BCH Graduate Survey of Biochemistry

Transcription

1 BCH 5045 Graduate Survey of Biochemistry Instructor: Charles Guy Producer: Ron Thomas Director: Glen Graham Lecture 30 Slide sets available at:

2 Gene Silencing by RNAi Initially what has become known as PTGS or interference RNA (RNAi) was a unusual genetic phenomenon observed in petunias and a few other plants. In the early 1990s, Carolyn Napoli, Rich Jorgensen and colleagues introduced a pigment-producing gene under the control of a strong promoter hoping to get more intense flower color. However, many of the flowers appeared variegated or even white. The phenomenon was termed "cosuppression", since the expression of both the introduced gene and the homologous endogenous gene were suppressed. Similarly, introduction of a viral gene for the TEV virus coat protein resulted in specific resistance to viral infection in transgenic plants. Lindbo et al., 1993 proposed that the resistant state and reduced steady state levels of transgene transcript accumulation were mediated at the cellular level by a cytoplasmic activity that targets specific RNA sequences for inactivation.

3 Carolyn Napoli received both her B.S. and Ph.D. degrees in microbiology from the University of Florida. What caused this gene silencing, in part, was a form of silencing that occurs at the post-transcriptional level (thus post-transcriptional gene silencing, or PTGS). Once activated, PTGS is mediated by a diffusible, trans-acting molecule. It is now known that the trans-acting factor responsible for PTGS in plants and other organisms is dsrna. The first evidence that dsrna could lead to gene silencing came from work in the nematode Caenorhabditis elegans and later was extended to plants. A key finding by Hamilton and Baulcombe in 1999 provided the first clue.

4 They identified RNAs of ~25 nucleotides in plants undergoing cosuppression that were absent in non-silenced plants. These RNAs were complementary to both the sense and antisense strands of the gene being silenced. Initiation of PTGS begins when input dsrna is degraded into nucleotide small interfering RNAs (sirnas). The sirnas are produced when a nuclease called Dicer, a dsrna-specific ribonuclease, processively cleaves dsrna in an ATP-dependent manner to bp duplexes (sirnas), with 2-nucleotide 3' overhangs figure on next page. Next the sirna duplexes bind to a nuclease complex and forms the RNA-induced silencing complex (RISC). ATP-dependent unwinding of the sirna duplex is required for RISC activation. The active RISC then targets homologous transcripts through base-pairing and cleaves the target mrna about 12 nucleotides from the 3' terminus of the sirna.

5 Gene silencing by dsrna has been firmly established to occur in plants. More recently, hairpin RNA (hprna) constructs for the efficient transformation and silencing of plant genes has been described. Constructs that encode for the production of self-complementary hairpin RNA containing sense/anti-sense arms of varying size has been shown to effectively silence a number of genes in a variety of plant species. Constructs that included a functional intron in the hprna could yield silencing rates in excess of 90% of transformed plants. Using the generic vector, phannibal, intron containing hprna constructs can be easily made using PCR products. Thus it has become a practical strategy to include gene silencing by RNAi for any gene of interest in any easily transformable species. The best part of this is that it does not require knowing the plant s genome sequence.

6 A brief schematic of RNAi gene silencing. Transgenes for transformation can be created with internal complementary sequences that can give rise to regions of double stranded RNA (dsrna). Once formed, dsrna is processed by a ribonuclease III called dicer. The process produces nucleotide fragments that can either be double stranded or become single stranded. These short RNAs are also called guide RNAs. The nucleotide fragments associate with a complex having four major activities which are helicase (HEL), homology searching activity (HSA), exoribonuclease (NUC) and endoribonuclease (NUC). Together with the guide RNA these four activities form the RNA induced silencing complex (RISC). The complex associates with target mrnas and rapidly catalyzes their destruction. Transgene RNAi Gene Silencing GACCAAGG GACCAAGG CUGGUUCC GACCAAGG CUGGUUCC Intron CCUUGGUC Cut by Processing Nuclease ( Dicer RNase III) Hel HSA Nuc mrna nt fragments forming guide RNAs RISC Nuc Hel HSA Hel HSA Nuc Nuc Nuc Nuc nt RNA AAA(n) RNAi Movie from Nature Review Genetics

7 RNAi Movie Terminology used in the video that you should know: Argonaute (AGO) proteins Dicer dsrnas dsrna viruses RDRP RNAi micrornas or mirnas PiWi Pre-mRNAs Ribonuclease RISC sirna RNA polymerase II Slicer RNase III The video also shows the following: cap, splicing, polya tail, nascent proteins, ribosomes, polyribosomes, and a ribonuclease

8 Sirna Therapeutics is a subsidiary of Merck and Protiva Biotherapeutics, Sirna Therapeutics, Inc. Research & Development 1700 Owens Street, 4th Floor San Francisco, CA Web Site: that develops therapeutics based on RNA interference (RNAi) technology. It engages in research, preclinical, and/or clinical development with product candidates in the various areas, including age-related macular degeneration (Sirna-027), asthma, chronic hepatitis, dermatology, diabetes, Huntington s disease and oncology. Sirna-027 has finished a phase 3 study involving a larger number of patients treated and observed over a longer period. A single intravitreal dose of Sirna-027 containing up to 1600 μg/eye appeared to be well tolerated in patients with neovascular AMD that had been refractory to other therapies. In some subjects, improvement in visual acuity and foveal thickness was observed, with no dose-response or dose-limiting effects. Sirna-034 for the treatment of hepatitis C virus is in a preclinical stage. The company, formerly known as Ribozyme Pharmaceuticals, Inc., was founded in 1992 and changed its name to Sirna Therapeutics, Inc. in 2003, and was later acquired by Merck and Protiva.

9 David L. Nelson and Michael M. Cox LEHNINGER PRINCIPLES OF BIOCHEMISTRY Fifth Edition CHAPTER 27 Protein Metabolism 2008 W. H. Freeman and Company

10 Protein synthesis and the genetic code. Nomura found that ribosomes have 2 subunits that are composed of RNAs (65%) proteins (35%) and. E. coli has 15,000 ribosomes which accounts for almost 25% of dry weight of the cells.

11 Prokaryotic 70S ribosome

12 The Nobel Prize in Physiology or Medicine 1968: A University of Florida Bachelor s (1948) and Master s degree (1952) Graduate in Zoology from Orlando, FL. His group added a synthetic poly-uracil RNA to a cell-free extract of Escherichia coli ribosomes and other cellular machinery that could possibly function in protein synthesis. DNase was used to remove DNA so that no additional proteins would be produced other than that from the synthetic polyu- RNA. They then added 1 radioactively labeled amino acid, and 19 unlabeled amino acids to each extract, varying the labeled amino acid in each sample. In the extract containing the radioactively labeled polyphenylalanine, the resulting polypeptide was also radioactive demonstrating that polyu contained the genetic code for phenylalanine.