Science - USA (2021-12-24)

methylation. By contrast, PNRPD1::NRPD1(DFHT;
M5)nrpd1exhibited the same low-DNA methyl-
ation level as thenrpd1mutant. Moreover, the
24-nt siRNAs associated with the RdDM loci
were substantially reduced in PNRPD1::NRPD1
(DFHT; M5)nrpd1plants, compared with wild
type and PNRPD1::NRPD1 nrpd1(fig. S12). These
results demonstrate that the Pol IV–RDR2
interaction is essential for production of the
dsRNA precursors of 24-nt siRNA and the sub-
sequent DNA methylation.
On the basis of our cryo-EM structures and
previous results from biochemical and genetic
studies, we propose a backtracking-triggered
RNA channeling model of dsRNA synthesis
by the Pol IV–RDR2 complex (Fig. 6): (i) Pol IV–
RDR2 is recruited to genomic RdDM loci by
SHH1- and CLASSY-family proteins to initi-
ate synthesis of Pol IV RNA ( 21 – 25 ); (ii) Pol
IV moves forward and elongates Pol IV RNA

toalengthof30to40nt( 6 , 7 ); (iii) Pol IV back-

tracks, and Pol IV RNA is threaded through

the interpolymerase RNA channel into the

RDR2 active site, where Pol IV RNA is trapped

andusedasthetemplateforRDR2RNAsyn-

thesis; (iv) RDR2 elongates RDR2 RNA result-

ing in further Pol IV backtracking; (v) RDR2

pulls Pol IV RNA out of the Pol IV active center

and releases Pol IV RNA, resulting in Pol IV

termination; and (vi) RDR2 releases dsRNA

from its active site. After completing a tran-

scription cycle, Pol IV returns to the position

where the Pol IV RNA synthesis initiates and

presumably could initiate another round of

Pol IV RNA synthesis to amplify small RNA

signals for local or long-distance action ( 47 ).

We predict that the lack of TFIIS interaction

renders Pol IV inefficient in cleavage of back-

tracked RNA during elongation and ensures

that completely backtracked RNA is delivered

into the RDR2 active center. Pol IV backtrack-

ing can be triggered by increased dwell time

in the pretranslocation state caused by dsDNA

unwinding incapability ( 20 ), torsion stress posed

by positively supercoiled downstream DNA ( 48 ),

and/or nucleosome roadblocks ( 49 , 50 ). A recent

report revealed that RDR2 RNA synthesis can-

not proceed unless Pol IV unwinds a down-

stream dsDNA of >17 bp in length ( 20 ). This

finding supports the minimal requirement of

backtracked nascent Pol IV RNA to reach the

RDR2 active center in our model, suggesting a

possible role of dsDNA unwinding in Pol IV

RNA backtracking. Further structural and bio-

chemical studies are required to elucidate how

Pol IV backtracking is achieved in vivo.

The structures of Pol IV–RDR2 complexes

provide atomic details for such an unprece-

dented two-RNA-polymerase assembly ( 18 ).

The interpolymerase RNA channel revealed

SCIENCEscience.org 24 DECEMBER 2021•VOL 374 ISSUE 6575 1585

(1)

PoI IV initiation

RNA transfer

channel

NTP

channel

dsRNA

channel

RDR2

upstream downstream

Pol IV

5'

3'

3'

5'

RNA ex

it

channe

l 5' 3'

NTP

channel

ddsRNAsRNA

channel

RDR2

upstream downstream

Pol IV

3'5' 5'3'

RNA

exit

cha

nnel

5' 3'

inter-polymerase RNA

channel

(5)

PoI IV termination

RDR2 elongation

NTP

channel

ddsRNAsRNA

channel

RDR2

upstream downstream

Pol IV

5'

3'

3'

5'

RNA

exit

cha

nnel 5'

inter-polymerase RNA

channel 3' 3'5'

(2)

PoI IV elongation

NTP ddsRNAsRNA

RDR2

upstream downstream

Pol IV

5'

3'

5'

RNAex 3'

it

chann

el

inter-polymerase RNA

channel

3'

5'

NTP

channel

ddsRNAsRNA

channel

RDR2

upstream downstream

Pol IV

3'

5'

5'

3' 3'5'

RNA

exit

chann

el

inter-polymerase RNA

channel

5'

3'

(3)

PoI IV backtracking

RDR2 initiation

NTP

channel

ddsRNAsRNA

cchannelhannel

RDR2

upstream downstream

Pol IV

5'

3'

3'

5'

3'

3'

5'

5'

RNA ex

it

cha

nnel

(4)

PoI IV backtracking

RDR2 elongation

inter-polymerase RNA

channel

RNA exitchannel

NTP

channelchannel

RDR2

upstream downstream

Pol IV

5'

3' 5'3'

5'

3'

3'

5'

(6)

RDR2 termination

inter-polymerase RNA

channel

Pol IV NT-DNA

Pol IV T-DNA

Pol IV RNA

RDR2 RNA

ddsRNAsRNA

channelchannel

Fig. 6. Backtracking-triggered RNA channeling model for dsRNA synthesis by Pol IV-RDR2.Black arrows indicate the direction of Pol IVÐRDR2 movement on DNA.

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