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8/9/2019 Paper RNAi in Yeast LS 3Oct09
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Budding Yeast:
Model Organism for Biological Research
Eukaryotic single cellular organism
Short generation time
Easy and less expensive to culture
Low cost of maintenance
High economic impact
Complete genome sequenced
Highly annotated genes
Well studied genetics and easy tomanipulate
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RNAi
Functional Genomics
Therapeutics
Biotechnology
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RNAi and Yeast
RNAi machinary is conserved across all the genera from
unicellular fission (S. pombe) yeast to multicellular plants and
animals
RNAi machinary could not be detected by the availablebioinformatic approaches in budding yeast
This feature limits the utility of budding yeast to use RNAi for functional
genomics and other studies (especially S.cerevisiae)
Ago proteins have been recently discovered in few species of budding
yeast - S. castelliiand K. polysporus. However, no other RNAi genes,
especially dicer, have not been found in these budding yeast.
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Other RNAi machinary was
lost in budding yeast during
evolution? (RNAi is non-
existant: a Pessimistic view)
OR
Traditional bioinformatics
approaches were unable to
trace the presence of other
RNAi proteins like dicer? (RNAi
exists: an Optimistic view)
Budding Yeast and RNAi Machinery
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RNAi in Budding Yeast
Ines A. Drinnenberg, David E. Weinberg, Kathleen T. Xie, Jeffrey P. Mower,
Kenneth H. Wolfe, Gerald R. Fink, David P. Bartel
Scienceexpress, 10th Sep 09
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Small RNA Isolation From Budding Yeast
Small RNA were enriched in 23mers (S. castelli, K. polysporus)
Small RNA begin with U (S. castelli, K. polysporus) or A/U (C. albicans)
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Small RNA Mapping to Budding Yeast Genome
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Phasing and Offset of small RNA
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Phasing and Offset of small RNA
Small RNA phasing was 23 nt
+/- strand small RNA offset was 2 nt
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What are these small RNA?
Small RNA are 22-23mer in size Their 5 ends are either A/U
They map to Transposons, Y elements, ORFs in addition to rRNA
and tRNA
Phasing is 23nt
Offset is 2nt
These are defenitely siRNAs
Where is the Dicer?
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Dicing Activity in Whole Cell Extracts
dsRNA was cleaved into 22-23mer RNA
Whole cell extracts possesses dicing activity
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Strategy for Identifying Dicer
Helicase PAZdsRBD dsRBD
RNAseIIIa
RNAseIIIb
Typical Structure of a Dicer:
Search for a Typical Dicer: No Result
Search for an RnaseIII protein: 2 results found in S. castellii, K. polysporus
and C. albicans
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Strategy for Identifying Dicer
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Is RNaseIII* the Budding Yeast Dicer
RnaseIII* is the budding yeast Dicer
(S. castelli)
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Phylogeny of the budding yeast Dicer
Budding yeast Dicer is close to Rnt1 rather than the cannonical Dicer
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S. castelliiDicer is functional in S. cerevisiae showed robust activity in E. coli
Biochemical Analyses of Budding Yeast Dicer
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The impact of RNAi on the S. castellii
transcriptome. (A) Strand-specific mRNA-Seq
analysis of annotated ORF transcripts in wild-
type (WT) and RNAi-mutant strains. Plotted is
the log2 ratio of transcript abundance in
ago1 versus wild-type (x-axis) and dcr1
versus wild-type (y-axis). Colors indicate the
density (reads/kb) of antisense small (2223-
nt) RNAs that co-purified with Ago1.
Impact of RNAi on S. castellii transcriptome
RNAi impacted the transposons and Y element
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Engineering RNAi in S. castellii
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GFP constructs are functional in budding yeast and correlate with
the presence ofAGO1 and DCR1
Engineering RNAi in S. castellii
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Engineering RNAi in S. castellii
Silencing GFP depended on DCR1 for siRNA production and AGO1 for siRNA function
Engineered siRNAs could silence a gene
Target transcript could originate from a locus distinct from that producing siRNAs
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DCR1 is enough to produce siRNA
AGO1 enhances the siRNA production
Reconstitution of RNAi in S. cerevisiae
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Reconstitution of RNAi in S. cerevisiae
DCR1 is enough to produce siRNA but AGO1 is required for silencing
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Silencing coincides with the decrease in mRNA
Reconstitution of RNAi in S. cerevisiae
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Silencing of Ty1 retrotransposons by RNAi in S. cerevisiae. (A) Immunoblot probing for Ty1
Gag protein (p45) and its precursor (p49) (39) in S. cerevisiae strains expressing the indicated
S. castelliigenes. Strains were grown under standard (30C) or transposition-inducing (20C)
conditions. The blot was reprobed for actin. RNA blot probing for Ty1 mRNA, analyzing the
same cultures as in (A). Ethidium bromide-stained rRNA is shown.
Reconstitution of RNAi in S. cerevisiae
Transposons are affected by RNAi reconstitution in S.
cerevisiae