Transcript RNA-dependent RNA polymerase is an essential

RNA-dependent RNA
polymerase is an essential
component of a self-enforcing
loop coupling heterochromatin
assembly to siRNA production
What is already known?
• Without rdp1, heterochromatin is lost
• Ago1 and other RITS (RNAi effector
complex) proteins bind constitutive
heterochromatin at all known loci
• Deletion of RNAi components impair
silencing at centromeres, initiation of
heterochromatin assembly at mat locus
• RdRPs thought to convert single stranded
to dsRNA, enhancing RNAi effect
What are they asking?
• Is RdRP (RNA dependent RNA Polymerase)
activity responsible for heterochromatin
formation, chromosome segregation,
centromere silencing?
• Is Rdp1 recruited to the centromeres?
• Is there a self-inducing pattern where siRNA are
generated by the RNAi pathway, then target
heterochromatin proteins to homologous
sequences. (This should maintain
heterochromatin)
How did they address these
questions?
• Model: schizosaccharomyces pombe
• ChIP analysis
– otr1R::ura4+ construct. Otr1R is not important
except that is usually kept as heterochromatin
Chromatin Immunoprecipitation
Otr1R
BEAD
Rdp1
O
t
r
1
R
Centrifugation pulls
down the beads,
chemically dissociate
Load result into
a gel, run
electrophoresis
Stain for reporter
gene (ura4)
Rdp1 on constitutive
heterochromatic domains
ChIP analysis for Rdp1 at the
centromeres, telomeres and
mat locus (constitutive
heterochromatin areas)
WCE= Whole Cell Extract
ChIP analysis of Rdp1 at
centromeric repeats with one
RITS constituent protein
knocked out in each; as well
as Dcr1 (involved in RNAi),
Clr4 (H3K9 methylation) and
Swi6 (heterochromatin
formation).
What have they shown?
• Rdp1 localized at heterochromatin
(centromeres, telomeres, mat locus)
• Rdp1 localization depends of RITS, Dcr1,
and heterochromatin assembly factors
S. Pombe
N Crassa
C. Elegans
C. Elegans
A. thaliana
expressing
ura4
sensitizes
cells to
FOA
Coomassie
Brilliant Blue
What have they shown?
• Rdp1 localized at heterochromatin
(centromeres, telomeres, mat locus)
• Rdp1 localization depends of RITS, Dcr1,
and heterochromatin assembly factors
• Their Rdp1 mutant construct is expressed
at the same level, but with the mutation
normally silent genes are expressed
There is a similar level of Swi6 and H3K9me in the whole cell, but less at the otr1R
region and centriole in the mutant; problem with heterochromatin assembly
What have they shown?
• Rdp1 localized at heterochromatin
(centromeres, telomeres, mat locus)
• Rdp1 localization depends of RITS, Dcr1, and
heterochromatin assembly factors
• Their Rdp1 mutant construct is expressed at the
same level, but with the mutation normally silent
genes are expressed
• There is a similar level of Swi6 and H3K9me in
the whole cell, but less at the otr1R region and
centriole in the mutant; problem with
heterochromatin assembly
Similar viability with no
treatment, but the mutant is
more sensitive to TBZ,
indicating failure of
centromeres
Wild types rarely show
“lagging chromosomes”-which fail to split in
anaphase, and don’t get
incorporated into the new
cells– but about 1/5 of rdp1
mutants
Taz1 is a telomere binding
protein, and colocalizes
with Swi6 because of its
heterochromatin state. This
shows problems in
telomere clustering (there
are too many in the
mutants)
What have they shown?
• Rdp1 localized at heterochromatin (centromeres,
telomeres, mat locus)
• Rdp1 localization depends of RITS, Dcr1, and
heterochromatin assembly factors
• Their Rdp1 mutant construct is expressed at the same
level, but with the mutation normally silent genes are
expressed
• There is a similar level of Swi6 and H3K9me in the whole
cell, but less at the otr1R region and centriole in the
mutant; problem with heterochromatin assembly
• Mutation causes mitosis problems, possibly due to
failure of heterochromatin assembly
A) Mutation of Rdp1 at D903 abolishes activity in affinity purified samples.
B) siRNAs were lost in mutants of RdRP activity, reducing it to background levels
C) Shows that this is exclusively an RNA effect; pattern not affected by DNase
What have they shown?
• Rdp1 localized at heterochromatin (centromeres,
telomeres, mat locus)
• Rdp1 localization depends of RITS, Dcr1, and
heterochromatin assembly factors
• Their Rdp1 mutant construct is expressed at the same
level, but with the mutation normally silent genes are
expressed
• There is a similar level of Swi6 and H3K9me in the whole
cell, but less at the otr1R region and centriole in the
mutant; problem with heterochromatin assembly
• Heterochromatin assembly problem causes mitosis
problems
• RdRP-affecting mutation removed ability to create siRNA
The RITS components, Ago1 Chp1
and Tas3, fail to localize with the
mutant strain. In this ChIP, they are
not associated with otr1R or the
centriole
Rdp1 mutant does not associate with
the heterochromatin either
What have they shown?
• Rdp1 localized at heterochromatin (centromeres,
telomeres, mat locus)
• Rdp1 localization depends of RITS, Dcr1, and
heterochromatin assembly factors
• Their Rdp1 mutant construct is expressed at the same
level, but with the mutation normally silent genes are
expressed
• There is a similar level of Swi6 and H3K9me in the whole
cell, but less at the otr1R region and centriole in the
mutant; problem with heterochromatin assembly
• Heterochromatin assembly problem causes mitosis
problems
• RdRP-affecting mutation removed ability to create siRNA
• RdRP-afecting mutation prevents the formation of the
RITS complex and association of the Rdp1 to
heterochromatin
So what is the point?
• Rdp1 required for creating dsRNAs that
activate Dicer, which in turn amplifies
siRNAs
• siRNA target heterochromatin in cis, help
maintain heterochromatin state: if the
heterochromatin opens and expresses, the
siRNA will silence it again
• siRNA needed for H3K9 methylation, RITS
localization
STEPS in cycle
• Bidirectional transcription of repeat
elements creates dsRNA
• Dicer cleaves the dsRNA into siRNA
• siRNA targets histone methyltransferase
and RITS to the repeat element
• RITS binds Rdp1, others at
heterochromatic loci
• Ago1 binds siRNA, uses Rdp1’s RdRP
activity to create more dsRNA
So what?
• Support idea that RNAi maintains
heterochromatin in a loop, not a line
• Rdp1 converts “aberrant” transcripts into
siRNA to resilence the gene
• Keeping certain genes off is critical for the
differentiation of cells or reactions to the
environment