Lecture 5 - RNAi - Biotechnology PDF

Lecture 5 Antisense RNA ( RNAi) BIOC 3260 Principles of Biotechnology Dr. Angela T. Alleyne 1 LEARNING OUTCOMES At the end of this lecture you will be able to: 1. Explain what is Antisense...

Lecture 5 Antisense RNA ( RNAi) BIOC 3260 Principles of Biotechnology Dr. Angela T. Alleyne 1 LEARNING OUTCOMES At the end of this lecture you will be able to: 1. Explain what is Antisense RNA 2. Describe the RNAi mechanisms and techniques used in vitro and in vivo 3. Discuss the role of gene silencing in cells 4. Describe the functions of Dicer 5. Explain the role of Argonaute and PIWI proteins 6. Discuss PTSG in plants 7. Review the use of gene silencing in biotechnology 8. Describe Antisense oligonucleotides and their uses 9. Consider bioethics of gene silencing applications in biotechnology 2 Gene structure A typical gene contains three main regions: a promoter, a 5ʹ untranslated region (5ʹ UTR), and an open reading frame (ORF). The 5ʹ UTR contains sequences and elements needed during translation but is not actually translated into protein. 3 Taken from Bioinformatics: An introduction 2nd edition Springer Selzer 4 et al RNA regulation Promoter regions of eukaryotic genes are more complex and contain more elements, e.g. initiator box, TATA box, and others that bind to transcription factors. Transcription factors and epigenetic changes contribute to the regulation of transcription The regulatory system for transcription and translation in both eukaryotes and prokaryotes rely on varying forms of RNA 5 This Photo by Unknown Author is licensed under CC BY  Antisense RNA A single strand of RNA that is complementary to mRNA forming a non- functional dsRNA strand In biotechnology can be used to artificially block protein synthesis ( translation) in the laboratory. Natural roles of antisense RNAs include: control of circadian rhythms in the fungus Neurospora, bacterial iron metabolism, HIV-1 gene expression, control of eukaryotic transcription factors, control of RNA editing, alternative splicing of certain genes, control of the bacterial ColE1 plasmid replication, and induction of heterochromatin formation. CAN YOU SUGGEST ANY OTHERS? 6  + - Antisense refers to the orientation of complementary strands during transcription. The two complementary strands of DNA are referred to as sense (= coding or plus) Non functional and antisense (= noncoding or minus Taken from: http://www.biology-pages.info/A/AntisenseRNA.gif 7  Antisense RNAs are synthesized using the coding strand (sense) as a template and are complementary to the mRNA. If antisense DNA binds to mRNA, the heteroduplex of RNA and DNA triggers RNase H to degrade the mRNA. Taken from Clark Biotechnology 8 Ribosome binding sites or splice junctions are blocked when Antisense DNA targets mRNA for Antisense and mRNA from a duplex- degradation no translation. Taken from Clark Biotechnology 9  Gene silencing Gene silencing – the process of degrading RNA into short RNA fragments that activate ribonucleases to target homologous mRNA. It was first observed in plants after introduction of an extra copy of an endogenous gene in plants. RNA silencing is a gene regulatory mechanism that limits transcription by: ̶ suppressing transcription (transcriptional gene silencing [TGS], or ̶ activating a sequence-specific RNA degradation process viz. (post transcriptional gene silencing [PTGS]/RNA interference [RNAi]). 9  RNA interference a biological process in microRNA (miRNA) and small interfering RNA (siRNA)—inhibit gene expression, usually by binding to messenger RNA (mRNA) and RNAi triggering its degradation. Used in Biotechnology to knock down gene expression in cell culture and in vivo in model organisms. 9  (in vivo) RNA interference ( RNAi) RNA interference (RNAi) ̶ mediates resistance to both endogenous parasitic and exogenous pathogenic nucleic acids, thereby protecting the genome against invasion by mobile genetic elements such as viruses and transposons ̶ Eukaryotes use RNA interference to protect the cell from RNA viruses by targeting double-stranded RNA. ̶ regulates the expression of protein-coding genes. ̶ RNA interference (RNAi) is present in many different organisms: plants, fungi, and many animals. ̶ GENE impedance (GENEi) is a new term that was coined to include all of these phenomena. 10  RNAi function/application RNAi has been used in C. elegans and Drosophila to in mammalian tissue is the determine the role of specific investigation of the specific proteins in the various stages roles of human proteins in of development. disease development. Curing some genetic diseases additionally, RNAi libraries aid in the identification of the role of unknown proteins in cells, where each clone in the library targets a specific protein in the cell and marks it 11 for silencing.  RNAi mechanism long dsRNA ( lncRNA- long noncoding RNA) is cut or "diced" into small fragments ~21 nucleotides long by an enzyme called "Dicer". Dicer transfers the siRNAs to the RNA-induced silencing complex (RISC). These siRNA then bind to a family of proteins called Argonaute proteins. 14  RNAi mechanism 1. "Dicer cuts or lncRNA into small fragments ~21 nucleotides 2. RISC, a 22–25 nt RNA component mediates sequence- specific RNA degradation in vitro. 3. RISC and a 130 kD protein component (Agronaute) are both required for sequence-specific RNA degradation 13 Taken from: https://i1.wp.com/farm6.static.flickr.com/5030/5815079788_5586b2faa0_o.png  RNAi mechanism After binding to an Argonaute protein, one strand of the lncRNA is removed, leaving the remaining strand available to bind to messenger RNA target sequences Once bound, the Argonaute protein can either cleave the messenger RNA, destroying it, or recruit other factors to regulate the target sequence. 12  Argonaute proteins Argonaute proteins are highly specialized binding modules that accommodate small RNA components such as microRNAs (miRNAs), short interfering RNAs (siRNAs) or PIWI-associated RNAs (piRNAs) , and coordinate downstream gene-silencing events by interacting with other protein factors Adapted from: https://images.nature.com/full/nature-assets/nrg/journal/v14/n7/images/nrg3462-f2.jpg 13 Argonaute protein structure in humans N terminus domain, Linkers, L1 and L 2, PIWI domain and a Mid domain 13  PIWI proteins Small RNAs regulate gene expression by guiding Argonuate (Ago) protein- containing complexes to different cellular sites for molecular action. Ago proteins have a variable N-terminal domain, a highly conserved PAZ domain, which together with the Mid-domain binds a small RNA. The C-terminal domain is called the PIWI domain Ago subfamily proteins bind microRNAs (miRNA) and small interfering RNAs (siRNA) 14  PIWI Interacting RNA (piRNA) PIWI ( P element induced wimpy testis) proteins bind PIWI-interacting RNAs (piRNAs). piRNAs are 25 to 33nt in length and are found in mammals in male germ cells and stem cells PIWI-interacting RNAs (piRNAs) protect germ cells from transposons in organisms as diverse as flies, fish or mammals. Biogenesis of piRNAs is independent of Dicer piRNAs guide PIWI proteins to complementary RNAs derived from transposable elements PIWI proteins cleave the transposon RNA, leading to silencing PIWI proteins maintain fertility by being involved in gametogenesis 15  DNA silencing is initiated by biogenesis of small RNAs from host precursors that are then bound by effector proteins which lead to transcriptional silencing or elimination of complementary sequences. Dicer- green RNA complexes- Blue Polymerase- Brown AGO yellow Piwi- purple Histones orange DNA methylase grey Taken from : http://www.cell.com/molecular- cell/pdf/S1097-2765(13)00138-X.pdf 16 ̶ Antisense oligonucleotides in vitro: In Vitro synthesis ̶ similar to that for constructing DNA primers for PCR applications, except with the use of ribonucleotides (less stable than deoxynucleotides) ̶ Modifications are made to increase the stability of RNA oligonucleotides include: ̶ use of peptides with attached nucleic acid bases as the backbone of the molecule The core region maintains RNase H (peptide nucleic acids, or PNAs), sensitivity, whereas the outer regions are RNase H resistant ̶ substitution of a sulfur for the oxygen in the phosphate groups (phosphorothioate oligonucleotide ), and ̶ modifications to the five-carbon ribose (O -alkyl group to the 2ʹ-OH of the ribose makes the oligonucleotide resistant to DNase and RNase H degradation. 17 Modifications are made to increase the stability of RNA oligonucleotides 18 Antisense oligonucleotides Single stranded oligonucleotides 21-24 nt long complementary to specific to mRNA Two types: 1. Occupancy mediated degradation- bind and cleave their RNA targets by using endogenous enzymes e.g Rnase H 2. Occupancy only- ASO can regulate the RNA transcripts without enzymes- stearic hinderance by alteration of splicing 1 ASO therapy 1 Antisense oligonucleotide therapies 1 Some Approved ASO drugs Drug Name Target Fomivisrsen CMV retinitis Nusinersen Muscular dystrophy Volanesorsen Dystrophin (liver) 27  In vitro synthesis Cloned into a vector (MCS) to give an antisense RNA upon transcription. The cloned gene can then be transformed into target cells and expressed into antisense RNA. The target antisense RNA is made only in the presence of specific signals or conditions. The template DNA is transcribed by a T7, T3 or SP6 RNA phage polymerase in the presence of ribonucleoside triphosphates (rNTPs). The polymerase traverses the template strand and uses base pairing with the DNA to synthesize a complementary RNA strand (using uracil in the place of thymine). 19  In vitro synthesis of antisense RNA The RNA polymerase travels from the 3ʹ → 5ʹ end of the DNA template strand, to produce an RNA molecule in the 5ʹ → 3ʹ direction. Taken from Clark Biotechnology 20  Delivery into cells Uptake of the oligonucleotides is mediated by endocytosis. Encapsulation of the oligonucleotides into liposomes, either within the liposome or attached to the surface To help prevent degradation, the oligonucleotides are attached to basic peptides. Attaching the oligonucleotides to basic peptides that function as transcription factors ensures delivery straight to the nucleus. Taken from Clark Biotechnology 21  Post transcriptional Gene silencing (PTGS) RNA interference (RNA i) also called Post-Transcriptional Gene Silencing ( PTGS) ̶ introduction of transgenes or double-stranded RNA (dsRNA) into a variety of hosts can trigger post-transcriptional silencing of all homologous host genes and/or transgenes ̶ PTGS results in the specific degradation of a population of homologous RNAs ̶ PTGS greatly reduces mRNA accumulation in plant cytoplasm but does not affect transcription ̶ PTGS in plants have three steps: initiation, propagation and maintenance 22  PTGS in plants PTGS is a viral defence mechanism in plants o In plants, mutants defective in PTGS are more susceptible to viral infection o viruses that infect plants encode a number of factors that can suppress PTGS PTGS in plants requires at least two genes – 1. SGS3 (which encodes a protein of unknown function containing a coil-coiled domain) 2. MET1 (which encodes a DNA-methyltransferase) – that are absent in RNAi in animals. PTGS in plants and RNAi in animals may be derived from a similar ancestral mechanism. 23  Micro RNA biotechnology microRNAs (miRNA) can guide Argonaute proteins to repress messenger RNAs that match the miRNA incompletely, allowing one miRNA to regulate hundreds of genes. Humans make more than 500 distinct miRNAs, and the inappropriate production of specific miRNAs has been linked to several diseases. Drugs to inhibit disease-causing miRNAs are now being tested as therapies for several human diseases e.g. cancer, Huntington disease. RNAi can be activated in mammalian cells by short hairpin RNAs ( shRNAs ) that mimic the structure of miRNAs 24  Gene silencing occurs following siRNA biotechnology interaction of the RNA effector precursors with the RNase III enzymes Drosha (for miRNA) and Dicer (for miRNA and siRNA) and subsequent formation of the RNA-interfering silencing complex (RISC) Biotech applications of silencing occur through synthesis of : 1. small interfering RNA (siRNA) ,or 2. vector based short hairpin RNA (shRNA)- o synthesized in the nucleus of cells o Bound to RISC for activity siRNA and shRNA pathways converge at the RISC complex o processed in the nucleus by a complex containing the RNase III enzyme Drosha shRNAs, as opposed to siRNAs, are, further processed and transported to the cytoplasm, and the incorporated into the RISC for activity. Taken from: 25 https://translationalneurodegeneration.biomedcentral.com/articles/10.1186/s40035-017-0101-9#Fig2  Engineering plant viruses Plant viruses can be engineered to produce dsRNA in plant tissues at high levels to cause gene silencing and mortality in insects after feeding. For example: construction of recombinant Tobacco mosaic virus (TMV) that produces RNAs in sense or antisense orientation targeting actin, chitin synthase 1, of the insect pest Planococcus citri that were initially inoculated to Nicotiana benthamiana (tobacco) plants through agroinfiltration. 26  This method was successful in causing silencing of the insect vector that is known to facilitate the transmission of Grapevine leafroll associated virus 3(Khan et al., 2013). The RNAi effects in the insect vectors were not caused by plant virus replication within the vectors, but by the large amounts of dsRNA produced in the plant cells. 27  Ribozyme action Ribozymes are catalytic RNA molecules or RNA-enzymes 28  Ribozymes Ribozymes are naturally occurring and have been exploited for medical and industrial applications. 29  Ribozymes Functions: the self-removal of introns from an mRNA with the help of snRNAs, replication of some viruses and sub-viral agents (viroids and satellite viruses), cleavage plus ligation of single-stranded RNA molecules, and modification of rRNA ribonucleotides by snoRNAs (small nucleolar RNAs). 30  Cleavage site Two main categories small e.g hairpin ribozymes and large ribozymes e.g RNase P and Group I introns Nature Publishing Group 31  Ribozyme biotechnology New ribozymes can be engineered in vitro by mixing random ribozyme sequences with known target RNA molecules for binding. In vitro evolution adds a mutagenesis step after each selection cycle, thus diversifying the outcome. Several engineered ribozymes are being used to inhibit replication of human immunodeficiency virus (HIV). an engineered ribozyme can be used for the treatment for viruses, diseases, or cancer because it is specific to a target mRNA. 32  Ethical considerations RNA interference can control normal gene expression and has recently begun to be employed as a potential therapeutic agent. Ethically suitable actions are those that do the greatest good for the greatest number of persons , nonmaleficence ( do no harm- intentionally refraining from actions that cause harm) and beneficence ( promoting good- prevent harm, remove harm and promote good) A risk-benefit analysis should consider: 1. Specificity of silencing effect at target site/s 2. toxicity and 3. Safety and efficacy of in vitro and in vivo delivery methods https://geneticliteracyproject.org/2018/08/28/gene-silencing-through-rna-interference-scores-first-drug- approval/ https://www.onpattro.com/ 33  References: Clark, D. and Pazdernik, N. J. 2016. Biotechnology. Elsevier http://www.cell.com/molecular-cell/pdf/S1097-2765(13)00138- X.pdf http://www.nature.com/nrg/multimedia/rnai/animation/index.h tml?foxtrotcallback=true https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4234069/#R97 34