1 / 17

RNA interference

RNA interference. Terms. RNAi: RNA interference . Degradation of mRNA molecules specified by complementary double-stranded RNA dsRNA: double-stranded RNA . Complementary RNA molecules which form a double helix

gerik
Télécharger la présentation

RNA interference

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. RNA interference

  2. Terms • RNAi: RNA interference. Degradation of mRNA molecules specified by complementary double-stranded RNA • dsRNA: double-stranded RNA. Complementary RNA molecules which form a double helix • siRNA: small interfering RNA: RNA duplexes of 21-23 nucleotide pairs with 2 unpaired nucleotides at each 3’ end. Identify mRNA molecules for degradation in RNAi. • RISC: RNA-induced silencing complex. Including proteins and siRNAs and degrades mRNA molecules • stRNA: small temporal RNA. Small RNA molecules that act to suppress translation. • PTGS: posttranscriptional gene silencing: A means of gene regulation in plants which occurs after the production of mRNA. • PPD: PAX and PIWI domain. Proteins, including those involved in RNAi, PTGS and quelling, that contain the above two protein domains.

  3. Discussion: What is RNAi? Describe the phenomena known as RNAi What are the major molecular players in RNAi? Why do cells have RNAi?* Why is RNAi important to researchers?

  4. Whole-genome knock-down • What are the major goals of whole-genome knock-down experiments? • What are the potential sources of error in such experiments? • Why might RNAi be a better or worse choice for this type of experiment than conventional knock-out experiments?

  5. Gene Knock-down by RNAi • In C. elegans, instead of completely knocking out every gene in the genome, genes’ expression was suppressed via RNAi Host Genome mRNA (single-stranded) dsRNA (fed) Degradation of complementary mRNA

  6. Some Details • > 16,000 genes were studied • Worms are feed on a specfic strain of E. coli expressing dsRNA complementary to one worm gene for >36 hours • The progeny of these animals are scored for lethality and other phenotypic defects

  7. Phenotypes are rare • Only ~10% of screened genes had a detectable phenotype • 5.5% of the total genes screened showed embryonic lethality • Other phenotypes included slow growth and movement defects • 64% of genes with phenotypes known from other experiments were detected

  8. Figure 3: Conservation of domains in genes with different RNAi phenotypes. All predicted genes were placed into one of four mutually exclusive classes on the basis of their InterPro domain content. The 'ancient' class comprises genes for which all predicted domains are also encoded in the S. cerevisiae, A. thaliana, D. melanogaster and H. sapiens genomes; the 'animal' class comprises genes that contain any domain present in the D. melanogaster or H. sapiens genomes, but not in S. cerevisiae or A. thaliana, and the 'worm' class comprises genes containing any domain present in the C. elegans genome, but not in the other four. The proportions of All, Nonv and Vpep genes that fall into each class are shown

  9. Lethal Duplication and Robustness • Experimental work in yeast and C. elegans has demonstrated that the expression of many genes can be suppressed without noticeable effect • One possible reason for this is that duplicate copies of these genes buffer that loss Host Genome Gene Knock-down Resulting Phenotype X Single Copy Gene X Duplicated Gene Viable

  10. Do genes with more duplicates show less severe knock-down effects? Do genes with closely related duplicates show less severe knock-down effects than those with more distant ones? Do highly expressed genes show more severe knock-down effects?

  11. Glossary of Sequence Evolution • Distances between sequences are conventionally reported in terms of the number of substitutions per nucleotide (or amino acid) site. • It is useful to distinguish synonymous substitutions from non-synonymous substitutions • Ks conventionally represents the number of synonymous substitutions per synonymous site • Ka represents the number of non-synonymous (amino-acid changing) substitutions per non-synonymous sites • There are a number of methods of calculating Ks and Ka

  12. Sequence Similarity: Significant association between duplicate amino acid sequence similarity and severity of knock-down No association between duplicate synonymous site similarity and severity of knock-down

  13. Duplicates, Sequences and Knock-down • Genes with duplicates are less likely to show a lethal knock-down effect and more likely to show no knock-down effect than are genes without duplicates • Gene duplicates with similar amino acid sequences have less severe knock-down effects than do other duplicates • Genes duplicates with similar synonymous sites do not show differences in knock-down effect compared to other duplicates

  14. Robustness and Duplication: Conclusions • Duplicate genes provide protection against loss of gene function • Sequence similarity and expression similarity both show a negative correlation to severity of knock-down effect • Sequence and expression similarity are only weakly correlated themselves (data not shown)

More Related