paper rnai in yeast ls 3oct09

8/9/2019 Paper RNAi in Yeast LS 3Oct09

1/24

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


2/24

RNAi

Functional Genomics

Therapeutics

Biotechnology


3/24

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.


4/24

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


5/24

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


6/24

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)


7/24

Small RNA Mapping to Budding Yeast Genome


8/24

Phasing and Offset of small RNA


9/24

Phasing and Offset of small RNA

Small RNA phasing was 23 nt

+/- strand small RNA offset was 2 nt


10/24

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?


11/24

Dicing Activity in Whole Cell Extracts

dsRNA was cleaved into 22-23mer RNA

Whole cell extracts possesses dicing activity


12/24

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


13/24

Strategy for Identifying Dicer


14/24

Is RNaseIII* the Budding Yeast Dicer

RnaseIII* is the budding yeast Dicer

(S. castelli)


15/24

Phylogeny of the budding yeast Dicer

Budding yeast Dicer is close to Rnt1 rather than the cannonical Dicer


16/24

S. castelliiDicer is functional in S. cerevisiae showed robust activity in E. coli

Biochemical Analyses of Budding Yeast Dicer


17/24

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


18/24

Engineering RNAi in S. castellii


19/24

GFP constructs are functional in budding yeast and correlate with

the presence ofAGO1 and DCR1



20/24


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


21/24

DCR1 is enough to produce siRNA

AGO1 enhances the siRNA production

Reconstitution of RNAi in S. cerevisiae


22/24


DCR1 is enough to produce siRNA but AGO1 is required for silencing


23/24

Silencing coincides with the decrease in mRNA



24/24

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.


Transposons are affected by RNAi reconstitution in S.

cerevisiae

paper rnai in yeast ls 3oct09

Documents