RNA interference

Figure 1. Both DNA strands of some genes are transcribed. However, in plants, most siRNAs are generated by RNA-dependent RNA polymerase [1]. Single strand RNA transcripts: ssRNA. RNA interference requires that two base pair-complementary strands of RNA to come together to form double stranded RNA[2].

The Nobel Prize in Physiology or Medicine in 2006 was awarded to Andrew Z. Fire and Craig C. Mello for their research on RNA interference [3]. The goal of this learning project is to complement the Wikipedia article about RNA interference in two ways. The first goal is to provide a user-friendly introduction to the topic. This means providing learning resources for people who would normally be unable to understand a technical Wikipedia article on the topic of RNA interference. The second goal is to provide learning resources that allow interested university students to collaboratively explore the science behind each awarded Nobel Prize in more detail than is possible with the related Wikipedia article. If you have not done so already, take a look at the Wikipedia article about RNA interference then select one of these learning paths:

1. Explore a user-friendly introduction to the practical medical implications of RNA interference that arise from the Nobel Prize-winning scientific research of Andrew Z. Fire and Craig C. Mello.
2. If you were able to read and appreciate the Wikipedia article about RNA interference then continue reading below and participate in further exploration of this subject.

RNA interference was discovered as a mechanism used by cells for regulating gene expression. This discovery has quickly resulted in the widespread use of artificial interfering RNAs as an important laboratory research technique for altering the amount of specific proteins inside cells. There is also active study of the potential value of RNA interference for medical applications[4].

## Short review: RNA interference in cells

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Figure 2. Cells can trim double stranded RNA to form small inhibitory RNA (siRNA). An siRNA can be processed to the single strand anti-sense RNA and used to target mRNAs for destruction. Several proteins (colored ovals) are required for efficient RNA interference. The protein-containing complex was named "RNA-induced silencing complex", RISC. [5].

This section and the next few sections briefly introduce RNA interference (RNAi) and will orient you towards the activities. See the Wikipedia article about RNA interference for more a more detailed introduction to RNAi.

The normal function of RNA interference inside cells depends on the production of double stranded RNA (dsRNA). Base pair-complementary RNA strands (ssRNA) can be produced by transcription of both template DNA strands of some genes (Figure 1). In other cases, the protein-coding RNA sense strand might be produced by a virus and the antisense RNA strand produced by the host cell. The sense and antisense RNA strands form double strand RNA (Figure 2, top) that is processed to small (about 20 base pairs long) inhibitory RNA (siRNA). The siRNA can form a molecular complex with proteins that first strip away the sense strand of RNA, making the antisense inhibitory RNA (iRNA) available for base pairing with messenger RNA (mRNA). This targets a specific mRNA for destruction (Figure 3), resulting in inhibition of the biological function that would otherwise be served by the protein coded for by the mRNA.

There had long been interest in the idea that it might be possible to alter gene expression by using single strands of nucleic acid that might base pair hybridize with a specific target in cells. However, it was only in 1998 that experiments were described showing the unexpected power of double stranded RNA to block gene expression [6]. The experimental system used was the worm Caenorhabditis elegans. This was a useful experimental system because the developmental orgin of all of this organism's cells is known and it is possible to inject RNA into early embryos and observe changes in the pattern of development.

Figure 3. Diagram showing how the anti-sense RNA (the yellow strand in this diagram) of the RISC complex targets destruction of complementary mRNA. The active site of the enzyme that cuts the mRNA has the amino acids Asp-Asp-His (DDH) at the active site[7]. Image source: Leemor Joshua-Tor.

### Protection against viruses

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An important aspect of RNA interference is its role in protecting organisms from some deleterious effects of viruses [8]. Such a role for RNA interference was first found in plants, but has also been found in some animals[9].

### Gene expression knockdown experiments

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RNA interference is now a widely used biology research technique that can be applied to both cultured cells [10] and whole animals[11]. RNA interference can be used to selectively reduce the level of expression of a specific protein. Clues to the function of the protein can be obtained by observing changes in cell or organism behavior after knockdown of expression.

### Medical applications

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It has been suggested that knockdown of expression of proteins by virus-delivered siRNA might be effective for future treatments of Amyotrophic lateral sclerosis and other neurodegenerative diseases. Working with laboratory mice as an experimental model system for the human disease ALS, Miller et al showed that loss of muscle function could be slowed using RNA interference[12].

## Detailed Review

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Figure 4. Statistics of RNAi publications in Google Scholar (When “RNAi” typed to search bar of Google Scholar, these results are gained).

RNAi is discovered as a defense system to prevent attack of viral and parasitic RNA [13]. Later on, other fuctions of its emerged, like endogeneous gene regulation [14]. This system is able to seen in Caenorhabditis elegans, Drosophila, yeast as well as can be done in mammals [15]. Main mechanism of RNAi is revealed by Fire and Melo [16]. It is used in many laboratories to induce gene silencing in organisms by delivering appropriate dsRNA (double stranded RNA) [17]. RNAi can be utilized as an analysis tool to understand function of genes therefore let us to obtain an evolutionary view. It is also possible to determine mutant genes, which cause death of the organism, via RNAi [18]. RNAi centered techniques for full genomic screen provides oppurtunity to identify specific genes via testing a given trait with high trustability [19]. CRISPR interference is an alternative for RNAi in prokaryotes. CRISPR and RNA interference mainly differ at structures of the components [20] , however, pathways share similarities [21]. Increased studies ulliminated the way that RNAi work and let scientist to utlize it better.

#### Basic Mechanism of RNAi in Mammals

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Three main components of RNAi silensing includes Drosha, Dicer and Argonaute (Ago) proteins. Both Drosha and Dicer are the members of RNAase III family. They have ability to cut dsRNA by leaving 5’ phosphate head and 2 nt (nucleotide) overhang at 3’ end. With this property, they are responsible for processing of newly transcribed small hairpin non-coding RNAs to smaller pieces. The pieces are 21- 25 nt dsRNAs [22][23][24]. Drosha only found in animal cells [25][26][27]. After, one of the strands of 21- 25 nt dsRNAs loaded into RISC (RNA induced silensing complex). This complex is the direct effector of mRNA translational inhibition and it is mainly based on members of Argonaute (Ago) protein family [28]. Slicing activity of Ago is dependent to perfect complementarity of guide RNA with target RNA [29]. Imperfectly matching leads to generation of several scenario containing translational inhibition, prevention of de- adenylation and nuclease degradation by P body. While each scenerio is possible alone itself, also combination of them can be observed [30]. Some Ago including complexes may provide inhibition of transcripition via affecting chromatin structure or causing modification on histone proteins [31].Basic mechanism of post-transcriptional gene silencing in mammals is given step by step in figures named Part 1, Part 2, Part 3, Part 4, Part 5.

Part 1: Firstly, transcripts of precurser of miRNA- micro RNA (green) and siRNA- small interfering RNA (red) is gained, by the help of RNA polymerase II and III, respectively. Than these transcripts are folded into hairpins.MiRNA bear a bulge and stem as differences from siRNA.

Part 2: Drosha found in nucleus cuts stem of pri- miRNA by leaving 2 nucleotide overhang at 3’ end.

Part 3: Pre- miRNAs and siRNAs (shRNA- small hairpin RNA) are exported from nucleus via Exportin 5 nucleus membrane protein.

Part 4: In cytoplasm, Dicer cuts precurser molecules to smaller parts (generally into 21- 25 nucleotide length) by leaving 2 nucleotide 3’ end. This enzyme also has ability to separate two strands of short dsRNAs from eachother. One of these strand called guide strand while the other one named as passenger strand.

Part 5: Only guide strand is loaded to RISC (RNA induced silencing complex) that targets mRNAs that are partially or fully complementary to guide stand sequence.

### RNAi in other Organisms

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RNA interference is an highly conserved mechanism but components of the system may be distinct from one organism to another, even some contributers of the mechanism may be deficient in some cases [32].

C. elegans: C. elegans is a transparent roundworm. It has 1mm length. Diet of its consists of bacteria. By means of the efforts of Dr. Sydney Brenner, this animal became one of the strongest model organism [33]. Ago- guide RNA interraction forms the base of RISC(RNA induced silencing complex)[34]. Ago has two extremely characteristic domain called PAZ(PIWI/ Ago/ Zwille) and Piwi. These domains are responsible for holding guide RNA in a way that does not close the side which base pairing occurs with target mRNA [35][36]. In PIWI, catalytic role is borne by DDH motif which is similar to RNase H family catalytic [37]. Problem on the residues of DDH on Ago2 results in cancellation of slicing of target [38][39].

Figure 5. Illustration of C. elegans response against exogeneous dsRNA. First five steps, endogeneous steps, are the similar with mammals endogeneous RNAi steps described at Part 1, 2, 3, 4, 5. SiRNAs enters the cell by means of SID-1. Then primary siRNAs are produced by the help of various proteins containing rde-1, rde-4, dcr-1, drh-1/2. And then RISC generated based on rde-1/ guide strand associated structure. Some of remaining parts of exogeneous RNA behave as primer for secondary siRNAs by the work of RNA dependent RNA polymerase (RdRp). PIR- 1 dephosphorylates 2nd siRNAs, produced by RdRP [40]. After that these siRNAs associate with Ago proteins that are different from first ones and called as secondary Ago proteins (SAGO). Further RISC is formed with SAGO and target molecules are processed.

RDE- 1 is a type of Ago in C. elegans, works with rde- 4 [41]. Without rde-4, decline on siRNA level happens [42][43]. RDE- 4 has a binding motif which provides better interraction with long dsRNA, instead of short ones like siRNA. This protein is not just linked with rde- 1 and dsRNA, also have connection with the other elements of RNAi. Rde- 4’ s main responsibility is recognition of long dsRNA and presentation of it to DCR- 1 [44].

SID- 1, a type of membrane protein of C. elegans [45], provides passage of exogeneous dsRNA, into C. elegans cytoplasm [46]. Homologs of SID- 1 found in mammals however not found in Drosophila [47]. MicroRNA has crucial effects on differentiation and time of development of this organism [48]. Different organisms have different anatomies and physiologies which cause application of separate techniques to force organism to accept service of dsRNA. Introduction of dsRNA to C. elegans can be achieved via several methods. These methods are:

a) Direct injection. dsRNA including solution is ejected to the body cavity. In this method, RNAi response arises throughout the body.

b) Production of trangenic amimal that express target dsRNA. The technique is limited with the tissue that the technique applied.

c) Immersion of the organism to the solution of dsRNA. Ingestion of RNAs leads generation of RNAi response.

d) Feeeding with bacteria which are genetically modified to produce target dsRNA [49].

Drosophila: Drosophila genus hosts one of the model organism named Drosophila melanogaster [50].

Properties of domains of Dicer belongs to Drosophila  Domain of Dicer | Main Job of the Domain

|

Double strand binding domain (at C terminus) | Connect to target dsRNA [51]
PAZ domain | Determination of the strand that will be used as guide [52]
RNase domains (RNase IIIa/ RNase IIIb) | Cleavage of the target to smaller peaces
DEXH- box helicase domain (at N terminus) | Separation of double stranded RNAs to single strand
DUF- 283 | Unknown [53]

In Drosophila, dicer- 1 and dicer- 2 found as paralogs of Dicer. As dicer- 1 plays role in generation of miRNAs, dicer- 2 included in synthesis of siRNAs. Explanation of this discrimination originates from structural differences. While dcr- 1 does not have helicase domain, dcr-2 suffers from absence of PAZ domain [54]. However, in terms of function, dicer- 1 and dicer- 2 don’ t have a thick boundary wall that separates them from each other. Both of the enzymes are obligatory for functioning of the siRNA mediated destruction of target mRNA and gene silencing [55]. PAZ domain of Dicer works with RNase III domains in order to provide determination of 3’ overhang ends of target RNAs [56]. RNase IIIa and RNase IIIb gather to take over catalytic process that dicer is responsible for [57]. Linkage of these domains with Ago occurs at the side of PIWI domain of Ago [58]. Efficiency of the enzyme is highly dependent to 3’ overhang nucleotide sequence [59] After Dicer processing, small noncoding RNAs passed into RISC[60].

List of members belongs to RISC of Drosophila [61]:

* Ago2
* dFXR (Drosophila Ortholog of Fragile X Mental Retardation Protein)
* VIG (Vasa Intronic Gene)
* Tudor- SN
* R2D2
* Aubergine
* Armitage- RNA helicase
* Others

While RISC loading occurs, guide strand selection is carried out by the help of R2D2 protein. As passenger strand is gong to be degraded, other one used as a guide [62]. Identification of passenger and guide strand of siRNA is achieved accourding to thermostability of ends of strands [63]. At the time of guide strand selection, Dicer connects to less stable 5’ end of siRNA as R2D2 binds to more stable one, in thermodynamicity. Less stable ended one loaded into RISC [64]. In Drosophila, separation of passenger and guide strand is carried out by Ago2 [65].

Usage of less stable strand on RISC lowers, even minimize, posibility of off targeting [66]. PAZ of Ago contact with 3’ end of siRNA while PIWI domain makes connection both with RNase III domains of Dicer and phosphate headed end of siRNA single strand [67]. PAZ affinity towards 3’ overhangs originated from aromatic residues on it [68]. Different members of Ago family bind to distinct siRNAs [69]. All of four Ago of mammalian cells can connect to miRNA [70]. As opposite to mammals, Drosophila melanogaster has only Ago1 in its miRNA pathway [71].

Drosophila has 3 type of siRNA based RISC[72] as shown in table located below.

Types of siRNA RISC in Drosophila RISC type | Composition | Mission

|
|

R1 | Dicer- 2/ R2D2/ others | Processor of long dsRNA and precursor of R2 [73]
R2 | Dicer- 2/ R2D2/ Ago2/ Armitage/ others | Unwinding of siRNA [74][75][76]
R3 (Holo- RISC)(Formation of R3 needs ATP) | Dicer- 1/ Dicer- 2/ R2D2/ Ago2/ siRNA/ VIG/ Tudor- SN/ dFXR/ others | Silencing, chromatin formation etc. [77]

The I Factor is the structure that is the cause of the disorder named as Hybrid Dysgenesis, in Drosophila melanogaster [78]. This disorder contains abnormalities mainly based on high mutation rate [79]. I Factor repression is maternal and have long term effect [80]. Repression of the factor carried out by the help of RNAi. One strong scenerio about it suggests siRNAs for repression originated from egg cytoplasm [81].

Another elements occurs to cause other types of Hybrid Dysgenesis. One of them is called P element, which are DNA transposons [82][83][84]. High level of transposible element activity drives cell to death [85]. P element is under the control of RNA mediated mechanism [86].

Figure 6. Propagation of methylation. HP1 has ability to recognize methylated H3K9. After recognition, it sits on methyl and call Su(var)3-9. Su(var)3-9 connects HP1 and add methyl to next histone.

PEV (Position Effect Variegation) is the result of chromosomal rearrangement of the place of euchromatic gene. The euchromatic gene transferred next to heterochromatin and euchromatic characteristics of the translocated gene disappears, gene become silenced. PEV is well studied in D. melaogaster eye and body pigments [87]. PEV is mainly related with propagation of methylation[88].

HP1 based silencing is achieved by the help of RNAi components [89][90]. Expression of transposable elements of Drosophila is majorly controlled at transcription level. RNAi connected to gene silencing via Polycomp proteins in Drosophila [91]. Polycomb response elements helps silencing via employing Polycomb complex [92]. Stuck at RNAi machinary provoke rise in transcription of heterochromatic genes[93].

S. pombe: Although Schizosaccharomyces pombe is a fungus, because of its high similarities with other eukaryotes, it became a good choice to work on some cellular events including RNAi based heterochromatin generation [94]. In S. pombe, also known as fission yeast, RNAi has considerable effects on the formation of heterochromatin. Centromers, telomeres, locus of mating- type and ribosomal DNA are the regions under the control of RNAi mechanism in terms of chromatin modification [95].

Heterochromatin is the name of sites exposured to methylation on lysine 9 of H3, a type of histone protein. Methylation occurs with the help of Clr4, a histone methyltransferase. Number of methyl inserted to H3 can vary from one to three [96]. Loss of Clr4 directly means that loss of H3K9 methylation. Clr4 has chromo and SET domain. SET plays role in the methylation. Abnormality on SET domain causes results in changes on the methylation level at some loci of S. pombe [97][98].

Clr4 also mediates Swi 6 to bind to related loci [99][100][101]. Methyl addition to lysine 9 residue lets Swi 6, a chromo domain protein in fission yeast, to link to H3 by the help of its chromo domain [102]. Dimerization of Swi 6 proteins may lead other proteins to associate with Swi 6 via interraction surface resulted from dimerization [103].

Acetylation of target residue blocks methyl insertion. Therefore histone deacetylase activity is needed. This requirement is carried out by the enzymes of HDACs (histone deacetylases) [104]. Sir2 and Clr3 are the examples of HDACs with Clr6, which has broad target catalog that makes it crucial [105][106][107].

Presence of RNAi mechanism is as significant as functioning of Clr4 and Swi6 [108]. RITS (RNA induced transcriptional silencing complex) and RDRC (RNA directed RNA polymerase complex) are the two complexes that are the components RNAi mediated generation of heterochromatin in Schizosaccharomyces pombe [109][110][111]. RITS composes of Ago1, Chp1 and Tas3 besides siRNA bound to Ago1 [112]. In RITS, Chp1 is responsible for interraction of the RITS with H3K9me2 (2 methyl added to lysine 9 of H3) via its chromo domain [113]. In the absence of one or more components of RITS, remainings can associate but activities of this “half” RITS is not capable of doing activities of fully equiped RITS [114][115][116]. Dcr1 and Clr4 are required, alongside Rdp1 (RNA dependent RNA polymerase 1) catalytic activity, for RDRC- RITS linkage [117][118][119]. Relation of RITS with both siRNA biogenesis and chromatin structural changes is an evidence of presence of feedback mechanism working between RNAi and chromatin modification [120]. H3K9 methylation at the telomeric, centromeric and mating- type region is not totally cancelled out in the absence of RNAi. This is because of RNAi independent pathways in S. pombe, like Taz1 protein in telomeres. Experiments suggest that other chromo protein Chp2 takes over the job of Chp1 at related loci [121].

Heterochromatin has impotance on mating- type determination. Abnormalities found in the heterochromation structure of mating- type switching genes, can drive haploid cell of S. pombe to death [122].

Arabidopsis: In plants, the place for Drosha is filled with the Dicer homolog protein called Dicer- like 1 [123]. Plants produce four Dicer Like Proteins, abbreviated as dcl. Four of each bears separate jobs. Processes of pre- miRNAs (precursor miRNAs) are carried out via dcl- 1 while dcl- 2 is responsible for production of siRNA used in defense mechanism against to viruses. Dcl- 3 generates siRNAs that are the part of chromatin modification and silencing at transcription level. The last dcl enzyme named dcl- 4 cares formation of trans- acting siRNAs which are involved in regulation of accumulation mRNAs [124][125][126][127][128][129][130].

Dcl- 1 is escorted by Hyponatic Leaves1 (HYL1) and Serrate (SE) to function in Arabidopsis. While HYL1 is a dsRNA binding protein [131][132], SE is the encoder of C2H2 zinc finger protein, which affects miRNAs’ relation with leaf polarity in Arabidopsis [133]. It is current truth that SE and HYL interracts[134][135]. Dcl- 2, 3 ,4 are mainly involved in siRNA generation pathway [136]. Dcl- 1 also has crucial missions in the formation of natural antisense short interfering RNAs. Thus, this event is an example demonstrates dcl- 1 is not restricted with miRNA genesis [137][138].

Before process of Dcl, Arabidopsis utilizes HASTY, homolog of exportin 5, in order to export miRNA from nucleus [139][140].

Methylation of miRNAs is carried out via HEN1 protein as a general step of miRNA biogenesis in plants. Methylation happens after Dcl functions [141][142]. 2 overhang of 3’ end of miRNA duplex is favored substrate for HEN1. Change on the size of overhang may cause to dramatically decline or stop of HEN1 methylation acitivity [143]. HEN1 functions regardless of the 5’ end characteristics. In addition to 3’ end nucleotides, 2’ OH of end nucleotide at 3’ end is significant determinant of methyl addition [144]. Methyl is added to this 2’ OH. Efficiency of methylation changes with the length of miRNA, in vitro [145]. Again in vitro, HEN1 has ability to methylate both siRNA and miRNA duplexes [146]. Methylated miRNA enters RISC after processed by Ago for separation of passenger strand from the guide one [147].

Most of the Ago from Arabidopsis are capable of slicing target RNA [148]. As opposite to animals, plants have miRNAs capable of destructing intended mRNA when perfect or barely perfect matching between mRNA and guide strand is provided [149]. At 3’ end of mRNA, just after translational termination codon, a region called 3’ untranslated region is localized, abbraveated as 3’ UTR [150]. This region contains miRNA response elements (MREs). In mRNA metabolism of plants, interraction between miRNA and MREs determines mRNA will be degradaded or not while in animals, this relation just may cause inhibition of translation [151].

MiRNA cleavage remains smaller RNA particles which will be degraded by exonucleases, like XRN4 [152]. Before exonuclease activity, a few modification like insertion of adeninosine or uridine to the end of target molecule can be required [153][154].

Plants utilizes its phloem or/and plasmodesmata to propagate RNAi response to other cells of the plant body [155]. Unlike animal miRNA genes, those ones belongs to plants are intergenic and do not exhibit co- transcription and/or translation [156]. Production of miRNA controlled via feedback mechanism that is mediated by means of miRISC activity on mRNAs of Dcl- 1 and RISC components. It is thought that miRNA evolves fast in plants. This idea is supported with the observation that most of miRNA of Arabidopsis are nonconserved in poplar and rice [157].

## Proteins of RNAi

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### Drosha

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In human, DGCR8 and Drosha associates with each other [158].

### Dicer

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Insertion of ATP triggers production of siRNA by interraction of ATP with helicase/ATPase domain located on one the Dicer of Drosophila and Dicer of C. elegans [159]. Although mammalian Dicer has helicase/ATPase domain, no influential effect of ATP occurs [160], in the absence of Dicer in mammalian cells does not inhibits RISC activity [161].

Two example of dsRNA bindind domained proteins, that associate with human Dicer, are TRBP and PACT [162]. TRBP is an effective component for optimum silencing by siRNAs and endogeneous miRNAs [163]. Additionally, the proten contributesto regulation of some other mechanisms like control of cell growth [164][165].

Dicer works under the presence of the Mg2+ which can be replaced with Co2+ or Mn2+. Dicer even can process dsRNA that have blocked ends with RNA tetraloop or DNA- RNA duplexes. It converts these ends to 2nt overhang 3’ ends. After that it continues its normal pathway [166].

Human Dicer works regardless of ATP [167][168][169]. This condition is more or less same for UTP, TTP, GTP etc. and nonhyrolyzable ATP analogs [170]. In chicken- human hybrid cells, lack of Dicer is ended up with the mislocation of RAD1 and BupR1 heterochromatin related proteins [171].

Efficieny of Dicer- 1 enzyme in Drosophila is dependent to Loqs, a type of protein[172][173].

Dicer- 2 from Drosophila melanogaster cannot function without ATP [174][175][176] as other RNA helicases are not able to do, too [177]. Functioning of Girdia intestinalis Dicer void of helicase domain [178] demonstrates dispensability of the domain for working of the enzyme [179].

An extremely conserved part of RNAase III family proteins is called endonuclease domain. It is found in the form of tightly associated dimer. Because of dimerization, a space form between monomers which dsRNA molecule sits. The space is named “catalytic valley”. The valley carries negative charge on itself [180] which lets magnessium ion to interract. Magnessium ions locate close to ends of this valley [181].

RNase IIIa domain cuts from 3’ end of one strand of dsRNA while RNase IIIb refers to cut from 5’ end of opposite strand [182].

In Girdia, distance between PAZ and RIIIDs domains is equivalent to the length of cleavage product [183]. Thus it is thought that length of miRNAs and siRNAs determined by structure of Dicer [184].

### Ago

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PIWI domain is the slicer of Ago in RISC [185]. PAZ of Ago contact with 3’ end of siRNA while PIWI domain makes connection both with RNase III domains of Dicer and phosphate headed end of siRNA single strand [186]. PAZ affinity towards 3’ overhangs originated from aromatic residues on it [187]. Different members of Ago family bind to distinct siRNAs [188].

Intrerraction between Ago and Dicer is achieved by the help of Hsp90 protein. This linkage can be disrupted via block of Hsp90 by means of Hsp inhibitors [189].

### RISC

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Size of overhangs is one of the most important factor affecting the RNAi activity. As even change in phoshate head of siRNA may result in severe effects on the mechanism of RNAi, its absence means directly loss of RISC stability [190].

Available Mg2+ is an obligation for catalytic activity of RISC [191].

### RDRP

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RdRP primarily belongs to viruses. It directly contributes to viral gene transcription and replication, in viral systems. In general, RdRP carries 4 main domain, palm ,fingers, thumb and N terminal domain that connects fingers to thumb domain [192].

It has cleft for sitting of template RNA strand. This template creates base pairing with small part of primer RNA [193][194]

RdRP, in general, known as a type of error prone polymerase [195].

## Details of RNAi in mammals

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Neither sense nor antisense strand of dsRNA alone can trigger RNAi response. Both strands together is needed to do it [196].

Generally, transcripts of miRNA genes, pri- miRNAs, have at least 1000 nt. After operation of DGRC8/DROSHA on pri- miRNA, pre- miRNAs are gained with 60- 70 nt length, 2nt overhang at both side and 10 nt loop at one side. All of these process happen in the nucleus [197][198][199].

Seed sequence is the region of off-target mRNA that match with 6-7 nt of siRNA sequence. If siRNA on RISC has seed sequence coomplementary to 3’ UTR of mRNA, it behaves as miRNA and prevents transcription of that mRNA, which is not real target RNA molecule [200]. Off target effects could drive cell to death by cellular toxicity [201]. H3K9 and H3K7 of mammalian cells are the targets of methyl addition via siRNA directed mechanism. With this methylation silencing at the level of transcription achieved at related si- RNA targetted promoter sequences [202][203][204][205].

Vigilin is one of the RNAi protein involved in the relation of RNAi with heterochromatin formation [206]. DNA repair protein DNA- PK (DNA Protein Kinase) behaves like a bridge between vigilin and HP1 (Heterochromatin Protein 1) [207][208]. With its kinase activity, vigilin complex promotes phosphorylation of HP1 [209].

DNA- PK is not only repair protein that vigilin interracts. Another “bridge” protein is ATM [210] which found in DNA Damage Response signaling [211]. Drosophila has ortholog of ATM called dATM that helps HP1 to sit true place on telomeres [212].

Synthetic shRNAs (small hairpin RNAs), which has 25- 29 nt long and are 2’ overhanged at 3’ ends, are more effective than siRNAs that have the same target sequence [213]. The main problem of the usage of these shRNAs and siRNAs is that they cannot generate permenant or inducible slincing on the cells of mammals. The reason for transient effect is lack steps of amplication process of RNAi involved in another systems. Each cell division and formation of each RISC declines the nnumber of siRNAs whose effect is not amplified. However some solutions are available so as to enable stable gene silencing in mammalian cells [214][215][216].

RNAi machinary is capable of realizing even one nucleotide difference between two mRNA molecule. Because of this high specifity, it can be used for allele- specific purposes means that while it affects mutated allele expression, normal allele expression remains fixed [217].

As a result of its weight and charge siRNA is not able to pass freely biological barriers [218]. In order to overcome this issue, different methods are developed. 4 main groups of delivery techniques can be counted as:

1. Lipid based techniques [219]
2. With high level similarity to lipid based ones, polymer based methods [220]
3. Naked delivery methods
4. Peptid based techniques [221]

Succesfull transfection of siRNAs can be reached by electroporation, which causes toxicity [222], or using microsponge [223], liposomes or dendrimers [224].

SiRNAs preferentially created with UU or TT overhangs to be recognized by RNAi mechanism [225].

2’ – O- (2- methoxyethyl) modification at second base importanly declines off targetting [226]. Utilization of LNAs (Locked Nucleic Acids) as a modification on siRNA is another option [227] to strengthen the RNAi inducer molecule [228][229].

Internal modifications are not sufficient at introduction of inducer RNA to central nervous system cells [230].

### Immune system and RNAi

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Long dsRNA also triggers sequence independent interferon response (Figure 7 and Figure 8) in addition to possibility of RNAi mechanism induction, in mammalian cells [231].

Interferons (IFNs) are the cytokines capable of influencing multiple cell types. IFN is dramatically significant at communication among cells throughout innate immune responses beside adaptive ones. It primarily targets bacterial and viral infections alongside neoplastic transformation [232]. INFs catogorized in to type I and type II IFN in terms of the way that they regulate immune components [233].

Figure 7. (RNA) Sequence Independent Intereron Response Figure 8. (RNA) Sequence independent interferon response blocks the production siRNA and miRNA from their precurser by degrading these precurcer before Dicer and/or Drosha.

Type II IFN contains IFN- γ. IFN- γ sits at the center of immune responses because expression of molecules used in intercellular communication and cellular interractions is under the control of its. At the time of the infection, Dendrite cells and monocytes are alerted by molecules located on the cell wall of the bacteria. Then alerted cells activates T and NK (Natural Killer) cells to produce and release IFN- γ [234].

MHC (Major Histocompatibility Complex) class I cell surface structures have responsibility to present an order of peptide epitopes to CD8+ T cells [235]. Cells carrying proper epitopes for CD8+ cell recognition interracts with T cell and cause release of IFN- γ [236].

Released IFN- γ connects its receptor on the target cells and leads dimerization of IFN- γ- R1 and IFN- γ- R2 subunits of the receptor. Further, dimerization causes phosphorylation of JAK1 and JAK2 receoptor associated kinases. Then JAK1 and JAK2 phosphorylates STAT1 monomers to make them proper for dimerization. Dimerized STAT1 goes to inside of nucleus so as to activate transcription of some components found in the immune response [237].

IFN- γ has a negative feedback mechanism based on SOCS1. SOCS1 which is awaken by IFN- γ, prevents JAK1 and JAK2 activity, thus further steps extending until transcripitional activation fails [238].

Other machinaries exists in order to regulate IFN- γ signaling pathway. These are mainly based on inhibiting the work of proteins included in IFN- γ signaling and MHC class I path. Block of MHC class I pathway means loss of presentation of abnormal cell to CD8+ T cells, therefore IFN- γ even is not secreted from the T cell [239].

MiRNAs are also said to be regulatory molecules of innate and adaptive immune system [240]. At many stages of tumor, aberrancies on miRNA metabolism is determined [241]. MiRNAs may contribute or repress the tumor [242]. They achieve this by influencing APM (Antigen Presenting Machinary) [243], which makes protein ready for presentation to immune cells [244], and regulating IFN- γ signaling proteins [245].

IFN- γ is able to interfere miRNA metabolism [246] whereas miRNA metabolism can disrupt IFN- γ synthesis, seen in NK cells [247].

Immune system disorders orginate from overexpression of immune factors. Targetting these factors are able to recover disorders related to immunity [248].

If the siRNA carries danger motif (5’- GUCCUUCAA – 3’) TLR7 dependent immune response happens via recognition of siRNA by TLR7 receptor on plasmocytoid dendritic cells [249][250]. Stoppage of this type immune system alertion can be provided by means of chemical modifications or nanopartical coating the inducer RNA [251]. Because TLRs expression is dependent to cell type, immune respose against siRNA is also depends on cell type [252].

### PiRNA

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PiRNAs has distinct size and end modification from siRNAs as well as from miRNAs [253].

PiRNAs’ biogenesis is dicer independent [254]. Activity area of piRNA includes germ- line development, stem cell recovery, transposon blocking and epigenetic regulation [255][256]. They play role in both posttranscriptional and chromatin level reactions [257]. Though it is not known how piRNAs are produced and amplified, existence of their slicing ability is a known fact [258][259][260].

### Long Noncoding RNAs

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Long noncoding RNAs (lncRNAs) differ from siRNAs, miRNAs and piRNAs (piwi- interracting RNAs) via their large size (<200 nt) [261].

LncRNAs participates in transcriptional activation, inactivation, silencing, by means of epigenetic modifications, and post- transcriptional gene silencing. Predictions and conclusions from experiments suggest that these RNAs functions by interracting with other RNAs, DNA and proteins, including transcription factors [262].

### Noncanonical RNAi Inducers

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While canonical siRNAs are being used, some diverge class of siRNAs discovered which do not alert innate immune system [263]. Noncanonical siRNAs, devoid of any modification, have restrictions in usage arises from their weakness against degradation, probability of undiserable guide strand election and induction of interferon response [264][265]. These siRNAs are larger than canonical ones while smaller than 30 nt. 27 nt length in these siRNAs gives the best performance at silencing and siRNAs with this length called dicer substrate siRNAs, abbreviated as dsiRNA [266]. DsiRNA are better at generating RISC Loading Complex as compared to canonical ones, which are 21 nt long [267]. One of the crucial issue for in vivo application of RNAi machinary stimulation by dsiRNA is fate of the siRNA in variety of body fluids [268]. Because of this issue RNAs can be modified in a way that does not disrupt Dicer association with modified RNA molecule [269]. 2’ – O- methyl modification is a good and not expensive path to jump over problems relate with stability in body fluids [270][271][272]. If dosage of modification is extremely high, mission fails in consequence of loss of Dicer related steps in RNAi system [273].

Lower concentration of dsiRNA is required for satisfactory silencing, compared to canonical siRNAs. Longer time influence and increased specificity provided via the use of dsiRNAs [274].

So as to decrease off- targetting, dsiRNA clusters enzymmatically produced via the use of Dicer [275].

In addition to dsiRNA, other alternatives to canonical siRNA occurs like shRNA, dumbell RNA and fork siRNA (Figure 9) [276].

Figure 9. Types of alternative noncanonical siRNA[277].

29 nt stemmed and 4 looped shRNAs are better than 19 nt stemmed ones at silencing of target gene. As loop length is also a significant factor however in high concentrations no critical differences in activity efficiency observed between short and long looped shRNAs [278]. Similar to loop length also side of loop (left or right) is not a determinant of silencing activity at high amounts [279].

Although shRNAs protected at one side via its loop, they even degraded fast in body fluids [280]. This problem overcome by Abe and Abe’s colleagues via invention of new type of siRNA that has two loop at both right and left, called RNA dumbell [281].

Fork siRNAs are generally preferred to knockout mutated or chimerical genes [282].

By the help of several strategies long dsRNAs also became a RNAi tool for mammalian cells. They are usually opted for multiple target silencing [283].

Two grups of long interfering RNA occurs, linear (Figure 10) and branched (Figure 11) longs dsRNA, preferentially with several modification to provide nuclease stability [284]. Some experiments validate these multi siRNAs utilize the same pathway with normal siRNAs [285].

Figure 10. Types of branched ldsRNAs[286].

Linear ones bear a gap on one strand. Devoid of gap, they are non- funtional. Branched ones have more variant shapes [287].

Figure 11. Structure of linear ldsRNAs

Although noncanonical RNAi inducers costs much for unit, low amounts give satisfactory results [288].

### Cancer

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RNAi theraphies offer to stop drug resistence in cancerous cells via switching on/off the relavant signaling paths [289][290]. Only RNAi based medication is not sufficient in order to defeat cancer, however, combination with other cancer approaches like radiotheraphy and chemotheraphy, cancer may be knockdown [291].

## Escape Strategies of Viruses

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Proteins produced because of viral attact leads formation of negative effects on the metabolism of miRNA [292]. FHV (Flock House Virus) produces B2 that inhibits RNAi via preventing Dicer cleavage [293]. B2 activity result in decline in the number of siRNA [294].

Some miRNAs are capable of contributing to virus replication while some of them may work for prevention of the replication [295][296].

## Agricultural utilization

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Major reason for agricultural damage is shown to be insect pests [297]. Although Bacillus thuringiensis toxin is highly influensive to combat with some of pests, most of the members of insect pests, especially Hempitera, are not affected by the toxin [298][299]. Because core RNAi mechinary found in all insect pests, RNAi became a strong option as a pestiside. Generally, one method to knock out agricultural pests is not sufficient because of that Integrated Pest Management (IPM) strategies are preferred [300].

However not all insect groups contain RNAi mechanism which totally limit RNAi inducer applications in RNAi lacking organisms [301].

It is essential to conserve beneficial insects, honey bee is one of them, from harmful biological factors, like pathoges and parazites, too [302].

## Conclusion

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P bodies also includes RNAi activity alongside cytoplasm [303].

When use of long dsRNA for silencing target gene unsucessfull in mammalian cells, by innate immune system, in non- mammalians these type of RNAs achieves silencing, because of absence of interferon response [304][305][306]. Short siRNA are option to bypass innate immune response [307]. With the shortening of normal siRNA, cleavage point of RISC complex alters, too [308]. However, it is observed that, in some cases, even 21 bp siRNA alongside longer ones can initiate interferon mechanism [309].

Because miRNA effects comparatively more limited than siRNA influence, usage of synthetic miRNA paralel to natural one also more restricted than synthetic siRNAs [310].

SiRNA theraphies seem to be more influencive among other antisense theraphies in terms of target specifity, longevity, stability in vivo, efficiency at low amounts and spreading of effect to other cells [311][312].

It is thought that usage of RNAi instead of transgenic reformation methods to generate genetically modified foods is safer because RNAi do not use the genes of other organisms. Thus organism genetic code remains orginal [313].

## Learning project: what next?

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Read about one of these topics and add what you learn to this page:

* What if inhibitory double stranded RNA could form from hairpins such as those found in tRNA molecules? (hint)
* What other medical applications have been proposed for RNA interference? (Hints: search here)
* Add questions and general discussion to the next sections.

### Discussions

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* Read RNAi: a novel strategy for the treatment of prion diseases by Qingzhong Kong and Lentivector-mediated RNAi efficiently suppresses prion protein and prolongs survival of scrapie-infected mice by Alexander Pfeifer et al. Discuss the idea that virus-mediated RNA interference can be used to treat neurological diseases.

#### Mice as a model system

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Are short-lived mice a good model system for study of human neurodegenerative diseases?

## See also

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* This lesson on RNA interference is part of the Nobel Prize in Physiology or Medicine learning project.
* Nobel Prize in Chemistry learning project
* RNA World
* Cell biology improvement drive

## External links

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* RNA interference article at Wikipedia.

## References

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113. ↑ Partridge, J. F., et al. (2002). "cis-acting DNA from fission yeast centromeres mediates histone H3 methylation and recruitment of silencing factors and cohesin to an ectopic site." Current Biology 12(19): 1652-1660.
114. ↑ Jia, S., et al. (2004). "RNAi-independent heterochromatin nucleation by the stress-activated ATF/CREB family proteins." Science 304(5679): 1971-1976.
115. ↑ Noma, K.-i., et al. (2004). "RITS acts in cis to promote RNA interference–mediated transcriptional and post-transcriptional silencing." Nature genetics 36(11): 1174.
116. ↑ Petrie, V. J., et al. (2005). "RNA interference (RNAi)-dependent and RNAi-independent association of the Chp1 chromodomain protein with distinct heterochromatic loci in fission yeast." Molecular and cellular biology 25(6): 2331-2346.
117. ↑ Motamedi, M. R., et al. (2004). "Two RNAi complexes, RITS and RDRC, physically interact and localize to noncoding centromeric RNAs." Cell 119(6): 789-802.
118. ↑ Verdel, A., et al. (2004). "RNAi-mediated targeting of heterochromatin by the RITS complex." Science 303(5658): 672-676.
119. ↑ Sugiyama, T., et al. (2005). "RNA-dependent RNA polymerase is an essential component of a self-enforcing loop coupling heterochromatin assembly to siRNA production." Proceedings of the National Academy of Sciences of the United States of America 102(1): 152-157.
120. ↑ Noma, K.-i., et al. (2004). "RITS acts in cis to promote RNA interference–mediated transcriptional and post-transcriptional silencing." Nature genetics 36(11): 1174.
121. ↑ White, S. A. and R. C. Allshire (2008). RNAi-mediated chromatin silencing in fission yeast. RNA Interference, Springer: 157-183.
122. ↑ Thon, G., et al. (2005). "The Clr7 and Clr8 directionality factors and the Pcu4 cullin mediate heterochromatin formation in the fission yeast Schizosaccharomyces pombe." Genetics 171(4): 1583-1595.
123. ↑ Kurihara, Y. and Y. Watanabe (2004). "Arabidopsis micro-RNA biogenesis through Dicer-like 1 protein functions." Proceedings of the National Academy of Sciences of the United States of America 101(34): 12753-12758.
124. ↑ Park, W., et al. (2002). "CARPEL FACTORY, a Dicer homolog, and HEN1, a novel protein, act in microRNA metabolism in Arabidopsis thaliana." Current Biology 12(17): 1484-1495.
125. ↑ Kurihara, Y. and Y. Watanabe (2004). "Arabidopsis micro-RNA biogenesis through Dicer-like 1 protein functions." Proceedings of the National Academy of Sciences of the United States of America 101(34): 12753-12758.
126. ↑ Vazquez, F., et al. (2004). "Endogenous trans-acting siRNAs regulate the accumulation of Arabidopsis mRNAs." Molecular cell 16(1): 69-79.
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