ISH a. No probe, b. No RNA c. antisense RNA, d. dsRNA 4 cell C. elegans embryos
Cell transfected with myc tagged Ago plus siRNA for luciferase. Test IP’s for cleavage. Knew Argo part of RISC
C. Swelling of pericardium D. E. Failure to close neural tube
Traphina (Asci of the peach leaf curl fungus atop a peach leaf) sacchro, truffle, penicillium Fission yeast was isolated in 1893 by Lindner from East African millet beer . The species name is derived from the Swahili word for beer (Pombe). It was first developed as an experimental model for studying the cell cycle by Murdoch Mitchison in the 1950s. The fission yeast researcher Paul Nurse , together with Lee Hartwell and Tim Hunt , won the 2001 Nobel Prize in Physiology or Medicine , for their work on cell cycle regulation.
TRBP Tar RNA binding protein RCK human homolog of yeast Dhh 1p. Interacts with eIF4e and represses translation
Last one Phil Sharp
RT PCR olio dt and 3’UTR gene specific primer. Hinf1 digest of cDNA labeled 32P. Hinf1 site 95 bp from polyA site.
Full blown RISC 80S
RNA Silencing RNAi Post transcriptional gene regulation by siRNA and miRNA Pete Burrows MIC 759 October 26, 2006
Small untranslated regulatory RNAs Eukaryotes ncRNA Prokaryotes sRNA
Guillier, et al. Genes and Development 20:2338, 2006
Which becomes guide strand in the RISC and which (passenger strand) is excluded?
Sequence and structure
Strand with the less-tightly base pared 5’ end is incorporated becomes guide strand
What is the endonuclease (Slicer) in RISC?
The ago1 mutant Arabidopsis develops abnormally because it does not produce an effector of silencing. The Argonaute genes were so named because the mutant plants look like an argonaute squid. The Sainsbury Laboratory John Innes Centre Colney Lane Norwich, NR4 7UH, UK Knew that Ago a RISC component
Identification of Argonaute 2 as Slicer in humans Published by AAAS J. Liu et al., Science 305, 1437 -1441 (2004)
Published by AAAS J. Liu et al., Science 305, 1437 -1441 (2004) Fig. 2. Argonaute2 is essential for mouse development
Published by AAAS J. Liu et al., Science 305, 1437 -1441 (2004) Fig. 3. Argonaute2 is essential for RNAi in MEFs
Sontheimer and Carthew, Science 2004 Sep 3;305(5689):1409-10
Multi domain RNase III enzyme the cleaves dsRNA or stem-loop pre-miRNA into siRNA and miRNA
TAR RNA Binding Protein, Cofactor for Dicer
RNA induced silencing complex
Argonaute ( AGO )
PAZ domain binds the characteristic two-base 3' overhangs of siRNAs
PIWI domain: dsRNA guided hydrolysis of ssRNA
Ago2 is slicer in mammalian RISC
Other Ago may function in miRNA silencing
Schizosaccharomyces pombe has DCR and AGO but not in S accharomyces cerevisiae Taphrina S. pombe S. cerevisiae Morel Penicillium http://www.glocalbeer.dk http://tolweb.org/tree?group=Ascomycota&contgroup=Fungi Swahili word for beer (Pombe)