METODOLOGIE INTEGRATE PER LA SELEZIONE GENOMICA DI PIANTE ORTIVE

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1 CORSO GENHORT METODOLOGIE INTEGRATE PER LA SELEZIONE GENOMICA DI PIANTE ORTIVE Marzo 2014 Docente: Pasquale Termolino

2 Posttranscriptional gene silencing Transcriptional gene silencing (TGS) Promoters silenced Genes hypermethylated in promoter region Purpose - Viral immunity? Posttranscriptional gene silencing (PTGS) Promoters active Gene hypermethylated in coding region Purpose - Viral immunity? This has recently been termed RNAi S. Grant (1999)

3 Short history of post-transcriptional gene silencing Definition: the ability of exogenous double-stranded RNA (dsrna) to suppress the expression of the gene which corresponds to the dsrna sequence 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

4 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

5 The discovery of RNA interference 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]

6 Short history of post-transcriptional gene silencing Mello and Fire: extension of above experiments, combination of sense and antisense RNA (= dsrna) was 10 times more effective than single strand RNA

7 The discovery of RNA interference 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 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

8 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.

9 RNAi Timeline 1990 co-suppression of purple color in plants 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" Zamone et al. Reported processing of long dsrna by Rnase III (Dicer) into shorter fragments of 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 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 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.

10 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 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 antiviral 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 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 nt sense and antisens strands hybridized to have 2 nt overhangs at both 3' ends. Small Interfering RNA Small Interfering RNA (sirna) is nt double-strand RNA. It guides the cleavage and degradation of its cognate RNA. Helicase Enzyme responsible for unwinding double stranded molecule

11 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.

12 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.

13 RNAi movie -

14 Presenting the cast:

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18 RISC 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

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21 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

22 Some features of RNAi 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

23 Types of known RNAi inducing molecules hprna (hairpin RNAs) mirna (micro RNAs)

24 THE SILENCING MECHANISM Two-step model to explain RNAi. I. dsrna is diced by an ATPdependent ribonuclease (Dicer) into short interfering RNAs (sirnas). duplexes of 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

25 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.

26 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

27 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

28 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.

29 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

30 microrna

31 The mirna pathway 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

32 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...

33 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 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

34 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

35 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

36 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

37 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.

38 Intermediate Summary: mirna vs. sirna mrna cleavage vs. Translational Repression Initiation Execution

39 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.

40 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 (RNAinduced 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 RNAdirected RNA polymerase (RdRP)-dependent synthesis of new dsrna.

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42 RNAi applications Genome-wide RNAi screening Done in C. elegans protein coding genes (predicted) 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

43 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

44 Just Scratching the Surface New roles for RNA added to our current paradigm for gene and protein regulation. A new buzz word?

45 Just Scratching the Surface New roles for RNA added to our current paradigm for gene and protein regulation. A new buzz word? Regulomics

46 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

47 Overview of small RNA molecules

48 Summary of differences between plant and animal mirna systems Plants Animals # of mirna genes: 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

49 Finding new mirna: problems 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.

50 How to use RNAi in plants Gene Insertion in a specific expression vector Plant cell A.tumefaciens

51 Examples of RNAi plants

52 PDS transgenes Partial transformation Silencing was LETHAL Complete transformation

53 Arabidopsis SPINDLEY homologue Mutants Wild type Wild type Mutants