REVIEW ARTICLE

RNA Interference: An ISSN No 2230-7885 CODEN JPBSCT Evolutionary Pathway NLM Title J Pharm Biomed Sci in Periodontal Tissue Swyeta Jain Gupta1,*, Amit Gupta2, Engineering Vivek Gautam3, Kaushik Kumar Pandey4, ABSTRACT Swati Choudhary5 RNA interference (RNAi) is an evolutionary conserved silencing pathway in which the 1  double-stranded RNA is broken down into small interfering RNA (siRNA) with the help of Senior Lecturer, Department of and RNA-induced silencing complex (RISC) complex by a series of steps. Pioneering Periodontics and Implantology, I.T.S. observations on RNAi were reported in plants, but later on RNAi-related events were Centre for Dental Studies and Research, described in almost all eukaryotic , including , flies, , Ghaziabad, Uttar Pradesh, India insects, parasites, and mouse and human cell lines. 2 Senior Lecturer, Department of Oral and Maxillofacial Pathology and Microbiology, KEYWORDS , RNAi, DICER, RISC I.T.S. Dental College, Hospital and Research Centre, Greater Noida, Uttar Pradesh, India INTRODUCTION 3 Prosthodontics (Private practitioner), Gautam Multispeciality Dental Clinic, The RNA-mediated silencing process is defined as RNAi, a discovery for P N Plaza Complex, Sigra, Varanasi, which Fire and Mellow received the 2006 Nobel Prize1. RNA interference Uttar Pradesh, India (RNAi) is a phenomenon primarily described as interference by RNA in the 4 Department of Prosthodontics Career expression of genetic information. It is a phenomenon primarily for the reg- Postgraduate Institute of Dental ulation of ; self or non self depending upon the surrounding and Hospital, Lucknow, Uttar Pradesh, India factors or conditions, with the help of RNA that are non coding 5 Oral and Maxilllofacial Pathology, in to control cellular metabolism and help in maintaining genomic Delhi, India 2 integrity by preventing the invasion of and mobile genetic elements .  Address reprint requests to The discovery of RNAi has opened the door to RNA-based therapeutics that *Dr. Swyeta Jain Gupta, Senior Lecturer, may prove to be an effective tool for the treatment of a large variety of dis- Department of Periodontics and Implantology, eases and for tissue regeneration, including periodontal tissue regeneration3. I.T.S. Centre for Dental Studies and RNA silencing a novel gene regulatory mechanism limits the transcript level Research, Ghaziabad, Uttar Pradesh, India E-mail: [email protected] by either suppressing (transcriptional gene silencing [TGS]) or by activating a sequence-specific RNA degradation process (posttran-  Article citation: Gupta SJ, Gupta A, scriptional gene silencing [PTGS]/RNA interference [RNAi]). RNAi works Gautam V, Pandey KK, Choudhary S. RNA interference: an evolutionary pathway in through small of approximately 20 to 30 . Although there periodontal tissue engineering. J Pharm is a mechanistic connection between TGS and PTGS, TGS is an emerging Biomed Sci 2016;06(01):39–43. 4,5 field while PTGS is undergoing an explosion in its information content . Available at www.jpbms.info RNA interference and its focus on the experimental results related to tissue 6 Statement of originality of work: The engineering have led to conceptual advancements in this field . manuscript has been read and approved by all the authors, the requirements for authorship have been met, and that each author believes that the HISTORY manuscript represents honest and original work. RNA molecules were earlier believed to serve only as messengers, bearing Source of funding: None. genetic information from DNA. But in the early 1980s it was revealed in Competing interest / Conflict of interest: The Escherichia coli that small RNA molecules (about 100 nucleotides in length) author(s) have no competing interests for financial can bind to a complementary sequence in mRNA and inhibit transla- support, publication of this research, patents, tion7. This inactivates mRNA and therefore prevents it from passing on and royalties through this collaborative research. the genetic information. The phenomena of RNAi first came into notice All authors were equally involved in discussed in the year 1990, when plant scientists were trying to intensify the hue research work. There is no financial conflict with the subject matter discussed in the manuscript. of red petunias for commercial benefits. Rich Jorgensen introduced a gene that controls the formation of red pigments in petunia but Disclaimer: Any views expressed in this in some cases it was observed that the colour disappeared altogether. The paper are those of the authors and do not reflect the official policy or position of the phenomenon was named as co-suppression. Co-suppression is a classical Department of Defense. form of eukaryotic post-transcriptional gene silencing. The reason behind such an observation was not clear at that time until it was reported in

Copyright © 2016

Received Date: 12 December 2015 – Accepted Date: 19 December 2015 – Published Online: 16 January 2016 40 Swyeta Jain Gupta transgenic petunia, where a sense meant to RNA (siRNA) and the -specific systemic trans- overexpress the host -A (CHS-A) gene mission of silencing from its site of initiation. caused the degradation of the homologous transcripts and the loss of pigmentation. Subsequently such MECHANISM OF RNA INTERFERENCE phenomenon was also observed in fungi, which was termed as quelling7,8. In animals, RNA silencing was Mechanism of RNA interferences as understood is first reported when Guo and Kemphues9 were attempt- that it comes into play when a double stranded RNA ing to use antisense RNA to shut down expression of is introduced either naturally or artificially in a cell. the par-1 gene in order to assess its function. More An endoribonuclease enzyme cleaves the long dsRNA recently, micro-RNA formation, heterochromatiniza- into small pieces of RNA. The small pieces could be tion, etc., have been revealed as other facets of natu- miRNA or siRNA depending upon the origin of long rally occurring RNAi processes of eukaryotic cells. As dsRNA i.e. endogenous or exogenous, respectively. A expected, injection of the antisense RNA disrupted double-stranded RNA may be generated by either RNA expression of par-1, but quizzically, injection of the dependent RNA polymerase or bidirectional transcrip- sense-strand control did too used antisense RNA to tion of transposable elements or physically introduced. block par-1 mRNA expression in but The mechanism of RNA interference can be divided found that the par-1 mRNA itself also repressed par-1. into two stages: (1) initiation and (2) effector. Their paradoxical observation—subsequently dubbed RNA interference (RNAi)—inspired the experiments Initiation of Fire, Mellow, and colleagues10,11. Fire et al.12 studied on the gene function especially This stage is characterised by the generation of siRNA on the gene responsible for movement in C. elegans. When mediated by type III Dicer. In , this nematods were injected with the mixture of sense Dicer, which is a large multidomain RNase III enzyme and antisense RNA, nematodes were observed with the has been identified in existence into two forms: Dcr-1/ impaired movement suggesting a defective muscle gene Loquacious (Loqs)-PB (also known as R3D1-L[long]): protein. They concluded that the dsRNA injected must generate miRNA and Dcr-2/R2D2 generates siRNA14,15. match the mature “trimmed” mRNA sequence for the Dcr-1 share a structural with Dcr-2, despite gene and the interference could not be elicited by that they display different sets of properties such as sequences. This implies that interference takes place after ATP requirements and substrate specifications. Dcr-1 is transcription, probably in the rather than in an enzyme that shows ATP independent functions and the cell nucleus12. affinity towards stem-loop form of RNA (precursor of During the occurrence of RNAi/PTGS, the mRNA miRNA). It has been identified that Dcr-1 requires a was revealed to be targeted with large complex double-stranded RNA binding protein partner14,16. In proteins to form RNA induced silencing complex, Drosophila Dicer-1 is seen to interact with the dsRBD commonly called RISC; in association with si RNA. protein Loquacious (Loqs). Immunoaffinity purifica- Subsequently, it has been shown that RISC contains tion experiments reveal that Loqs resides in a functional at least one member of the protein family, pre-miRNA processing complex, and stimulates and which is likely to act as an endonuclease and cut the directs specific pre-miRNA processing activity. These mRNA (often referred to as the Slicer function). It results support a model in which Loqs mediates miRNA was also demonstrated that a like nucle- biogenesis and, thereby, the expression of genes regu- ase, called Dicer, is responsible for the processing of lated by miRNAs. Loquacious protein is supposed to be dsRNA to short RNA. In certain systems, in particu- composed of three dsRNA binding domains, which is lar plants, worms and fungi, an RNA dependent RNA encoded by loqs gene into two types of proteins PA and polymerase (RdRP) plays an important role in gener- PB; isoform to each other, by alternate splicing, among ating and/or amplifying siRNA13. which PB is known to enhance the affinity of Dcr-1 towards pre-miRNA. Dcr-2 shows ATP dependent activ- IMPORTANT FEATURES OF RNA SILENCING ity with substrate specificity to double stranded RNA. Structurally homologous to Dcr-1, also requires a The critical common components of the paradigm double-stranded RNA binding protein, namely R2D2, are that which function in association with specific RNase enzyme Dcr-2 forming a heterodimeric complex. i. the inducer is the dsRNA, R2D2, unlike Loq is supposed to be composed of two ii. the target RNA is degraded in a homology dsRNA binding domains that interact with long double dependent fashion, and the degradative machinery stranded RNA but does not regulate siRNA generating requires a set of proteins which are similar in activity of Dcr-2. Studies of the physiological functions structure and function across most organisms. of R2D2 by the design of mutant strain have shown In most of these processes, certain invariant features are that R2D2 protein plays an important in develop- observed, including the formation of small interfering ment. It has also been shown to be responsible for the

J Pharm Biomed Sci | Vol. 06 No. 01 | 39–43 RNA interference 41 stability of Dcr-2 and decrease in the concentration of (AaAgo) was determined and further R2D2 in cells reduces the concentration of Dcr2 and confirmed the relationship to RNase H. Among the four vice versa. These results indicate that both dsRNA bind- domains of Ago proteins, Eukaryotic Ago proteins are ing domains of Dcr-2 and R2D2 are critical for Dcr-2/ found to be characterised by two functional domains R2D2 complex to bind and load siRNA into siRISC PAZ and . complex. Dcr-2 contains an RNA domain, a The RNA interference model has taken a shape as; DUF283 domain and a PAZ domain at the N terminus Dcr-2/R2D2 binds with siRNA, it forms RDI com- as well as tandem RNase III motifs and a dsRBD motif plex (R2D2 Dcr Initiator complex). RDI orients siRNA at the C terminus. It is unclear which of these domains duplex so that the 5 prime ends with lower melting physically contact siRNA. Since neither Dcr-2 nor R2D2 temperature preferentially interacts with Dcr-2 the less bind siRNA alone, it is possible that siRNA is bound at stable end, R2D2 binds the more stable end of siRNA21. the interface between Dcr-2 and R2D2, which triggers This leads to thermodynamic asymmetry which helps a conformational change in either or both proteins, the separation of guide strand from the passenger strand allowing them to bind siRNA cooperatively17. (other strand). Studies have shown that small change in the structure of siRNA effects on determination of the Effector guided strand of the RISC complex which concludes, enzyme that governs the RISC assembly selects the siRNA The second step of RNA interference mechanism is strand incorporated into RISC on the basis of struc- referred as effectors step; the incorporation of guide ture22. Also the orientation of Dicer2/R2D2 on siRNA strand. This is characterized by the formation of silenc- determines the guide strand. It suggests that structure ing complex. siRNA determines the orientation of Dcr2/R2D2 which Single strands of siRNA and miRNA duplexes, in turn determines the strand that will be incorporated referred to as guide strands, are incorporated into RNA into RISC as guide strand. Ago2-dependent complex, silencing effecter complexes such as the RNA-induced pre-RISC, was identified and Ago2 was found to be silencing complex (RISC), or the RNA-induced initia- responsible for conversion of pre-RISC to holo-RISC i.e. tion of transcriptional gene silencing (RITS) complex. the effective removal of passenger strand. The pre-RISC complex contains duplex siRNA whereas holo-RISC 1. RNA induced silencing complex (RISC) cleaves m contains just the guide strand of the siRNA, the passen- RNA or represses their by homology ger strand having dissociated from holo-RISC. 5 prime dependent m-RNA degradation. end of siRNA acts as a target recognition guide which is 2. RNA-induced initiation of transcriptional gene required for the efficient RNA interference mechanism. silencing complex (RITS) regulates heterochro­ Here the PIWI domain of the catalytic subunit of RISC matin assembly. (holo-RISC), Ago-2, acts as RNase H (highly conserved The core of these RNA silencing effecter complexes, in ) and cleaves the target m-RNA backbone. RISC was found to be composed of PPD proteins (PAZ It has been observed that a small induction was PIWI domain proteins) which are highly conserved enough for silencing a particular gene in the whole super-family. Members of PPD proteins contains cen- organism. This indicates that RNA interference silenc- trally located PAZ (100 amino acids) and C-terminal ing mechanism could amplify its signal intensity. This located PIWI (300 amino acids). amplification mechanism was termed as transitivity; Subsequently, it was revealed that the PPD protein siRNA bearing new homology sequences (upstream/ in RISC was Ago proteins. Ago proteins are members downstream) of the target zone are synthesised. These of PPD protein super-family. Not all PPD proteins are newly synthesised siRNA were used to destroy the target. known to perform the same task in all systems but when A role of RNA dependent RNA polymerase (RdRP) was studies were performed and compared the number of assumed in this mechanism. The siRNA derived in RNA PPD in different systems; Nematodes were found to have interference pathway may have two different fates, either more specific function of PPD than others. it may be converted into holo-RISC for the destruction The purification of complete eukaryotic Ago protein of the target m-RNA, or it may contribute to the ampli- was difficult so experiments were performed with their fication step by the generation of secondary siRNA. Two prokaryotic homolog which revealed that it has a struc- models were proposed as the mode of its action for the ture similar to RNase H ribonuclease18. The Ago protein generation of secondary siRNA. (1) RdRP generates the was found to be comprised of four major domains: newly synthesised siRNA directly. (2) RdRP use siRNA N-terminal, PAZ domain, middle and PIWI domain. The as a template for the production of long dsRNA, which purification and study of RISC revealed that it consist of are then cleaved into siRNA. It was found that the newly Ago proteins in a major concentration which was sus- synthesised RNA was of 24–26 nucleotides long instead pected to be the catalytic subunit part of this protein19. of 20–22 nucleotides. The reason behind this is unex- Structure directed mutagenesis experiments implicate plained. The newly synthesised siRNA again joins the Ago protein as the catalytic subunit of RISC20. A crystal cycle for the production of long double stranded RNA structure of a full-length eubacterial Ago protein from and sustains the amplification process23.

J Pharm Biomed Sci | Vol. 06 No. 01 | 39–43 42 Swyeta Jain Gupta

RNA induced silencing of new approaches. Third-generation periodontal therapies involve nanoscale science and moldless manufacturing technology commonly known as rapid prototyping (RP or solid free-form fabrication). These scientific and technologic innovations will make it possible to fabri- cate complex scaffolds that mimic the different struc- tures and physiologic functions of natural fibroosseous tissues, including those, such as periodontium, which consist of hard and soft tissues. The advancement of such technology might also make it possible to produce patientspecific cell-scaffold constructs with optimal distribution of cells and high vascular permeability24.

CONCLUSION AND FUTURE PROSPECTS A conserved biological response to double-stranded RNA, known variously as RNA interference (RNAi) or post-transcriptional gene silencing regulates the expres- sion of protein-coding genes. RNAi has been cultivated as a means to manipulate gene expression experimen- tally and to probe gene function on a whole- scale. As we gain more insight into the mechanisms, more effective methods for analysis of gene functions may evolve. The knockdown technology might improve vastly with better-designed - or -based vec- tors for delivery of siRNAs to the appropriate tissues at the appropriate time. Such technology is bound to give a new shape to therapeutic gene silencing as well. The science and technology of RNAi finally, has not only provided us with a powerful new experimental tool to study the function of genes but also has given us a cultural ocean of virtually bottomless depth for array of APPLICATION OF RNA INTERFERENCE IN applications related to tissue engineering. PERIODONTAL REGENERATION REFERENCES The first siRNA-based therapeutic tested in human clin- ical trials was the VEGF-targeted RNA for the treatment 1. Intini G. Future approaches in periodontal regeneration: gene of of the retina. As RNAi emerges therapy, stem cells and RNA interference. Dent Clin North Am. as a promising technology for the treatment of several 2010 Jan;54(1):141–55. diseases, one may also envision RNAi to be an effec- 2. Slavkin HC, Bartold PM. Challenges and potential in tissue engi- tive tool for tissue regeneration, including periodontal neering. Periodontol 2000. 2006;41:9–15. 3. Timmons L, Fire A. Specific interference by ingested dsRNA. regeneration. Nature. 1998 Oct 29; 395(6705):854. Through the silencing of genes that negatively con- 4. Baulcombe DC. Fast forward based on virus induced trol cell proliferation and cell differentiation or genes gene silencing. Curr Opin Plant Biol. 1999 Apr;2(2):109–13. that induce inflammation or apoptosis, RNAi may favour 5. Bender J. A vicious cycle: RNA silencing and DNA methylation in tissue regeneration. Cathepsin B–targeted siRNA also plants. Cell. 2001 Jul 27;106(2):129–32. prevents chondrocytes from dedifferentiation, provid- 6. Hammon SM, Caudy AA, Hannon GJ. Post-transcriptional ing a potential tool to prevent cartilage degeneration. gene silencing by double-stranded RNA. Nat Rev Genet. 2001 Tumor necrosis factor-α-targeted siRNA can suppress Feb;2(2):110–9. osteolysis induced by metal particles in a murine cal- 7. Cogoni C, Macino G. Conservation of transgene-induced varia model, opening the way to the application of post-transcriptional gene silencing in plants and fungi. Trends RNAi in orthopaedic and dental implant therapy. In Plant Sci. 1997;2:438–43. 8. Romano N, Macino G. Quelling: transient inactivation of gene terms of bone regeneration, Gazzerro and colleagues expression in by transformation with homol- have demonstrated that downregulation of Gremlin ogous sequences. Mol Microbiol. 1992 Nov;6(22):3343–53. by RNAi in ST-2 stromal and MC3T3 osteoblastic cells 9. Guo S, Kemphues KJ. par-1, a gene required for establish- increases the BMP-2 stimulatory effect on disciplines ing polarity in C. elegans , encodes a putative Ser/ in which the introduction of biologic and engineer- Thr kinase that is asymmetrically distributed. Cell. 1995;81: ing knowledge and skills are allowing the development 611–20.

J Pharm Biomed Sci | Vol. 06 No. 01 | 39–43 RNA interference 43

10. Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC. 17. Xiang L, Feng J, Savitha K, Dean S, Qinghua L. Dicer-2 and R2D2 Potent and specific genetic interference by double-stranded coordinately bind siRNA to promote assembly of the siRISC RNA in C. elegans. Nature. 1998;391:806–10. complexes. RNA. 2006;12(8):1514–20. 11. Bernstein E, Caudy A, Hammond SM, Hannon GJ. Role for a 18. Song JJ, Smith SK, Hannon GJ, Joshua-Tor L. Crystal structure of bidentate ribonuclease in the initiation step of RNA interfer- Argonaute and its implications for RISC slicer activity. Science. ence. Nature. 2001 Jan 18;409(6818):363–6. 2004;305:1434–7. 12. A, Si Qun Xu, Mary K. Montgomery, Steven A. 19. Martinez P, Amemiya CT. Genomics of the HOX gene clus- Kostas, Samuel E. Driver and Craig C. Mello. Potent and specific ter. Comp Biochem Physiol B Biochem Mol Biol. 2002;133(4): genetic interference by double-stranded RNA in C. elegans. 571–80. Nature. 1998, 391: 806–810. 20. Liu J, Carmell MA, Rivas FV, Marsden CG, Thomson JM, Sung JJ, 13. Montgomery MK, Xu S, Fire A. RNA as target of double-stranded et al. Argonaute 2 is the catalytic engine of mammalian RNAi. RNA-mediated genetic interference in Caenorhabditis elegans. Science. 2004;305:1437–41. Proc Natl Acad Sci U S A. 1998 Dec 22;95(26):15502–7. 21. Kim K, Lee YS, Carthew RW. Conversion of pre-RISC to holo- 14. Jiang F, Ye X, Liu X, Fincher L, McKearin D, Liu Q. Dicer-1 and RISC by Ago2 during assembly of RNAi complexes. RNA. R3D1-L catalyze microRNA maturation in Drosophila. Genes 2007;13(1):22–9. Dev. 2005;19:1674–9. 22. Khvorova A, Reynolds A, Jayasena SD. Functional siRNAs and 15. Forstemann K, Tomari Y, Du T, Vagin VV, Denli AM, Klattenhoff miRNAs exhibit strand bias. Cell. 2003;115(2):209–16. BC, et al. Normal microRNA maturation and germ-line stem 23. Hamilton A, Voinnet O, Chappell L, Baulcombe D. Two classes cell maintenance requires Loquacious, a double stranded RNA- of short interfering RNA in RNA silencing. The EMBO J. 2002; binding domain protein. PLoS Biol. 2005;3:e236. 21(17):4671–9. 16. Saito K, Ishizuka A, Siomi H, Siomi MC. Processing of pre-­ 24. Nakahara T. A review of new developments in tissue engi- by the Dicer-1-Loquacious complex in Drosophila neering therapy for periodontics. Dent Clin N Am. 2006;50: cells. PLoS Biol. 2005 Jul;3(7):e235. 265–76.

J Pharm Biomed Sci | Vol. 06 No. 01 | 39–43