metodologie integrate per la selezione genomica di … · • dicer is a member of the rnase iii...

CORSO GENHORT

Docente: Pasquale Termolino e-mail: termolin@unina.it Marzo 2014

METODOLOGIE INTEGRATE PER LA SELEZIONE GENOMICA DI PIANTE ORTIVE

•  Promoters active •  Gene hypermethylated

in coding region •  Purpose - Viral

immunity?

S. Grant (1999)

Transcriptional gene silencing (TGS) Posttranscriptional gene silencing (PTGS)

This has recently been termed “RNAi”

•  Promoters silenced •  Genes hypermethylated

in promoter region •  Purpose - Viral

immunity?

Posttranscriptional gene silencing

• Definition: the ability of exogenous double-stranded RNA (dsRNA) to suppress the expression of the gene which corresponds to the dsRNA sequence.

• 1990 Jorgensen :

• Introduction of transgenes homologous to endogenous genes often resulted in plants with both genes suppressed! •  Called Co-suppression •  Resulted in degradation of the endogenous and the transgene mRNA

Short history of post-transcriptional gene silencing

The discovery of RNA interference • An Unexpected Result…

•  petunias surprisingly developed areas of hypopigmentation when transduced with the gene encoding an enzyme required for pigment synthesis.

•  The phenomena was called Co-suppression

•  Similar effects seen in fungi. called “Quelling”

•  Later, in the C. elegans Camp...

• Antisense RNA injection method for gene inactivation

• Sense RNA injections gave same result!

• Remained a mystery... “The basis for the sense effect is under investigation ...” [Guo and Kemphues, 1995]

The discovery of RNA interference

Short history of post-transcriptional gene silencing -3

•  1998 Mello and Fire:

•  extension of above experiments, combination of

sense and antisense RNA (= dsRNA) was 10

times more effective than single strand RNA

• Some Sharp Reasoning •  Both sense and antisense RNAs sufficient for silencing •  Silencing can persist, even though RNA is easily degraded

•  Could dsRNA be mediating a new silencing mechanism?

The discovery of RNA interference

•  The RNAi revolution begins… •  Fire and colleagues found that introducing long double-stranded

RNA (dsRNA) into C. elegans led to the targeted degradation of homologous mRNA.

•  Coined the term “RNA interference” - RNAi

• RNAi in C. elegans • Silencing of a green fluorescent

protein (GFP) reporter in C. elegans occurs when animals feed on bacteria expressing GFP dsRNA (a) but not in animals that are defective for RNAi (b). •  Note that silencing occurs

throughout the body of the animal, with the exception of a few cells in the tail that express some residual GFP.

•  The lack of GFP-positive embryos in a (bracketed region) demonstrates the systemic spread and inheritance of silencing.

RNAi Timeline •  1990

•  co-suppression of purple color in plants. •  1995 Guo S, and Kemphues KJ.

•  First noticed that sense RNA was as effective as antisense RNA for suppressing gene expression in worm C. elegans

•  1998 Fire et al. •  First described RNAi phenomenon in C. elegans by injecting dsRNA into C. elegans which

led to an efficient sequence-specific silencing and coined the term "RNA Interference". •  2000 Zamone et al.

•  Reported processing of long dsRNA by Rnase III (Dicer) into shorter fragments of 21-23-nt intervals in Drosophila extracts

•  2001 Bernstein et al. •  Cloned Dicer, the RNase III enzyme that is evolutionarily conserved and contains helicase

and PAZ domains, as well as two dsRNA-binding domains. •  2002 Tuschl T and colleagues

•  First described RNAi in mammalian cells •  2003 Paddison et al. Sui et al. Paul et al.

•  Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. •  2003 Song et al.

•  First reported that siRNAs can be used therapeutically in whole animals •  2004 Kawasaki and Taira Morris et al.

•  First observed that siRNA silences gene at transcriptional level possibly through directing de novo DNA methylation.

RNAi Glossary •  Dicer –

•  Dicer is a member of the RNase III family of nucleases that specifically cleave double-stranded RNAs. Dicer processes long dsRNA into siRNA of 21-23 nt.

•  Micro-RNA – •  Micro-RNAs (miRNA) are single-stranded RNAs of 22-nt that are processed from ~70-nt hairpin RNA precursors by

Rnase III nuclease Dicer. Similar to siRNAs, miRNAs can silence gene activity via destruction of homologous mRNA in plants or blocking its translation in plants and animals.

•  Post-Transcriptional Gene Silencing – •  Post-transcriptional gene silencing (PTGS) is a sequence-specific RNA degradation system designed to act as an anti-

viral defense mechanism. A form of PTGS triggered by transgenic DNA, called co-suppression, was initially described in plants and a related phenomenon, termed quelling, was later observed in the filamentous fungus Neurospora crassa

•  Ribozyme – •  Ribozymes are RNA molecules that act as enzymes in the absence of proteins.

•  RNA Interference – •  RNA Interference (RNAi), a term coined by Fire et al in 1998, is a phenomenon that small double-stranded RNA

(referred as small interference RNA or siRNA) can induce efficient sequence-specific silence of gene expression. •  RNA-Directed DNA Methylation –

•  RNA-directed DNA methylation (RdDM) is an RNA directed silencing mechanism found in plants. Similar to RNA interference (RNAi), RdDM requires a double-strand RNA that is cut into short 21-26-nt fragments. DNA sequences homologous to these short RNAs are then methylated and silenced.

•  RNA-Induced Silencing Complex – •  RNA-induced silencing complex (RISC) is an siRNA-directed endonuclease, catalyzing cleavage of a single

phosphodiester bond on the RNA target. •  RNAi Trigger –

•  RNAi triggers are double-stranded RNAs containing 21-23 nt sense and antisens strands hybridized to have 2 nt overhangs at both 3' ends.

•  Small Interfering RNA – •  Small Interfering RNA (siRNA) is 21-23-nt double-strand RNA. It guides the cleavage and degradation of its cognate

RNA. •  Helicase –

•  Enzyme responsible for unwinding double stranded molecule

What is RNAi? •  RNA interference (RNAi) is an evolutionally highly conserved

process of post-transcriptional gene silencing (PTGS) by which double stranded RNA (dsRNA) causes sequence-specific degradation of mRNA sequences.

•  It was first discovered in 1998 by Andrew Fire and Craig Mello in the nematode worm Caenorhabditis elegans and later found in a wide variety of organisms, including mammals.

RNAi is a conserved mechanism •  RNAi is a universal, omnipresent conserved mechanism in

eukaryotic cells.

•  The cellular mechanism of RNAi predates evolutionary divergence of plants and worms.

•  key proteins involved in RNAi in disparate organisms are highly conserved.

RNAi movie -

Presenting the cast:

q RISC is a large (~500-kDa) RNA-multiprotein complex, which triggers mRNA degradation in response to siRNA

q  some components have been defined by genetics, but function is unknown, e.g.

q  – unwinding of double-stranded siRNA (Helicase !?)

q  – ribonuclease component cleaves mRNA (Nuclease !?)

q  – amplification of silencing signal (RNA-dependent RNA polymerase !?)

q  cleaved mRNA is degraded by cellular exonucleases

RISC

RNAi Amplification

q A small amount of dsRNA can silence a vast amount of target mRNA in C. elegans.

q Mechanistic explanations for this observations: q Each siRNA fragment can target the homologous

mRNA q Catalytic mechanism: each siRNA fragment can be

used several times. q RNA directed RNA synthesis

Ø dsRNA needs to be directed against an exon, not an intron in order to

be effective

Ø  homology of the dsRNA and the target gene/mRNA is required

Ø  targeted mRNA is lost (degraded) after RNAi

Ø  the effect is non-stoichiometric; small amounts of dsRNA can wipe out an excess of mRNA (pointing to an enzymatic mechanism)

Ø  ssRNA does not work as well as dsRNA

Some features of RNAi

Types of known RNAi inducing molecules

hpRNA (hairpin RNAs) miRNA (micro RNAs)

THE SILENCING MECHANISM • Two-step model to explain RNAi.

•  I. dsRNA is diced by an ATP-dependent ribonuclease (Dicer) into short interfering RNAs (siRNAs). •  duplexes of 21 23 nucleotides bearing

two-nucleotide 3' overhanging ends.

•  II. siRNAs are transferred to a second enzyme complex, designated RISC for RNAi-induced silencing complex. The siRNA guides RISC to the target mRNA, leading to its destruction. •  the antisense strand of the siRNA is

perfectly complementary

•  The classical RNA interference (RNAi) pathway in Drosophila •  Long double-stranded RNAs

(dsRNAs) are processed by the R2D2/Dicer heterodimer into small interfering RNAs (siRNAs).

•  The duplexed siRNA is unwound in an ATP-dependent manner*. •  *starting at the 5' terminus that has

the lowest relative free energy of base pairing.

•  This strand of the siRNA, the guide strand, is also preferentially taken up by the RNA-induced silencing complex (RISC).

•  The single-stranded siRNA guides the endonuclease activity of the activated RISC ("holoRISC") to the homologous site on the mRNA, cleaving the mRNA.

RNAi Amplification

•  A small amount of dsRNA can silence a vast amount of target mRNA in C. elegans.

•  Mechanistic explanations for this observations: •  Each siRNA fragment can target the homologous mRNA •  Catalytic mechanism: each siRNA fragment can be used several times. •  RNA directed RNA synthesis

• RNA Dependent RNA Polymerase (RdRP) • RdRP activity found in plants and C. elegans • Required for RNAi?

•  Not found in mammals or drosophila •  RdRP deficient plants and worms... Results not decisive

• Proposed mechanism: Random degenerative PCR [Lipardi et al., 2001] •  siRNA acts as primer for elongation on target mRNA

Immunity via RNAi •  RNAi is used as a form of primitive immunity to protect the genome

from invasion by exogenous nucleic acids introduced by mobile genetic elements, such as viruses and transposons.

microRNA

q miRNAs are small endogenous RNA molecules (~22nt) found in plant and animals

q Regulate gene expression through targeted mRNA cleavage or translational repression

q Play critical roles in a variety of cellular processes such as development, cell proliferation, apoptosis, and stress response, etc

microRNA

q Transcribed from endogenous gene as pri-miRNA q Primary miRNA: long with multiple hairpins q Imperfect internal sequence complementarity

q It is processed into 70-nt hairpins by the RNase III family member Drosha to become the pre-miRNA. q Note: How does it identify pri-miRNA?

q Hairpin terminal loop size q Stem structure q Hairpin flanking sequences

q exported to the cytoplasm by Exportin 5. q cleaved by the R2D2/Dicer heterodimer into the

mature miRNA. q It is cleaved by the R2D2/Dicer heterodimer into

the mature miRNA. q Symmetric 2nt 3’ overhangs, 5’ phosphate

groups

The miRNA pathway

The plot thickens… The Discovery of Endogenous Effectors for RNAi

•  Discovery of the first naturally occurring small RNA specie , lin-4 •  Non-coding, 22nt RNA

•  Identified in screen for defects in timing of larval development •  lin-4 partially complementary to conserved sites in lin-14 3’UTR

[Lee et al., 1993] •  lin-4 binds these sites •  lin-4 negatively regulates lin-14 translation

•  The naturally occurring small RNA designated microRNAs (miRNAs) (only later)

•  No other miRNAs found for 7 years! •  Second miRNA – let-7 [Reinhart et al., 2000]

•  Non coding, 21nt RNA •  Regulates lin-14 in same way as lin-4

•  Note: Homologues of lin-4 escaped bioinformatics Let-7 Homologs were easily detected [Pasquinelli et al., 2000] •  Drosophila, sea urchins, mice, humans...

Endogenous RNAi: miRNA in the Genome • Characteristic Properties

•  Highly conserved, particularly 5’ end •  All from hairpin precursors

• Genome Wide miRNA Identification •  Most has been done experimentally (Cloning and sequencing)

•  Over 100 novel miRNAs identified from C. elegans, Drosophila, and mammals

•  Expected to represent ~1% of predicted genes [Lim et al., 2003] •  Same as other gene families with regulatory roles •  200-255 miRNAs in humans •  >175 have now been experimentally confirmed [Griffiths-Jones, 2004]

•  Functional Characterization •  Lewis et al., (2003) estimate average of five mRNA targets per

miRNA •  Many targets are transcription factors - miRNAs regulate the

regulators •  Suggests major role in highly regulated processes •  Thousands of proteins may be regulated by miRNA

miRNA vs. siRNA •  miRNA: microRNA.

•  Encoded by endogenous genes. •  Hairpin precursors - pre-miRNAs

•  The pre-miRNAs are hairpins with imperfect complementarity in their stems and frequent bulges, mismatches and G:U wobble base pairings.

•  Recognize multiple targets. •  siRNA: short-interfering RNA.

•  Mostly exogenous origin. •  dsRNA precursors •  May be target specific

•  Discovered in different ways •  Similar biogenesis •  Share common pathway components and outcomes •  Understanding of miRNA comes from research on siRNA and vice

versa •  Maybe current understanding does not allow us to distinguish them

MULTIPLE MECHANISMS OF SMALL-RNA-MEDIATED GENE SILENCING

• The endogenous RNAi pathway contributes significantly to regulating cellular gene expression. •  Silencing of endogenous genes regulates basic biological

processes, including the transition from one developmental stage to the next. •  the archetype miRNAs, let-7 and lin-4, regulate C. elegans larval

development •  miRNAs are expressed in a specific spatial and temporal pattern

during development in D. melanogaster or differentiation of mouse embryonic stem cells

• The function of most miRNAs remains unknown…

• miRNA Biogenesis •  Transcribed from endogenous gene as pri-miRNA

•  Primary miRNA: long with multiple hairpins •  Imperfect internal sequence complementarity

•  It is processed into 70-nt hairpins by the RNase III family member Drosha to become the pre-miRNA. •  Note: How does it identify pri-miRNA?

•  Hairpin terminal loop size •  Stem structure •  Hairpin flanking sequences

•  The pre-miRNA is exported to the cytoplasm by Exportin 5. •  It is cleaved by the R2D2/Dicer heterodimer into the mature miRNA.

•  Symmetric 2nt 3’ overhangs, 5’ phosphate groups

• The miRNA pathway •  pri-miRNA •  processed by Drosha to become

the pre-miRNA. •  exported to the cytoplasm by

Exportin 5. •  cleaved by the R2D2/Dicer

heterodimer into the mature miRNA. •  The miRNA is loaded into RISC and

guides it to sites on the mRNA that have only partial sequence complementarity to the miRNA, leading to repression of translation.

•  Intermediate Summary: • miRNA

vs. siRNA • mRNA cleavage

vs. Translational Repression Initiation

Execution

An additional mechanism: Heterochromatin formation “long non coding”

•  The repeat-associated siRNA (rasiRNA) pathway •  Transcription from opposing

promoters found in repetitive DNA elements, such as centromeric repeats and satellite DNA, leads to the formation of long dsRNAs.

•  These long dsRNAs are cleaved by Dicer, presumably the R2D2/Dicer heterodimer, into siRNAs.

•  These are unwound and taken up by the RNA-induced transcriptional silencing complex (RITS)

•  RITS directs the establishment of silenced chromatin over the region of DNA homologous to the siRNAs.

•  This silenced chromatin is characterized by sequence-specific DNA methylation and histone methylation and by recruiting heterochromatin-associated proteins.

•  A model for the mechanism of RNAi •  Silencing triggers in the

form of double-stranded RNA may be presented in the cell as synthetic RNAs, replicating viruses or may be transcribed from nuclear genes.

•  These are recognized and processed into small interfering RNAs by Dicer.

•  The duplex siRNAs are passed to RISC (RNA-induced silencing complex)

•  The complex becomes activated by unwinding of the duplex.

•  Activated RISC complexes can regulate gene expression at many levels: •  promoting RNA

degradation •  translational inhibition •  chromatin remodelling

•  Amplification of the silencing signal in plants may be accomplished by siRNAs priming RNA-directed RNA polymerase (RdRP)-dependent synthesis of new dsRNA.

RNAi applications • Genome-wide RNAi screening

•  Done in C. elegans •  19 757 protein coding genes (predicted) •  16 757 inactivated using RNAi

•  New standard for systematic genome wide functional studies

• RNAi mutants for genomic selection • Advanced plant breeding • RNAi as a solution for mammalian genetics • Potential therapeutic use

New Frontiers for RNA… • Small RNAs likely to have bigger impact on gene and protein regulation

• New classes of small RNAs: •  Tiny non-coding RNA [Ambros et al., 2003]

•  tncRNA – 20-22nt •  Discovered in C. elegans •  Not likely generated from hairpin loops •  Not conserved among species •  Many complementary to mRNAs •  Function unknown

• RNA as a Molecular Switch: Small Modulatory RNA – smRNA [Kuwabara et al., 2004] •  Discovered in mice •  Conserved in vertebrates •  Interacts with regulatory protein •  Turns transcriptional repressor into activator

Just Scratching the Surface… • New roles for RNA added to our current paradigm for

gene and protein regulation. • A new buzz word?

Just Scratching the Surface… • New roles for RNA added to our current paradigm for

gene and protein regulation.

• A new buzz word?

“Regulomics”

miRNA vs. siRNA •  miRNA: microRNA.

•  Encoded by endogenous genes. •  Hairpin precursors - pre-miRNAs

•  The pre-miRNAs are hairpins with imperfect complementarity in their stems and frequent bulges, mismatches and G:U wobble base pairings.

•  Recognize multiple targets.

•  siRNA: short-interfering RNA. •  Mostly exogenous origin. •  dsRNA precursors •  May be target specific

•  Discovered in different ways •  Similar biogenesis •  Share common pathway components and outcomes •  Understanding of miRNA comes from research on siRNA and vice

versa •  Maybe current understanding does not allow us to distinguish them

Overview of small RNA molecules

Plants Animals # of miRNA genes: 100-200 100-500 Location in genome: intergenic regions Intergenic regions, introns Clusters of miRNAs: Uncommon Common MiRNA biosynthesis: Dicer-like Drosha, Dicer Mechanism of repression mRNA-cleavage Translational repression Location of miRNA target in a gene: Predominantly Predominantly the 3′-UTR

the open-reading frame # of miRNA binding sites in a target gene: Generally one Generally multiple Functions of known target genes: Regulatory genes Regulatory genes—crucial

crucial for development, for development, structural enzymes proteins, enzymes

Summary of differences between plant and animal miRNA systems

There are three problems q How to find microRNA genes?

q Given a microRNA gene, how to find its targets?

q Target-driven approach: q Xie et al. (2005) analyzed conserved motifs that are

overrepresented in 3’ UTRs of genes q Found out they are complementing the seed

sequences of known microRNAs. q They predicted 120 new miRNA candidates in

human.

Finding new miRNA: problems

How to use RNAi in plants

Gene

Insertion in a specific expression vector

A.tumefaciens

Plant cell

Examples of RNAi plants

PDS transgenes

Partial transformation

Complete transformation

Silencing was LETHAL

Wild type

Wild type

Mutants

Mutants

Arabidopsis SPINDLEY homologue