pageGene Silencing tools. dsRNA = double stranded RNAScience 296: 1265 May 17, 2002

C.elegans RNAi databasehttp://nematoda.bio.nyu.edu/

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