RNA folding and its importance. Mitesh Shrestha

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1 RNA folding and its importance Mitesh Shrestha

2 Diseases Caused due to Protein Misfolding Alzheimer s Disease Parkinson s Disease Cataracts Sickle Cell Disease Prion Diseases Cystic Fibrosis

3 Ribozymes Ribonucleic Acid Enzymes Catalyze phosphate-group transfer and peptidebond formation Ribonuclease P

4 Factors influencing RNA folding (Thirumalai and Hyeon., (n.d.)) In the cell RNA folding is likely to be influenced by the speed and directionality of transcription and, in prokaryotes, of translation. In addition, bimolecular interactions with other RNA molecules, proteins, metabolites and ligands (i.e., polyamines) as well as ion homeostasis play a key role in successful folding of a functional transcript.

5 Problems associated with RNA folding (Zemora and Waldisch., 2010) In principle, RNA encounters two major folding problems: (i) RNA molecules are prone to misfold, thereby becoming trapped in inactive, often long-lived conformations, the escape from which becomes rate limiting during the folding process; (ii) the native, functional RNA conformation might not be thermodynamically favored over other intermediate structures, thus requiring the assistance of a specific RNA-binding protein (or high salt) for stabilization of the tertiary structure.

6 RNA Viruses Rota Virus Hepatitis A Bird Flu (H5N1) Swine Flu (H1N1) HIV Ebola

7 RNA as hereditary material Double stranded RNA (Rota Virus) Single stranded +ve Sense RNA (Picorna Virus) Single stranded ve sense RNA (Influenza Virus) Retroviruses (HIV)

8 Stability of RNA genomes High genetic variability and rapid evolution (Combe and Sanjuan., 2014) High mutation rate (Domingo and Holland., 1997) Lack of proofreading activity of RNA virus polymerases (Elena and Sanjuan., 2005)

9 RNA interference (Hammond., 2005) Andrew Fire and Craig C. Mello shared the 2006 Nobel Prize in Physiology or Medicine for their work on RNA interference in the nematode worm Caenorhabditis elegans, which they published in RNA-dependent gene silencing process that is controlled by the RNA-induced silencing complex (RISC) and is initiated by short double-stranded RNA molecules in a cell's cytoplasm, where they interact with the catalytic RISC component argonaute. Could be endogenous (mirna or sirna) or exogenous (dsrna)

10 Phenomena first observed in petunia Attempted to overexpress chalone synthase (anthrocyanin pigment gene) in petunia. (trying to darken flower color) Caused the loss of pigment.

11 RNA interference (Contd.)

12 RNA interference (Contd.)

13 RNA interference (Contd.) RNA induced Silencing Complex

14 RNA interference (RISC) The RNA-induced silencing complex, or RISC, is a multiprotein complex, specifically a ribonucleoprotein, which incorporates one strand of a double-stranded RNA (dsrna) fragment, such as small interfering RNA (sirna) or microrna (mirna). The single strand acts as a template for RISC to recognize complementary messenger RNA (mrna) transcript. Once found, one of the proteins in RISC, called Argonaute, activates and cleaves the mrna.

15 RISC-loading complex The RISC-loading complex (RLC) is the essential structure required to load dsrna fragments into RISC in order to target mrna. The RLC consists of dicer, the human immunodeficiency virus transactivating response RNAbinding protein (TRBP) and Argonaute 2. Dicer is an RNase III endonuclease which generates the dsrna fragments to be loaded that direct RNAi. TRBP is a protein with three double-stranded RNA-binding domains. Argonaute 2 is an RNase and is the catalytic centre of RISC. Dicer associates with TRBP and Argonaute 2 to facilitate the transfer of the dsrna fragments generated by Dicer to Argonaute 2. More recent research has shown the human RNA helicase A could help facilitate the RLC

16 The RISC-loading complex allows the loading of dsrna fragments (generated by Dicer) to be loaded on to Argonaute 2 (with the help of TRBP) as part of the RNA interference pathway.

17 RNA interference (RISC Contd.) Functions: Loading of dsrna Gene regulation mrna degradation Translational repression Heterochromatin formation DNA elimination

18 RNA interference (Transcriptional Silencing) Components of the RNAi pathway are used in many eukaryotes in the maintenance of the organization and structure of their genomes. Modification of histones and associated induction of heterochromatin formation serves to down regulate genes pre-transcriptionally; this process is referred to as RNA-induced transcriptional silencing (RITS), and is carried out by a complex of proteins called the RITS complex.

19 RNA interference (Transcriptional Silencing) The RITS complex in S. pombe contains at least a piwi domain-containing RNase H-like argonaute, a chromodomain protein Chp1, and an argonaute interacting protein Tas3 which can also bind to Chp1, while heterochromatin formation has been shown to require at least argonaute and an RNAdependent RNA polymerase. Loss of these genes in S. pombe results in abnormal heterochromatin organization and formation of the chromosome centromeres, resulting in slow or stalled anaphase during cell division. Not applicable in Mammalian Cells

20 RNA interference (Summary)

21 RNA interference (Contd.): Applications Gene Knockdown Functional Genomics Medicine Antiviral Cancer Biotechnology Food Other crops Transgenic plants Genome-scale screening

22 Summary RNA folding and importance RNA as a genetic material Stability of RNA genome Introduction to RNAi, Mechanism and Applications

23 Readings Thirumalai, D., & Hyeon, C. (n.d.). Theory of RNA Folding: From Hairpins to Ribozymes. Non-Protein Coding RNAs Springer Series in Biophysics, Molecular Cell Biology,4 th Ed., Section 6.3 Zemora, G., & Waldsich, C. (2010). RNA folding in living cells. RNA Biology, 7(6), Domingo, E., & Holland, J. J. (1997). Rna Virus Mutations And Fitness For Survival.Annual Review of Microbiology Annu. Rev. Microbiol., 51(1), Combe, M., & Sanjuán, R. (2014). Variation in RNA Virus Mutation Rates across Host Cells. PLoS Pathog PLoS Pathogens, 10(1). Elena, S. F., & Sanjuan, R. (2005). Adaptive Value of High Mutation Rates of RNA Viruses: Separating Causes from Consequences. Journal of Virology, 79(18), Hammond, S. M. (2005). Dicing and slicing. FEBS Letters, 579(26),

24 Assignments How, on early earth, was it possible for replication to occur in the absence of protein enzyme? [2.5] Why RNA folding is a difficult problem? [2.5] How is RNA helpful as a genetic material? [2.5] Explain in detail about RNA interference. [7.5] Differentiate between RNAi and Antisense technology. [2.5]

25 Central Dogma Upcoming Lecture Accessing of genome, Assembly of the transcription initiation complex synthesis of RNA, Processing of RNA, RNA degradation Assembly of the translation initiation complex, Protein synthesis, protein folding and protein processing, protein degradation