Agricultural Biotechnology RNA interference (RNAi) technology: Part I

Agricultural Biotechnology RNA interference (RNAi) technology: Part I
RNAi – Part I- Appl II. MSA-Shtayeh (Prof.) 4/28/2017

RNA interference (RNAi) technology
INTRODUCTION What is RNAi? RNA silencing and plant virus defense Epigenetics Discovery of RNAi Genes regulation at either the transcriptional or post-transcriptional levels Roles of gene silencing What is PTGS? Examples of RNAi (PTGS) Some General Features of PTGS What triggers (induces) the PTGS response when a virus infects a plant? PTGS pathway phases The mobile silencing signal Initiation of silencing spread Types of silencing movement in plants Short range silencing spread: Movement way of the silencing signal Plant genes involved in short range silencing Other genes A speculative model for short-range spreading of RNA silencing 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

INTRODUCTION - What is RNAi?
Interruption or suppression of the expression of a gene at transcriptional or translational levels is called gene silencing. This type of RNA interference (RNAi) is a process of dsRNA-mediated gene silencing in which only the mRNA cognate (related) to dsRNA is specifically degraded. RNAi mediated gene silencing is referred to as cosuppression or posttranscriptional gene silencing in plants; Quelling in fungi and gene silencing in animals. 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

RNAi -Appl II. MSA-Shtayeh (Prof.)
The term “RNA interference (RNAi)” was initially coined by Fire et al. (1998). RNA silencing is a novel gene regulatory mechanism that limits the transcript level by either suppressing transcription (TGS) or by activating a sequence-specific RNA degradation process [PTGS/RNA interference (RNAi)] (Agrawal et al., 2003). 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

RNA silencing and plant virus defense
Gene silencing is a general term describing epigenetic processes of gene regulation. The term gene silencing is generally used to describe the "switching off" of a gene by a mechanism other than genetic modification. That is, a gene which would be expressed (turned on) under normal circumstances is switched off by machinery in the cell. 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

Epigenetics Is the study of inherited changes in phenotype (appearance) or gene expression caused by mechanisms other than changes in the underlying DNA sequence, hence the name epi- (Greek: επί- over, above) -genetics. These changes may remain through cell divisions for the remainder of the cell's life and may also last for multiple generations. However, there is no change in the underlying DNA sequence of the organism; instead, non-genetic factors cause the organism's genes to behave (or "express themselves") differently. 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

Discovery of RNAi 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

Gene silencing Discovery OF RNAi/cont. Recovery in tobacco plants infected with tobacco ringspot virus. Note the gradual decline in the development of ringspot symptoms on the upper leaves until finally the top leaves appear perfectly normal. We now know that the virus causing the initial symptoms had activated viral RNA silencing that inhibited spread of the infection into the upper leaves, and caused them to be specifically immune to tobacco ringspot virus secondary infection. Figure. Turkish tobacco plant 23 days after inoculation with ringspot virus. RNAi -Appl II. MSA-Shtayeh (Prof.)

Discovery OF RNAi/cont.
During the 1990s, a number of gene silencing phenomena that occur at the post-transcriptional level were discovered in plants, fungi, animals and ciliates. 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

Discovery of PTGS (RNAi) cont.
First discovered in plants (Jorgensen, 1990) Introduction of a transgene homologous to an endogenous gene resulted in both genes being suppressed! Also called Co-suppression involved enhanced degradation of the endogenous and transgene mRNAs 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

Discovery of PTGS (RNAi) cont.
The silencing effect was first observed in plants in 1990, when the Jorgensen laboratory introduced exogenous transgenes into petunias in an attempt to up-regulate the activity of a gene for chalcone synthase, an enzyme involved in the production of specific pigments. Unexpectedly, flower pigmentation did not deepen, but rather showed variegation with complete loss of color in some cases. This indicated that not only were the introduced trangenes themselves inactive, but that the added DNA sequences also affected expression of the endogenous loci. This phenomenon was referred to as “cosuppression”. 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

Discovery of RNAi (cont.)
Jorgensen work involved attempts to manipulate pigment synthesis genes in petunia Genes were enzymes of the flavonoid/ anthocyanin pathway: CHS: chalcone synthase DFR: dihydroflavonol reductase When these genes were introduced into petunia using a strong viral promoter, mRNA levels dropped and so did pigment levels in many transgenics. 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

Discovery of RNAi (cont.) Effects of expression of CHS sense and antisense* RNA on flower pigmentation in Petunia van der Krol et al., Plant Cell 2:291 (1990); Plant Mol Biol 14:457 (1990). *Antisense RNA is a single-stranded RNA that is complementary to a messenger RNA (mRNA) strand transcribed within a cell. Antisense RNA may be introduced into a cell to inhibit translation of a complementary mRNA by base pairing to it and physically obstructing the translation machinery. RNAi -Appl II. MSA-Shtayeh (Prof.)

Discovery of RNAi (cont.) Observation: Simultaneous silencing of transgene and endogenous gene Name of phenomenon: Co-Suppression A similar phenomenon in the fungus Neurospora crassa was named quelling . Definitions: negative-sense RNA viral RNA with a base sequence complementary to that of mRNA; during replication it serves as a template for the transcription of viral complementary RNA. positive-sense RNA viral RNA with the same base sequence as mRNA; during replication it functions as mRNA, serving as a template for protein synthesis. Transgene: an additional gene introduced (in contrast to endogenous gene) 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

Discovery of RNAi (cont.) Co-Suppression a process that results in sequence-specific degradation of RNA gene corresponding to RNA is down-regulated no effect on transcription rate, high RNA turnover 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

The discovery of RNA interference / cont.
Fire et al. (1998) identified a related mechanism, RNA interference (RNAi) in animals (Caenorhabtitis elegans ). Antisense technology has been used for > 20 years, based on introducing an antisense gene (or antisense RNA) into cells to try to block translation of the sense mRNA. The “antisense effect” was probably due to RNAi rather than inhibiting translation. 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

The discovery of RNA interference / cont
Andrew Fire and Craig Mello published their break-through study on the mechanism of RNA interference in Nature in 1998. It was earlier known that antisense RNA, but also sense RNA could silence genes, but the results were inconsistent and the effects usually modest. 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

However, due to the fact that both sense and antisense RNA could cause silencing, Mello argued that the mechanism could not just be a pairing of antisense RNA to mRNA, and he coined the term RNA interference for the unknown mechanism. 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

Discovery of RNAi (cont.)
In their Nature paper, Fire and Mello tested the phenotypic effect of RNA injected into the worm C. elegans. They established that annealed sense/antisense RNA, but neither antisense nor sense RNA alone, caused the predicted phenotype (Fig.).

The discovery of RNA interference / cont Furthermore, only injection of double-stranded RNA (dsRNA) led to an efficient loss of the target mRNA (Fig. 2). 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

Double-stranded RNA (dsRNA) induced interference of the Mex-3 mRNA in the nematode Caenorhabtitis elegans. the mex-3 (mRNA) was injected into C. elegans ovaries, and then mex-3 mRNA was detected in embryos by in situ hybridization with a mex-3 probe. (a) control embryo (b) control embryo hyb. with mex-3 probe (c) Antisense RNA or (d) dsRNA Figure from Molec. Biology by Weaver. Conclusion: dsRNA reduced mex-3 mRNA better than antisense mRNA. Also, the suppression signal moves from cell to cell. RNAi -Appl II. MSA-Shtayeh (Prof.)

Fire and Mello could present a series of straightforward conclusions in their study. The main results can be summed up as follows: 1. silencing was triggered efficiently by injected dsRNA, but weakly or not at all by sense or antisense single stranded RNAs. 2. silencing was specific for an mRNA homologous to the dsRNA; other mRNAs were unaffected. 3.the dsRNA had to correspond to the mature mRNA sequence; neither intron nor promoter sequences triggered a response. This indicated a post-transcriptional, presumably cytoplasmic mechanism. 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

4. Fourth, the targeted mRNA disappeared suggesting that it was degraded. 5. only a few dsRNA molecules per cell were sufficient to accomplish full silencing. This indicated that the dsRNA was amplified and/or acted catalytically rather than stoichiometrically. 6. the dsRNA effect could spread between tissues and even to the progeny, suggesting a transmission of the effect between cells. 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

Furthermore, Fire and Mello made the remark that RNAi could provide an explanation for a phenomenon studied in plants for several years: posttranscriptional gene silencing (PTGS). Finally, they ended their paper by speculating about the possibility that “dsRNA could be used by the organism for physiological gene silencing”. 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

Within a year, the presence of RNAi had been documented in many other organisms, including fruit flies, trypanosomes, plants, planaria, hydra and zebrafish. In initial experiments with mammalian cultured cells, it was not possible to elicit a potent and specific RNAi response because of a predominant nonspecific physiological reaction of these cells to long dsRNA. 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

However, when the cells were exposed to a short, 21 nucleotide long, dsRNA, an efficient targeted silencing was also obtained in these cells. Thus, the generality of the RNAi phenomenon among eukaryotes was proven very rapidly; a remarkable exception is the budding yeast, Saccharomyces cerevisiae. 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

Discovery of RNAi (cont.) Since its discovery, the molecular machinery involved in RNAi has been further elucidated through a large number of studies. Has been detected in fission yeast, S. pombe 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

Discovery of RNAi (cont.) Genes are regulated at either the transcriptional or post-transcriptional levels. Transcriptional gene silencing is the result of histone modifications, creating an environment of heterochromatin around a gene that makes it inaccessible to transcriptional machinery (RNA polymerase, transcription factors, etc.). 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

Post-transcriptional gene silencing is the result of mRNA of a particular gene being destroyed or blocked. The destruction of the mRNA prevents translation to form an active gene product (in most cases, a protein). A common mechanism of post-transcriptional gene silencing is RNAi. 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

Discovery of RNAi (cont.) Transcriptional gene silencing (TGS) BL BR Promoter Coding Region pA Post-transcriptional gene silencing (PTGS) BL BR Promoter Coding Region pA M M M M M Promoter methylation mRNA degradation + X BL BR M M M M M M Block of primary transcription Methylation of the transcribed region RNAi -Appl II. MSA-Shtayeh (Prof.)

Discovery of RNAi (cont.) transcriptional gene silencing (TGS) inactivation of (trans)gene-specific nuclear RNA synthesis induced by a DNA-mediated or RNA-mediated DNA methylation methylation of promoter => reduced transcription post-transcriptional gene silencing (PTGS) reduction in steady-state mRNA levels without affecting the nuclear transcription normal transcription followed by RNA degradation As a result of the two mechanisms of gene-silencing, methylation and / or RNA degradation of specific DNA / RNA sequences occurs; 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

Discovery of RNAi (cont.) Roles of gene silencing: Regulation of endogenous gene expression: Both mechanisms of gene silencing are used to regulate endogenous genes expressions: For developmental purposes Stem cell homeostasis Methylation of heterochromatin Defense against pathogens and transposable elements: Mechanisms of gene silencing also protect the organism's genome from transposon and viruses. Gene silencing thus may be part of a defense system in plants. RNAi -Appl II. MSA-Shtayeh (Prof.) Appl. Biotech II gene-silencing & Viral suppressors of gene silencing MSAShtayeh

Stem cells homeostasis: The ability or tendency of stem cells to maintain internal equilibrium by adjusting its physiological processes. A stem cell is a cell that has the ability to continuously divide and differentiate (develop) into various other kind(s) of cells/tissues. 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

What is PTGS? PTGS is a sequence-specific RNA degradation process that targets foreign RNA. This includes: viral RNA Retrotransposons work by copying themselves and pasting copies back into the genome in multiple places. Initially retrotransposons copy themselves to RNA (transcription) but, in addition to being transcribed, the RNA is copied into DNA by a reverse transcriptase (often coded by the transposon itself) and inserted back into the genome. dsRNA, etc. 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

Why is PTGS significant with respect to plant viruses? PTGS is a mechanism that plants have developed for protection from virus infection (i.e., the plant PTGS system degrades viral RNA) 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

Note that… PTGS is also significant in many other organisms across all kingdoms PTGS-like phenomena have been studied for many years in several diverse organisms but it wasn’t until recently that these phenomena were found to be related to each other and to PTGS. The PTGS-like systems seen in other organisms have had different names (i.e., quelling, RNAi, co-suppression, RNA-mediated resistance, etc.), but now all of them are generally referred to as RNA silencing. 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

(dsRNA form) RNAi = RNA interference Note different terms used for same phenomenon: PTGS = quelling = co-suppression = VIGS = RNA interference VIGS, virus-induced silencing RNAi -Appl II. MSA-Shtayeh (Prof.)

Examples of RNAi (PTGS): Silencing of a plant gene by agro-infiltration of the plant with a cloned copy of that gene. by agro-infiltration of a GFP (green fluorescent protein) transgenic plant with GFP…. 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

Non-transformed plant GFP transgenic plant
The GFP in transgenic plants can be silenced by agroinfiltration with GFP Agroinfiltration: describes a means for expressing a protein at a high level in plants. The gene for the protein is cloned into a plasmid and the chimeric plasmid is placed in Agrobacterium. A culture of the Agrobacterium is grown and this culture is used to infiltrate leaves of a plant using a syringe (without the needle). The plasmid from Agrobacterium will then drive the synthesis of the protein at a high level in the infiltrated plant cells. Non-transformed plant GFP transgenic plant UV light UV light Fluoresces red under UV light Fluoresces green under UV light (note that some UV lamps will not cause the leaves to fluoresce red - instead, the leaves stay dark green) RNAi -Appl II. MSA-Shtayeh (Prof.) Appl. Biotech II gene-silencing & Viral suppressors of gene silencing MSAShtayeh

(dpi = days post-infiltration) -Agro-infiltrate leaf with GFP -Examine plant under UV light at 2 dpi -Examine again after 7dpi non-transgenic plant 2 dpi 7 dpi -Agro-infiltrate leaf with GFP -Examine plant under UV light at 2 dpi -Examine again after 7dpi GFP-transgenic plant 2 dpi 7 dpi -note that infiltrated area in the non-transgenic plant fluoresces green at 2 dpi but fluorescence fades by 7dpi -note that GFP infiltration of transgenic plant increases GFP fluorescence at 2 dpi, but after that the infiltrated area as well as the other leaves of the plant no longer fluoresce green – loss of green fluorescence is due to silencing of the GFP (both the GFP in the transgenic plant and the GFP that was co-inoculated) RNAi -Appl II. MSA-Shtayeh (Prof.)

Detailed Model for RNA silencing pathway in plants
hairpin RNA Viral ssRNA Transposon RNA Transgene RNA dsRNA Cleavage of dsRNA by Dicer-like enzyme (Dicer contains dsRNA cleavage activity, helicase and dsRNA binding activity) p OH HO siRNA (short interfering RNAs, nt long) p OH HO p OH HO p OH HO Enzymatic machinary: 1. RdRP conversion of ssRNAs into dsRNAs, 2. dicer or dicer-like complexes cleavage of the dsRNAs, 3. RISC degradation of the target RNAs 4. RNA-directed DNA-methylation (RdDM complexes) and different histone-modifying and chromatin remodeling activities. p OH HO siRNA/protein complex siRNA is unwound, single-stranded RNA formed (note that both the + and – strands of siRNA can anneal to viral RNA - therefore both + and – strand viral RNAs can be targeted) RISC complex (RNA-induced silencing complex) p OH (Specificity is due to the fact that the siRNA can only bind to complementary RNA (which can only be viral RNA ) sequence-specific target recognition p OH Viral ssRNA target (a) (continued on next slide) and/or (b) RNAi -Appl II. MSA-Shtayeh (Prof.)

(a) (b) ………etc Degraded viral RNA Newly synthesized viral RNA
Single-stranded siRNAs can also be used as primers to make more dsRNA by host RNA dependent RNA polymerase (b) Degraded viral RNA Newly synthesized viral RNA Host RNA dependent RNA polymerase single-stranded siRNA ………etc p Newly synthesized viral dsRNA Dicer (as above) New siRNAs The siRNAs feed back in to the cycle and further degradation of viral RNA occurs. Model was modified from Roth et al., (2004) Virus Research 102: RNAi -Appl II. MSA-Shtayeh (Prof.)

Some General Features of PTGS
Homology dependent – once viral RNA triggers PTGS, the PTGS system is directed only at that particular viral RNA DsRNA of the virus is the strongest inducer of PTGS (but the double-stranded regions of both + and – sense ssRNA also induce PTGS Viral RNA is degraded by the PTGS system (results in the loss of RNA to make protein and therefore the virus cannot continue to multiply and spread) Silencing signal is mobile (i.e., it can be transmitted to other parts of plant) Most plant viruses encode “suppressors” of gene silencing to combat the host PTGS system. 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

What triggers (induces) the PTGS response when a virus infects a plant?
Inducers of PTGS: 1. Viral dsRNA (recall from first lecture… the double-stranded replication intermediate that arises during virus infection) 2. Viral plus sense RNA (the double-stranded regions) 3. Viral minus sense RNA (the double-stranded regions) (+) (-) (+) (-) ds regions in (+) sense RNA Viral dsRNA replication intermediate ds regions in (-) sense RNA 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

PTGS pathway phases: The PTGS pathway has two distinct phases: 1. Initiation – the viral RNA triggers the PTGS system to degrade viral RNA into small pieces (called siRNA or small interfering RNA) Maintenance-the siRNA binds to complementary regions in viral RNA and this is either: a. degraded by a complex called RISC (RNA-Induced Silencing Complex) or b. or is used to make more viral RNA via the host RNA dependent RNA polymerase. The resulting dsRNA then feeds back into the system at the point where dsRNA is degraded to siRNA and the cycle continues to repeat. 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

The two main phases of the RNAi system
Viral ssRNA Initiation dsRNA Cleavage by “Dicer” siRNAs Maintenance siRNA anneals with viral RNA Synthesis of viral dsRNA (via the host RNA dependent RNA polymerase) and/or Cleavage (degradation) of viral RNA (by RISC) 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

RNAi on video: 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

The mobile silencing signal
In plants, the silencing signal can be sent to other parts of the plant The mobile signal moves along the same route the virus takes. Thus, the virus and plant are in a race. If the virus moves ahead of the signal, it is able to establish infection in other parts of the plant. If the signal is ahead of the virus, the viral RNA will be targeted by silencing and the virus will not be able to establish a further infection. 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

Evidence for a mobile silencing signal
(All plants are coloured as they would look under UV light ) Graft leaf from NS plant to S stock GFP transgenic, not-silenced (NS) Silenced GFP transgenic (S) Wait 7-9 days NS shoot NS stock S scion S stock Note that leaf from non-silenced plant becomes silenced when grafted onto a silenced stock. This implies that silencing signal from the silenced stock plant traveled to the grafted leaf and silenced it. Beheaded NS NS leaf on S stock 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.) For details, see Palauqui et al., (1997) EMBO J 16: 4738.

What is the nature of the mobile silencing signal? How is it sent to other plant parts? 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

Initiation of silencing spread
Silencing spread follows the initiation of silencing by over expression of sense or antisense RNA but most efficiently by the expression dsRNA. What ever the inducer is, in plants there is always some spreading of silencing taking place, from a few cells to the whole plant. 4/28/2017 RNAi – Part I- Appl II. MSA-Shtayeh (Prof.)

Types of silencing movement in plants
Short range spread. Extensive local spread. Systemic silencing spread. 4/28/2017 RNAi – Part I- Appl II. MSA-Shtayeh (Prof.)

4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

Short range silencing spread:
Occurs in a thin layer of cell wide as a result of cell to cell silencing movement without silencing signal amplification. occurs in endogenes not transgenes. when endogenes are targeted the effect of silencing is observed only in cells where the inducer (dsRNA) is active and in few surrounding cells. 4/28/2017 RNAi – Part I- Appl II. MSA-Shtayeh (Prof.)

Short RNA silencing signal is 21-nt siRNA;
as cell-to-cell spread of silencing is lost in plants with lesion in the DCL4 genes (DCL 4 responsible for the generation of 21-nt siRNA ) 4/28/2017 RNAi – Part I- Appl II. MSA-Shtayeh (Prof.)

Movement way of the silencing signal:
Spread takes place through the plasmodesmata (Fine cytoplasmic tubes connecting adjacent plant cells) Evidence : During short range silencing the only cells that escape silencing are guard cells of stomata with no opened plasmodesmata. 4/28/2017 RNAi – Part I- Appl II. MSA-Shtayeh (Prof.)

Plant genes involved in short range silencing
RDR6 (RNA-directed RNA polymerase 6) not needed (no amplification) DCL4 to produce the signal 21-nt siRNA NRPD1a (DNA-dependent RNA polymerase IV large subunit) (the gene encoding the large subunit for Pol4. RDR2 gene encoding RDR2 associated with Pol4. 4/28/2017 RNAi – Part I- Appl II. MSA-Shtayeh (Prof.)

Pol4 and RDR2 are proposed to assist in the generation of dsRNA then processed by DCL4 to generate 21-nt siRNAs which is the short range silencing signal. NRPD1a* (Nuclear RNA polymerase D), and RDR2 are proposed to affect silencing down stream by affecting the reception of the signal in the cells where silencing signal enters. * maintain PTGS. 4/28/2017 RNAi – Part I- Appl II. MSA-Shtayeh (Prof.)

Other genes Classy genes (CLSY1) is proposed to have a role at a step between NRPD1a and RDR2. (RDR2 severely disrupted in CLSY1 mutants). HEN1; stabilization of siRNA by methylation. DCL1; processing of miRNA. AGO1; sequence specific degredation. 4/28/2017 RNAi – Part I- Appl II. MSA-Shtayeh (Prof.)

A speculative model for short-range spreading of RNA silencing
The primary transcript produced by the transgene is processed by DCL1 in a Drosha-like fashion, facilitating its subsequent processing by DCL4 and/or DCL3. The DCL proteins act in concert with respective DRBs (dedicated dsRNA-binding protein 4), and may be competing with each other for the same dsRNA substrate. Processing of the hairpin by DCL3 gives rise to 24-nt-long RNAs, which are then stabilized by HEN1 through 2-O-methylation of their 3 ends, and can be used in the AGO4 pathway negatively affecting the transgene activity. Processing of the hairpin by DCL4 will give rise to 21-nt-long small RNAs which are also stabilized by HEN1. DCL4-produced siRNAs are then loaded into the AGO1-containing RISC, mediating cleavage of complementary mRNAs. The 21-nt-long siRNAs have been proposed to mediate the cell-to-cell movement either alone or in a ribonucleoprotein complex moving through the plasmodesmata (P) connecting neighbouring cells. The 21-nt siRNAs may also be produced from dsRNA generated by RDR2, once the NRPD1A-related TGS (transcriptional gene silencing) pathway has been initiated through the activity of the AGO4-containing RISC. Cell-to-cell silencing spread was shown to require the activity of NRPD1A, CLSY1 and RDR2 either at the donor or/and at the recipient cell.

A speculative model for short-range spreading of RNA silencing
1. The primary transcript produced by the transgene is processed by DCL1 in a Drosha-like fashion, facilitating its subsequent processing by DCL4 and/or DCL3. The DCL proteins act in concert with respective DRBs (dedicated dsRNA-binding protein 4), and may be competing with each other for the same dsRNA substrate. 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

Short range silencing RNAi -Appl II. MSA-Shtayeh (Prof.)

2. Processing of the hairpin by DCL3 gives rise to 24-nt-long RNAs, which are then stabilized by HEN1 through 2-O-methylation of their 3 ends, and can be used in the AGO4 pathway negatively affecting the transgene activity. 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

3. Processing of the hairpin by DCL4 will give rise to 21-nt-long small RNAs which are also stabilized by HEN1. DCL4-produced siRNAs are then loaded into the AGO1-containing RISC, mediating cleavage of complementary mRNAs. 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

4. The 21-nt-long siRNAs have been proposed to mediate the cell-to-cell movement either alone or in a ribonucleoprotein complex moving through the plasmodesmata (P) connecting neighbouring cells. 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

5. The 21-nt siRNAs may also be produced from dsRNA generated by RDR2, once the NRDP1A-related TGS (transcriptional gene silencing) pathway has been initiated through the activity of the AGO4-containing RISC. 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

6. Cell-to-cell silencing spread was shown to require the activity of NRDP1a, CLSY1 and RDR2 either at the donor or/and at the recipient cell. 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

Abbreviations used: AGO, Argonaute; CLSY1, CLASSY1; DCL, Dicer-like; dsRNA, double-stranded RNA; DRB, dedicated dsRNA-binding protein; GFP, green fluorescent protein; HEN1, HUA enhancer 1; miRNA, microRNA; nat-siRNA, natural antisense transcript-derived siRNA; NRPD1A, DNA-dependent RNA polymerase IV large subunit; POLIV, DNA-dependent RNA-polymerase IV; RDR, RNA-directed RNA polymerase; RISC, RNA-induced silencing complex; RNAi, RNA interference; SGS, suppressor of gene silencing; siRNA, short interfering RNA; S-silencing, sense-induced silencing; SDE3, suppressor of defective silencing 3; tasiRNA, trans-acting siRNA; VIGS, virus-induced gene silencing. 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

End of Part I of RNAi 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

Thank you 4/28/2017 RNAi -Appl II. MSA-Shtayeh (Prof.)

Agricultural Biotechnology RNA interference (RNAi) technology: Part I

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