Transposable Elements (TEs)

Transcription

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2 Transposable Elements (TEs) Box 1. Differences between transcriptional gene silencing and post-transcriptional gene silencing Transcriptional gene silencing (TGS) Silencing is caused by a blockage of transcription. TGS occurs in the nucleus. Usually promoters of silenced genes are methylated. TGS is inherited stably after meiosis. TGS is not involved in systemic silencing. ost-transcriptional gene silencing (TGS) Silencing is caused by a post-transcriptional RNA degradation. TGS is conducted in the cytosol. Methylation of the protein-coding region is often associated with TGS. TGS is reset every generation. One feature of TGS is systemic silencing, although the exact nature of the signal has not been established.

3 DNA Methylation

4 DNA methylation addition of a methyl group (CH 3 ) to cytosine In mammals methylation mainly occurs on the cytosine in a CpG context, In plants the cytosine can be methylated in the CpG, CpNpG, and CpNpN context, where N represents any nucleotide but guanine. In animals: ~2-7% of Cs are methylated In plants: ~25% of Cs are methylated DNA methylation absent in C. elegans, S. cervisiae and S. pombe This situation has possibilities for controlling development, because it clearly produces stable, yet reversible, changes in the genome. In addition, methylated DNA is found most often associated with inactive DNA regions (centromeres). Mutant Arabidopsis (reduced ability of methylation) - Meristem identity - Changes in organ number - Female sterility

5 DNA methylation Chemical structure of cytosine and 5-methylcytosine. Methyltransferases add a methyl group onto the 5 carbon of cytosine to generate 5- methylcytosine. Hydrolytic deamination converts cytosine to uracil and 5-methylcytosine to thymine (5- methyluracil). Treatment of single stranded DNA with sodium bisulfite leads to sulfonation of cytosine to form cytosine sulfonate, which is then deaminated to uracil sulfonate and desulfonated by alkali treatment to uracil. The methyl group protects 5- methylcytosine from sulfonation.

6 INTRODUCTION

7 Treatment of DNA with bisulphite converts cytosine residues to uracil, but leaves 5-methylcytosine residues unaffected. Thus, bisulphite treatment introduces specific changes in the DNA sequence that depend on the methylation status of individual cytosine residues, yielding single- nucleotide resolution information about the methylation status of a segment of DNA. Various analyses can be performed on the altered sequence to retrieve this information. The objective of this analysis is therefore reduced to differentiating between single nucleotide polymorphisms (cytosines and thymidine) resulting from bisulphite conversion

8 Cytosine methylation and inheritance of methylation patterns. Methylation at the 5 position of cytosine can occur at CpG and CpA/TpG residues in plants and is often correlated with the activity of the gene. The pattern of methylation of a gene is a combination of the action of maintenance methylases and de novo methylase activity on one hand, and the methylation and replication activities on the other.

9 In plants The principal Arabidopsis DNA methyltransferase enzymes, which transfer and covalently attach methyl groups onto DNA, are DRM2, MET1, and CMT3. There are currently two classes of DNA methyltransferases: 1. de-novo class, or enzymes that create new methylation marks on the DNA: DRM2 2. maintenance class that recognizes the methylation marks on the parental strand of DNA and transfers new methylation to the daughters strands after DNA replication: DRB2, MET1 and CMT3.

10 cytosine 5-methyltransferases in Arabidopis: minimum 10 genes in 4 families MET (metyltransferase) family MET1 (ddm2, met1), MET2a, MET2b (MET3) maintainance methylation of symmetric CG asociated with replication involvement of nucleosom remodeling complex CMT (chromomethyltransferase) family CMT3,(CMT1), CMT2 unique for plants maintainance methylation of CNG and certain asymmetric sequences DRM (domain rearranged methyltransferase) family DRM2 (DRM1) de novo methylation of all sequences (CG, CNG, asymmetric) maintainance methylation of asymetric and CNG regulation of gene expression, silencing Targed sequences determined by presence of homologous sirna At5g25480: related to mammalian Dnmt2

11 Determination of location of de novo DNA methylation Currently, it is not clear how the cell determines the locations of de-novo DNA methylation, but evidence suggests that for many, though not all locations, RNAdirected DNA methylation is involved. This sort of mechanism is thought to be important in cellular defense against RNA viruses and/or transposons both of which often form a double-stranded RNA that can be mutagenic to the host genome. By methylating their genomic locations, they are shut off and are no longer active in the cell, protecting the genome from their mutagenic effect.

12 DNA methylation RNA directs DNA methylation. A) Transcription of a locus gives rise to ol IVa-dependent aberrant RNA, which is shuttled to the nucleolus and is B) converted into double-stranded RNA by the RNA-dependent RNA polymerase RDR2. DICER-LIKE 3 (DCL3) processes the dsrna into 24 nt small interfering RNAs (sirnas), which are loaded into AGO4 complexes that also contain NRD1b. C) The sirna complex guides the de novo DNA methyltransferase DRM2. The chromatin remodeller DRD1 is also required. (Red lollipops = 5-methylcytosine, short squiggly lines = sirnas).

13 Methylation mutants Strong phenotype connected with developmental alterations observed only in triple mutant in all three genes. It may be due to the partial redundancy. Silencing of gene expression is not neccessarily connected with DNA methylation (remember also species without methylation)

14 Role of DNA methylation heritable modulation of transcription (predominantly methylation of promoters) defence against invasive DNA (transposons) regulation of gene expression parental imprinting modulation of chromatin structure timing of DNA replication regulation of homologous recombination tool of evolution polyploid plant genomes (large changes in methylation) new genes (mutagenesis)

15 RNA Silencing In an attempt to alter flower colors in petunias, researchers introduced additional copies of a gene encoding chalcone synthase, a key enzyme for flower pigmentation into petunia plants of normally pink or violet flower color. The overexpressed gene was expected to result in darker flowers, but instead produced less pigmented, fully or partially white flowers, indicating that the activity of chalcone synthase had been substantially decreased (1990)

16 Transformation 35S chalcone synthase etunia has own chalcone synthase Further investigation of the phenomenon in plants indicated that the downregulation was due to posttranscriptional inhibition of gene expression via an increased rate of mrna degradation (1990) mrna chalcone synthase from transgene

17 Finding gene silencing mechanism David Baulcombe (1989)

18 Host Silencing of Virus Symptoms Mild symptoms Severe symptoms

19 Host Silencing of BMV Symptoms Mild symptoms Severe symptoms Inoculated leaf

20 ost Transcriptional Gene Silencing (TGS) Vpg AAAAAAA Dcer Viral or Cellular RdRp Dicer Dicer Dicer BMV AT OH OH OH OH OH target recognition BMV AAAAAAA Native sirna duplex AT 21-23nt sirna/protein complex (sirn) sirna unwinding and RISC assembly (RNA-induced Silencing Complex)

21 Symptom recovery (Radhamani et al., 1998)

22 Cellular and Viral RdR Vpg Vpg Cellular and Viral RdR AAAAAA A Dicer Dicer Dicer Dicer Dicer Dicer TEV 1/HC- ro Dicer Dicer AT OH OH OH OH OH Native sirna duplex (russ et al. 1998)

23 ost Transcriptional Gene Silencing (TGS) Vpg AAAAAAA Dicer Viral or Cellular RdRp Dicer Dicer Dicer dsrna (Chachol syn.) AT OH OH OH OH OH target recognition AAAAAAA Chachol syn. Native sirna duplex AT 21-23nt sirna/protein complex (sirn) sirna unwinding and RISC assembly (RNA-induced Silencing Complex)

24 ost-transcriptional gene silencing (TGS) Anti-viral defense system rocess results in down-regulation of a gene at the RNA level (i.e., after transcription) dsrna initiates a sequence-specific RNA degradation pathway RNaseIII-like enzyme cleaves dsrna into nucleotide pieces (silencing signal, micro RNA (mirna)) Short RNA provide specificity for degradation TGS degrades target RNA - viral and transgene

25 In lants, fungi, Drosophila, & C. elegans, a RNAdependent RNA polymerase (RDR) is involved in initiation or amplification of silencing. CB and AB block access for RDR. AB missing. D. Baulcombe 1998 Nature 431:356

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27 mirna(micro RNA)

28 Gene regulated by mirna

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30 MicroRNAs (mirnas) are posttranscriptional regulators that bind to complementary sequences in the three prime untranslated regions (3' UTRs) of target messenger RNA transcripts (mrnas), usually resulting in gene silencing. mirnas are short ribonucleic acid (RNA) molecules, on average only 22 nucleotides long. The human genome may encode over 1000 mirnas, which may target about 60% of mammalian genes and are abundant in many human cell types. Each mirna may repress hundreds of mrnas. MiRNAs are well conserved in eukaryotic organisms and are thought to be a vital and evolutionarily ancient component of genetic regulation

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32 mirna genes are usually transcribed by RNA polymerase II (ol II).The polymerase often binds to a promoter found near the DNA sequence encoding what will become the hairpin loop of the pre-mirna. The resulting transcript is capped with a specially-modified nucleotide at the 5 end, polyadenylated with multiple adenosines (a poly(a) tail), [31][35] and spliced. The product, called a primary mirna (pri-mirna), may be hundreds or thousands of nucleotides in length and contain one or more mirna stem loops. When a stem loop precursor is found in the 3 UTR, a transcript may serve as a primirna and a mrna. RNA polymerase III (ol III) transcribes some mirnas, especially those with upstream Alu sequences, transfer RNAs (trnas), and mammalian wide interspersed repeat (MWIR) promoter units.

33 Function of mirna: gene regulation A mirna is complementary to a part of one or more messenger RNAs (mrnas). Animal mirnas are usually complementary to a site in the 3' UTR whereas plant mirnas are usually complementary to coding regions of mrnas In animals, micrornas more often only partially base pair and inhibit protein translation of the target mrna Animal micrornas target in particular developmental genes

34 short hairpin RNA

35 Dicer Dicer AAAAAAA AAAAAAA MiR171 sirna H H H O O O O O O HH H H O O O O H H Native mirna duplex 21-23nt sirna/mir NA/protein complex H O H O SCL family or target RNA (transcriptional activator, promoter binding protein etc on) H O H O H O mirna unwinding and RISC assembly (RNA-induced Silencing Complex) AAAAAAA AAAAAAA Flower induction Enlarge leave Shorten internodes environmental changing?? romoter inducer

36 Level of mirna 172 depending on the photoperiod mirna 172 Up-regulate flower inducing gene

37 mirna 172 Down-regulate flower inducing gene

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41 Expression of SMV HC-RO in Transgenic Soybean Alters Leaf Morphology, SMV Symptom Severity and Seed roduction

42 G5 HC-ro transgenic soybean; ΔC T value; Actin ΔC T - HC-ro ΔC T

43 GUS transgenic soybean

44 henotypes of HC-ro Transgenic Soybean (Flower) HC-ro high expressed line GUS

45 Seed production was inversely proportional to HC-ro expression level GUS transgenic plant HC-ro transgenic plant 14 hr day light; μem -

46 Seed pods production in transgenic soybeans grown in 20 cm diameter pots Lines GUS a LG5-1 LG5-2 LG5-3 Average of Seed ods 39 a b 27 b 29 b 27 b a Ten plants were analyzed per line b LSD (α = 0.05) = 9.17

47 Seeds/plant 10 plants from LG5-1 and 10 plants form GUS transgenic lines grown in the 30 cm diameter pots (one plant per pot) Log(-ΔCt) GUS transgenic soybeans

48 Seeds/plant 10 plants of each transgenic line from LG5-1, LG5-2, LG5-3, and GUS in 20-cm diameter pots ( two plants per pot) Log(-ΔCt)

49 mir171: SCL6 family Control wide range of developmental processing; root and hormone signaling Radial patterning in root Signaling by the phytochormone gibberellin Light signaling