Lecture 18 Flashcards
RNAi
Technique to decrease mRNA levels of a target gene
o siRNAs (small inhibiting RNA) can be used to knockdown gene function
can have off-site problems
o very useful for studying gene function
o gene therapy
o fight off infection
o number of products in clinical trials
miRNAs
Non-coding transcribed genes
o mutation of miRNAs can lead to cancer (CLL) and other diseases
o mutation that creates/abolishes a miRNA binding site can cause disease (TS)
o possible targets for gene therapy
o defects in miRNAs cause disease
RNAi Clinical Application
• Target rev gen in HIV w/ siRNAs
• Use siRNAs to target specific oncogenic variants (wild type copy unaffected) (ex. K-ras in human pancreatic carcinoma)
• Respiratory diseases
o Including asthma and CF – lead to chronic inflammation
o Reduce levels of interferon via siRNA leads to reduced inflammation
RNAi Problems
o Delivery
o Off-site effects
May target >1 mRNA b/s of sequence homologies (at least one mismatch)
o May induce subsets of genes in the immune response
Genetics of C. Elegans
o Cascade of regulatory genes that are temporally controlled
o Mutations cause skipping or reiteration of stage-specific events
o Lin-4 RNA is complementary to sequences in the UTR of lin-14 and lin-28
As lin-4 is transcribed, 14&28 decline
o Same thing was seen with let-7 and that it was in humans
Let-7 mutant leads to many types of cancer
miRNAs are found
o Humans (2588 = ~1-5% of known genes) o Mice – 1915 o Arabidopsis thaliana – 337 o Chicks – 994 o C. elegans – 434 o Drosophila m. – 466 o None in yeast and bacteria (pundai has miRNA, cerevisiae has only RNAi)
Tourette’s Syndrome
o Single point mutation (led to a stop codon, nonsense mutation) in SLITKR1 – caused a truncation (rare 1/174 TS pts)
o 2 additional identical mutations in SLITRK1’s 3’ UTR; arose independently
o Mutation produces a binding site for the miRNA miR-189
Results in downregulation of SLITRK1
o Usually the mutation for miRNA binding doesn’t cause a phenotype b/c its location has to overlap where the protein is made.
o Rare sequence variants associated w/ TS
o Example of how a random point mutation resulting in the production of a novel miRNA binding site can cause disease
Myostatin
o GDF8 on chr 2, discovered by mapping a quantitative trait locus
o TGF beta protein family member that inhibits muscle differentiation and growth
o Myostatin is produced primarily in skeletal muscle cells
o Circulates in the blood and acts on muscle tissue
o Texel sheep, Belgian Blue cattle, german baby boy, mighty mouse
• World anti-doping agency banned substances that inhibited myostatin
Myostatin and Whippets
o Myostatin mutation increased muscle mass and enhanced racing performance in whippets
Bully whippets have no health abnormalities other than muscle cramps, usually euthanized since they don’t conform to AKC standards
Whippets that are heterozygous for the mutation are best for racing (sprints) but not greyhounds w/ mutation b/c their track is longer
Uses for Myostatin Inhibitors
o Currently no clear drug candidates, very limited effectiveness in larger clinical trials
o A # of smaller clinical trials has demonstrated that inhibition of myostatin/ActRII signaling may help to improve muscle mass in patients with muscle wasting
o Some progressed into early stage of clinical testing, further testing still required
o Evidence of positive effects on muscle wasting through exercise training and nutritional supplementation are emerging
Myostatin and Sheep
• Myostatin in Texel sheep
o GA bp change in 3’ UTR
created potential binding site for miR-1 and miR-206
-Are known for the exceptional meatiness
Familial pleuropulmonary blastoma
o Rare pediatric lung tumor that is often part of an inherited cancer syndrome
o 11 multiplex PPB families harbor heterozygous germline mutations in DICER1
o Loss of DICER1 in the epithelium of the developing lung alters the regulation of diffusible factors that promote mesenchymal proliferation
