11BIS1012012MicroRNALect12

11BIS1012012MicroRNALect12 - BIS101-001: Genes and Gene...

Info iconThis preview shows page 1. Sign up to view the full content.

View Full Document Right Arrow Icon
This is the end of the preview. Sign up to access the rest of the document.

Unformatted text preview: BIS101-001: Genes and Gene Expression Transcription, Gene Expression RNAi Lecture #11 Chapter 8, 17 March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 1 In this lecture DNA is copied into an RNA by the process of transcription. RNA is used to make protein (messenger RNA) or used as structural components for other macromolecules like ribosomes (next lecture). Transcription can be "cosuppressed" or "knocked down" by small interfering RNAs (siRNAs), complementary to the mRNA Although it was thought that a single stranded anti-sense RNA was responsible for cosuppression, we now know that a dsRNA is needed. Extensive processing of the dsRNA is needed to produce the siRNA. siRNA can inhibit gene expression by promoting ribonuclease degradation or blocking the translation of the mRNA target. This whole process is called RNA interference (RNAi) and it has great potential for health and agriculture. BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 2 March 19, 2012 Sense and Antisense Strands 5'A G C C T A T T G G G A T C T A T T A A T C C C3' non-template, nontranscribed, sense or RNA-like strand template, transcribed or antisense strand 3'T C G G A T A A C C C T A G A T A A T T A G G G5' 5'A G C C T A T T G G G A T C T A T T A A T C C C3' sense strand RNA 5'A U U G G G A U C U A U U A A U C C 3'T C G G A T A A C C C T A G A T A A T T A G G G5' 5'A G C C T A T T G G G A T C T A T T A A T C C C3' C C C U A G A U A A U U A G G G5' antisense strand RNA 5'A U U G G G A U C U A U U A A U C C 3'T C G G A T A A C C C T A G A T A A T T A G G G5' March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 3 RNA Interference (RNAi) Story Co-suppression Antisense RNA(aRNA) Double-stranded RNA RNA interference (siRNA) (DsRNA = nuclear microRNA, long-hairpin dsRNA, or infectious dsRNA) March 19, 2012 Post-transcriptional silencing (knockdown) 4 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 Cosupression and purple petals in petunias Extra dose of mRNA for purple petal color Upon injection of the gene encoding mRNA responsible for purple coloring in petunias, the flowers became variegated or white rather than deeper purple as was expected. What happened? BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 5 March 19, 2012 RNA Interference (RNAi) 1998: Fire and Mello published a seminal paper on RNAi. Working with C. elegans, they tested the hypothesis that the trigger for gene silencing was not single-stranded RNA (ssRNA) but double-stranded RNA (dsRNA). December 2, 2006, Andew Fire and Craig Mellow (shown below) receive Nobel Prizes RNAi. March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 6 Antisense Mechanism March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 7 Biological function comes from proteins and RNAs http://www.youtube.com/watch?v=cK-OGB1_ELE March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 8 Sources of dsRNA and their processing March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 9 MicroRNAs and Primary MicroRNAs Shown below are two genes (A and B) transcribed from different strands (mRNA). Primary (pri-miRNA) and miRNA are shown as blue arrows. Notice that the pri- and miRNAs are non-coding (ORF-less) and can be made from either strand either between genes or within introns. It is unlikely that miRNAs inhibit (after dicer processing) the mRNAs of the genes they are made from. Why? Gene A pri-miRNA miRNA mRNA mRNA miRNA March 19, 2012 Exons Gene B 10 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 MiRNAs are highly conserved and processed March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 RNA Interference (RNAi): 5 Points 1. 2. 3. 4. 5. March 19, 2012 The silence the expression of a mRNA with siRNA requires the "RNA-Induced Silencing Complex"(RISC) and its 3 enzymatic activities"Dicer," Helicase and "Slicer." Other enzymes may also be involved (e.g., Drosha, exoribonuclease). The endonuclease activity of "Dicer"converts ds-shRNA into 2123 bp RNA (siRNA) making it competent for interference. The helicase activity of RISC, unwinds the "passenger" strand of siRNA from the "guide" strand. The guide strand is bound by Argonaut (Ago2) which facilitates its binding to its complementary target mRNA and the endonuclease activity of RISC ("slicer") cleaves the mRNA. Exoribonucleases degrade the RNA from its ends. In plants, the RNA-dependent, RNA polymerase activity of Dicer synthesizes new siRNA so it can pass from one cell to the next and from one cell division to the next (i.e. infectious). BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 12 miRNA Processing and Mode of Action Immediately after transcription, pri-miRNAs are enzymatically cropped by Drosha into short hairpin RNAs (shRNA) or pre-miRNAs. The shRNAs undergo active transport from nucleus to cytoplasm where they are recognized by Dicer. Dicer reduces the shRNA into a short-lived double strand siRNA The non-siRNA (passenger strand) is separated from the antisense or guide strand and degraded, releasing a functional siRNA/RISC complex. Drosha miRNA gene Transcription Nucleus Cytoplasm Dicer RISC Cropping Nuclear export Dicing miRNA separation ds-siRNA Functional RISC pri-miRNA March 19, 2012 pre-miRNA (shRNA) siRNA BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 13 RNA Induced Silencing Complex (RISC) Dicer/R2D2 binds and trims the small dsRNA (miRNA). This dsRNA is transferred to Argonaut (Ago2). The helicase of Ago2 unwinds the "passenger" and "guide" strands. The guide strand binds the target RNA and the endonuclease (Slicer) cuts the target RNA, followed by exonuclease digestion. http://helicase.pbworks.com/w/page/17605619/Emily-Devol March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 14 Processing of Small Interfering RNA (siRNA)* dsRNA Double stranded RNA is introduced into a cell and gets trimmed by the enzyme "dicer" to form siRNA. siRNA then binds to the RNA-Induced Silencing Complex (RISC) complex and is unwound. The anitsense RNA complexed with RISC binds to its corresponding mRNA which is the cleaved by the RNase "slicer" rendering it inactive. SiRNA can be replicated and become infectious (#6, 7, 5). BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 15 March 19, 2012 Antisense Therapy March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 16 Flavr Savr Tomatoe and Antisense* Antisense RNA methods have been used for commercial food production. The Flavr Savr tomato was developed by Calgene Inc. of Davis, California in 1991 and was approved by the U.S. FDA in 1994. The tomato was the first whole food created by biotechnology that was evaluated by the FDA. One of the problems associated with tomato farming is that the fruit must be picked while still green in order to be shipped to market without being crushed. The enzyme that causes softening in tomatoes is polygalacturonase (PG). This enzyme breaks down pectin as the tomato ripens, leading to a softer fruit. Calgene suppressed the expression of the gene encoding PG by introducing a gene encoding the antisense strand of the mRNA. When the introduced gene was expressed, the antisense strand bound to the PG mRNA, suppressing the translation of the enzyme. The Flavr Savr tomatoes therefore had low PG levels and remained firmer when ripe. This meant the Flavr Savr tomatoes could be ripen on the vine and then be shipped to market. Although the Flavr Savr tomatoes were approved for sale in the U.S., production problems and consumer wariness stopped the production of this fruit in 1997. BIS101003, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 17 March 19, 2012 Biological function comes from proteins and RNAs COMPANY OVERVIEW: FIRST IN HUMAN CLINICAL TRIALS for a chemically optimized siRNA -- a major milestone in the development of RNAibased therapeutics. Sirna-027, for the treatment of the wet-form of age-related macular degeneration (AMD), a condition in which the cells of the macula lutea degenerate, resulting in blurred vision and ultimately blindness. The macula is a small area at the back of the eye, near the center of the retina, at which visual perception is most acute. AMD occurs most often with increasing age (age-related macular degeneration or AMD), but there are hereditary forms of the disease that affect children and teenagers as well. ADM is seen in adults 15 million people in the United States over 60 years of age. Our therapeutic programs encompass a broad range of therapeutic areas including: Anti-Viral, Dermatology, Metabolism, Neurology, Ocular, Oncology, and Respiratory. Specific preclinical and clinical programs include: viral hepatitis, an infection of the liver that is caused by a variety of RNA viruses (hepatitis A virus, hepatitis B virus, hepatitis C virus, etc.).(HBV & HCV), hair removal, asthma and COPD, diabetes, Huntington's disease, and oncology. These programs represent large market opportunities and significant unmet medical needs. March 19, 2012 BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 18 Summary DNA is copied into an RNA by the process of transcription. RNA is used to make protein (messenger RNA) or used as structural components for other macromolecules like ribosomes (next lecture). Transcription can be "cosuppressed" or "knocked down" by small interfering RNAs (siRNAs), complementary to the mRNA Although it was thought that a single stranded anti-sense RNA was responsible for cosuppression, we now know that a dsRNA is needed. Extensive processing of the dsRNA is needed to produce the siRNA. siRNA can inhibit gene expression by promoting ribonuclease degradation or blocking the translation of the mRNA target. This whole process is called RNA interference (RNAi) and it has great potential for health and agriculture. BIS101001, Spring 2012--Genes and Gene Expression, R.L. Rodriguez 2012 19 March 19, 2012 ...
View Full Document

This note was uploaded on 03/18/2012 for the course BIS 101 taught by Professor Simonchan during the Winter '08 term at UC Davis.

Ask a homework question - tutors are online