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

Rdp1

Otr1R

BEADOtr1R

Centrifugation pulls down the beads, chemically dissociate

Load result into a gel, run electrophoresis

Stain for reporter gene (ura4)

Chromatin Immunoprecipitation

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

Coomassie Brilliant Blue

expressing ura4 sensitizes cells to FOA

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