Metabolism of Nuclear RNAMetabolism of Nuclear RNA

Coding and nonCoding and non--coding RNAcoding RNA

Coding Coding RNAsRNAs (4%(4% ) ) -- transcriptometranscriptomemRNAs : rapid turnovermRNAs : rapid turnoverhalfhalf--life life -- few minutes (bacteria) ~ few hours few minutes (bacteria) ~ few hours

(eukaryotes)(eukaryotes)NonNon--coding coding RNAsRNAs (96%(96% ))

rRNAsrRNAs : 80% in actively dividing bacteria: 80% in actively dividing bacteriatRNAstRNAssnRNAssnRNAs : included in mRNA processing (: included in mRNA processing (spliceosomespliceosome), ), eukaryotes onlyeukaryotes onlysnoRNAssnoRNAs : included in : included in rRNArRNA processing, eukaryotes processing, eukaryotes onlyonlymimi--RNAsRNAs: RNA: RNA--induced transcription silencinginduced transcription silencing

RNARNA processingprocessing

EndEnd--modifications : 7modifications : 7--methylguanosine cap, poly methylguanosine cap, poly (A) tail(A) tail

Splicing : removal of Splicing : removal of intronsintrons

Cutting events : Cutting events : rRNArRNA, , tRNAtRNA processing processing

Chemical modifications : addition of chemical Chemical modifications : addition of chemical groupsgroups,, RNA editing RNA editing

RNA Pol II is an RNA Factory

Capping of RNA pol II Capping of RNA pol II transcriptstranscripts

occurs early in all RNA pol II occurs early in all RNA pol II transcripts when the nascent RNA transcripts when the nascent RNA is in 20is in 20--35 nt stage (hnRNA, pre35 nt stage (hnRNA, pre--snRNA)snRNA)

after cleaving the after cleaving the γγ--phosphate phosphate ((RNARNA--triphosphatasetriphosphatase) a) a GMP GMP is is added in inverted orientationadded in inverted orientation (5(5´́ -- 55´́linkage) (linkage) (RNARNA--guanylyltranspheraseguanylyltranspherase))

Terminal inverted Terminal inverted guanosineguanosine is is methylatedmethylated at the 7 positionat the 7 position ((77--metyltranspherasemetyltranspherase))

Functions of CapFunctions of Cap

Protects mRNA from degradation by Protects mRNA from degradation by RNAsesRNAses

Enhances splicing efficiency of mRNAsEnhances splicing efficiency of mRNAs

Enhances transport of mRNAs from nucleus to cytoplasm Enhances transport of mRNAs from nucleus to cytoplasm

In the cytoplasm, the capIn the cytoplasm, the cap--structure is important for the initiation of structure is important for the initiation of translationtranslation

The eukaryotic translation initiation factor The eukaryotic translation initiation factor eIF4EeIF4E binds directly to the binds directly to the capcap--structurestructure

snRNAssnRNAs mm77G G -- nuclear export, mnuclear export, m33G G -- nuclear importnuclear import

PolyadenylationPolyadenylation

Requires the sequence 5'Requires the sequence 5'--AAUAAAAAUAAA--3' plus3' plus

the sequence 5'the sequence 5'--CACA--3' 103' 10--20 20 ntnt downstream, plusdownstream, plus

a 5'a 5'--(GU)(GU)--3' rich sequence further downstream3' rich sequence further downstream

Six factors are needed (PAP, CPSF, CSF, CFI, CFII, PAB), Six factors are needed (PAP, CPSF, CSF, CFI, CFII, PAB),

Two stage reaction (cleavage and AMP addition)Two stage reaction (cleavage and AMP addition)

100100--200 A residues added to give 200 A residues added to give ‘‘poly(Apoly(A) tail) tail’’

mRNA encoding histones are not polyadenylatedmRNA encoding histones are not polyadenylated

Main steps in polyadenylationMain steps in polyadenylation

CPSFCPSF((cleavagecleavage--polyadenylationpolyadenylationspecificity factorspecificity factor) ) binds AAUAAA in binds AAUAAA in

hnRNAhnRNACStFCStF ((cleavage stimulatory factorcleavage stimulatory factor) ) binds G/U rich sequence; CFI, CFII binds G/U rich sequence; CFI, CFII bind in betweenbind in betweenPAPPAP (PolyA polymerase) binds & (PolyA polymerase) binds & cleaves 10cleaves 10--35 b 3' to AAUAAA35 b 3' to AAUAAA at slow at slow rateratePAPPAP adds adds about 12 about 12 AA residues residues slowly, slowly, CFI, CFII and CPSF fall offCFI, CFII and CPSF fall off

PABIIPABII ((polyApolyA--binding protein II)binding protein II)binds, now As are added rapidly until binds, now As are added rapidly until reach 250, then stopsreach 250, then stops

Purpose of Purpose of PolyadenylationPolyadenylation

Protects mRNA Protects mRNA –– In cytoplasm In cytoplasm poly(Apoly(A) size decreases due to ) size decreases due to RNAsesRNAses, however , however poly(Apoly(A) polymerase continues rebuilding) polymerase continues rebuilding

Necessary for translation (site of binding by Necessary for translation (site of binding by poly(Apoly(A))--binding protein I binding protein I (PABI)(PABI)

Recruits mRNA to Recruits mRNA to polysomespolysomes

Required for splicing out of the last Required for splicing out of the last intronintron

prepre--mRNA splicingmRNA splicing

IntronsIntrons are intervening sequences that are intervening sequences that ““interruptinterrupt”” eukaryotic genes eukaryotic genes and must be removed before uninterrupted and must be removed before uninterrupted exonsexons coding for coding for proteins leave the nucleus as mRNAproteins leave the nucleus as mRNA

Three types of Three types of intronintron are known:are known:

1.1. Group I Group I intronsintrons -- Found in organelle and bacterial genes along with Found in organelle and bacterial genes along with some lower eukaryotes (fungi, some lower eukaryotes (fungi, tetrahymenatetrahymena) nuclear genes) nuclear genes

•• Can self splice without the aid of proteinsCan self splice without the aid of proteins

•• Require free GTP for splicingRequire free GTP for splicing

2.2. Group II Group II intronsintrons -- Found in organelle and bacterial genesFound in organelle and bacterial genes

•• Can self splice without the aid of proteinsCan self splice without the aid of proteins

•• Differ from Group I Differ from Group I intronsintrons in sequence and mechanismin sequence and mechanism

3.3. Nuclear Nuclear intronsintrons -- Found in eukaryotic nuclear genesFound in eukaryotic nuclear genes

•• Require proteins and other Require proteins and other RNAsRNAs for splicingfor splicing

Self splicing intronsSelf splicing introns

S.Altmann and T. Cech S.Altmann and T. Cech

group I requires binding of guanosine into active sitegroup I requires binding of guanosine into active site

results of splicing are mature RNA and linear intron RNAresults of splicing are mature RNA and linear intron RNA

group II does not bind any nucleotide it uses activated group II does not bind any nucleotide it uses activated OH of adenosine in active in branch site.OH of adenosine in active in branch site.

results of splicing are mature RNA andresults of splicing are mature RNA and lariat lariat intronintron

SSplicingplicing of nuclear of nuclear intronsintrons

Requires Requires spliceosomespliceosome

SpliceosomeSpliceosome consists of consists of snRNPssnRNPs and at least 70 participating and at least 70 participating proteinsproteins

TransesterificationsTransesterifications are facilitated by baseare facilitated by base--pairing of pairing of snRNAssnRNAs

Most mRNA are derived from the corresponding preMost mRNA are derived from the corresponding pre--mRNAmRNA

TrypanosomasTrypanosomas and and EuglenoidsEuglenoids construct mRNA from separate construct mRNA from separate precursors precursors –– trans splicingtrans splicing

Standard Standard intronintron boundary boundary –– ‘‘GUGU--AGAG’’ rulerule

SR Proteins Direct Splicing SR Proteins Direct Splicing by Defining by Defining ExonsExons

SR proteins family of 8 well conserved proteins.SR proteins family of 8 well conserved proteins.

SR proteins have NSR proteins have N--terminal RNP domains rich in ser terminal RNP domains rich in ser and and argarg..

Function to align 5Function to align 5’’ splice site and the branch point splice site and the branch point by interactions with by interactions with snRNPsnRNP U1 and U1 and snRNPsnRNP U2.U2.

Regulators of alternative splicing:Regulators of alternative splicing:

Alternative splicingAlternative splicing

Walter Gilbert Walter Gilbert -- 19771977

40 40 -- 60% of pre60% of pre--mRNA transcripts mRNA transcripts undergoes alternative splicing undergoes alternative splicing

Allow for tissue specific expressionAllow for tissue specific expression

ImmunoglobinImmunoglobin genes, Drosophila sex genes, Drosophila sex determinationdetermination

TranTranssport of mRNA: Balbianport of mRNA: Balbianii ringsrings

mRNA export andmRNA export and itsits alternate fates in the cytoplasmalternate fates in the cytoplasm

What is RNA Interference What is RNA Interference ((RNAiRNAi))

““The Process by which The Process by which dsRNAdsRNA silences silences gene expression...gene expression...”” [[MittalMittal, 2004], 2004]

Generally: Post transcriptional Generally: Post transcriptional gene gene silencing silencing (PTGS)(PTGS)

Results in : Degradation or translation Results in : Degradation or translation inhibitioninhibition

: : Heterochromatin formationHeterochromatin formation

A field with many unknownsA field with many unknowns

Handy Handy RNAiRNAi TermsTerms

dsRNAdsRNA: double stranded RNA, longer : double stranded RNA, longer than 30 than 30 ntnt

miRNAmiRNA: : microRNAmicroRNA, 21, 21--25 nt. 25 nt. Encoded by Encoded by endogenousendogenous genes. genes.

Hairpin precursorsHairpin precursors

Recognize multiple targets.Recognize multiple targets.

siRNAsiRNA: short: short--interfering RNA, 21interfering RNA, 21--25 nt.25 nt.Mostly Mostly exogenousexogenous origin.origin.

dsRNAdsRNA precursorsprecursors

May be target specificMay be target specific

RNAiRNAi: Two Phase Process: Two Phase Process

InitiationInitiationGeneration of matGeneration of matuure re siRNAsiRNA or or miRNAmiRNA

ExecutionExecutionSilencing of target geneSilencing of target gene

Degradation or inhibition of translationDegradation or inhibition of translation

miRNAmiRNA BiogenesisBiogenesisTranscribed from endogenous gene as Transcribed from endogenous gene as pripri--miRNAmiRNA

Primary Primary miRNAmiRNA: long with multiple hairpins: long with multiple hairpins

Imperfect internal sequence Imperfect internal sequence complementaritycomplementarity

Cleaved by Cleaved by DroshaDrosha into preinto pre--miRNAmiRNA

Precursor Precursor miRNAmiRNA: ~70nt imperfect hairpins: ~70nt imperfect hairpins

Exported from nucleusExported from nucleus

Cleaved by Dicer into mature Cleaved by Dicer into mature miRNAmiRNA

2121--25nt25nt

Symmetric 2nt Symmetric 2nt 33’’ overhangsoverhangs, 5, 5’’ phosphate groupsphosphate groups

Novina and Sharp, 2004

siRNAsiRNA BiogenesisBiogenesisDicer cleaves long Dicer cleaves long dsRNAdsRNA into into siRNAsiRNA 2121--25nt25nt

dsRNAdsRNA from exogenous sourcesfrom exogenous sources

Symmetric 2nt Symmetric 2nt 33’’ overhangsoverhangs, 5, 5’’phosphate groupsphosphate groups

Evidence for amplification in Evidence for amplification in C. C. eleganselegansand plantsand plants

Allows persistence of Allows persistence of RNAiRNAi??

Novina and Sharp, 2004

Protein Protein machinerymachinery of of RNAiRNAiDroshaDrosha -- Processes Processes pripri--miRNAmiRNA into preinto pre--miRNAmiRNA

-- Leaves Leaves 33’’ overhangsoverhangs on preon pre--miRNAmiRNA

DicerDicer -- Cleaves Cleaves dsRNAdsRNA or preor pre--miRNAmiRNA

-- Leaves 3Leaves 3’’ overhangs and 5overhangs and 5’’ phosphatephosphategroups groups

RNA Dependent RNA Polymerase (RNA Dependent RNA Polymerase (RdRPRdRP) ) --siRNAsiRNA acts as primer for elongation on acts as primer for elongation on target mRNA target mRNA –– more more dsRNAdsRNA producedproduced

Argonaute Argonaute (Ago)(Ago) -- lies at the heart all RNA lies at the heart all RNA silencing silencing effectoreffector complexescomplexes

RNA Induced Silencing Complex (RISC) RNA Induced Silencing Complex (RISC) -- RNAiRNAieffectoreffector complexcomplex

RNA induced transcriptional silencing (RITS) RNA induced transcriptional silencing (RITS) -- RISCRISC--like complex responsible for heterochromatin formation in like complex responsible for heterochromatin formation in S.pombeS.pombe..

Proposed Biologic RolesProposed Biologic Roles

• Antiviral Defense• Suppress Transposon Activity

• Gene Regulation (Silencing) (e.g. MicroRNAs, Heterochromatin)

‘Immune System’ of the Genome

RNAiRNAi ApplicationsApplications

GENETIC TOOL

GENE THERAPY

Probing Gene Function

Combat Viral InfectionTreat Genetic Diseases

(New expression strategies)

Setting up an Setting up an RNAiRNAi experimentexperiment

design of RNA design of RNA oligosoligos

siRNAsiRNA synthesissynthesis

Introducing Introducing siRNAsiRNA into the cellsinto the cells

monitoring effectsmonitoring effects

Design of specific Design of specific siRNAsiRNA

Multiple Multiple siRNAsiRNA to single targetto single targetConsider: Length: usually 19Consider: Length: usually 19--23 23 ntnt (27(27--mers mers --Kim Kim et al. et al. Nature Biotechnology, 2005),Nature Biotechnology, 2005),

Content of G/C: (30Content of G/C: (30--52%), 52%), Positions of A/U: in the sense strand Positions of A/U: in the sense strand Position in target mRNA: at least 100 Position in target mRNA: at least 100

bases downstream AUG, bases downstream AUG, Avoid 5Avoid 5´́or 3or 3´́ UTRUTR ((BoeseBoese et al., et al.,

Methods in Methods in enzymologyenzymology, 2005), 2005)......Scrambled controlScrambled controlPositive controlPositive control (GAPDH)(GAPDH)

siRNAsiRNA synthesissynthesis

Chemical synthesis Chemical synthesis

In vitro transcription In vitro transcription

Digestion of long Digestion of long dsRNAdsRNA by an by an RNaseRNase III family enzyme III family enzyme (e.g. Dicer, (e.g. Dicer, RNaseRNase III) III)

Expression in cells from an Expression in cells from an siRNAsiRNA expression plasmid or expression plasmid or viral vectorviral vector

Expression in cells from a PCRExpression in cells from a PCR--derived derived siRNAsiRNAexpression cassetteexpression cassette

Introducing Introducing siRNAsiRNA into the cellsinto the cells

TransfectionTransfection

ElectroporationElectroporation

Concentration ranging from 5 to 50 Concentration ranging from 5 to 50 nMnM

Monitoring effectsMonitoring effects

qRealqReal--Time PCRTime PCR

Western blotWestern blot

ImmunofluorescenceImmunofluorescence

Monitoring of interferon response: 2'5'Monitoring of interferon response: 2'5'--oligoadenylate oligoadenylate synthetasesynthetase, , dsRNAdsRNA--dependent protein dependent protein kinasekinase, PKR , PKR

Thank you.Thank you.