a genomewide rnai screen for genes that affect the stability, … · 2009-12-11 · rnai...

Copyright � 2009 by the Genetics Society of AmericaDOI: 10.1534/genetics.109.110171

A Genomewide RNAi Screen for Genes That Affect the Stability, Distributionand Function of P Granules in Caenorhabditis elegans

Dustin L. Updike and Susan Strome1

Department of Molecular Cell and Developmental Biology, University of California, Santa Cruz, California 95064

Manuscript received September 22, 2009Accepted for publication September 28, 2009

ABSTRACT

P granules are non-membrane-bound organelles found in the germ-line cytoplasm throughoutCaenorhabditis elegans development. Like their ‘‘germ granule’’ counterparts in other animals, P granulesare thought to act as determinants of the identity and special properties of germ cells, properties thatinclude the unique ability to give rise to all tissues of future generations of an organism. Therefore,understanding how P granules work is critical to understanding how cellular immortality and totipotencyare retained, gained, and lost. Here we report on a genomewide RNAi screen in C. elegans, whichidentified 173 genes that affect the stability, localization, and function of P granules. Many of these genesfall into specific classes with shared P-granule phenotypes, allowing us to better understand how cellularprocesses such as protein degradation, translation, splicing, nuclear transport, and mRNA homeostasisconverge on P-granule assembly and function. One of the more striking phenotypes is caused by thedepletion of CSR-1, an Argonaute associated with an endogenous siRNA pathway that functions in thegerm line. We show that CSR-1 and two other endo-siRNA pathway members, the RNA-dependent RNApolymerase EGO-1 and the helicase DRH-3, act to antagonize RNA and P-granule accumulation in thegerm line. Our findings strengthen the emerging view that germ granules are involved in numerousaspects of RNA metabolism, including an endo-siRNA pathway in germ cells.

GERM granules are large, non-membrane-bound,ribonucleoprotein (RNP) organelles found in

the germ-line cytoplasm of most, if not all, animals(Eddy 1975; Saffman and Lasko 1999). The term‘‘germ granule’’ encompasses what are known as Pgranules in Caenorhabditis elegans, polar granules inDrosophila melanogaster, germinal granules in Xenopuslaevis, and the perinuclear nuage in mouse and humangerm cells. These large RNP complexes contain aheterogeneous mixture of RNAs and proteins. To date,most of the known germ granule proteins acrossspecies, and all of the known P-granule componentsin C. elegans, are associated with RNA metabolism,which suggests that a main function of germ granules ispost-transcriptional regulation (Strome 2005; Seydoux

and Braun 2006). Germ cells are unique in their abilityto give rise to all tissues of future generations of anorganism. Consequently, germ cells are considered to beboth totipotent and immortal. The widespread presenceof germ granules in germ cells across species and theability of germ granule transplantation to inducefunctional germ cells suggest that germ granules are

key determinants of the identity and special propertiesof germ cells (Smith 1966; Illmensee and Mahowald

1974; Ephrussi and Lehmann 1992). As more compo-nents and regulators of germ granules are identified, wewill better understand their role in conferring germ cellidentity and properties.

One of the earliest identified constitutive compo-nents of C. elegans P granules is PGL-1 (Kawasaki et al.1998). Identification of genes whose loss alters the leveland/or distribution of PGL-1 offers an avenue toidentify other P-granule components and componentsthat regulate P-granule assembly and stability. Forexample, the C. elegans VASA homolog GLH-1, anotherconstitutive P-granule component, acts ‘‘upstream’’ ofPGL-1; in glh-1 loss-of-function mutants, PGL-1 is notproperly localized to P granules and instead a significantportion of PGL-1 is diffusely distributed in the germ-linecytoplasm (Kawasaki et al. 1998; Spike et al. 2008a).More recently another constitutive P-granule compo-nent, DEPS-1, was identified in a forward genetic screenfor mutants that display PGL-1 localization defects similarto those seen in glh-1(lf) mutants (Spike et al. 2008b).Targeted studies have demonstrated that mutation orRNAi of other genes also disrupts PGL-1 in the germ line.These include genes encoding the nuclear transportinsIMB-2, IMB-3, IMB-5, and IMA-3 ( J. Ahringer, personalcommunication; Geles and Adam 2001); the maternalzinc finger protein MEX-1 (Mello et al. 1992; Guedes

Supporting information is available online at http://www.genetics.org/cgi/content/full/genetics.109.110171/DC1.

1Corresponding author: Room 329, Sinsheimer Labs, Molecular, Cell andDevelopmental Biology, 1156 High St., Santa Cruz, CA 95064.E-mail: [email protected]

Genetics 183: 1397–1419 (December 2009)

http://www.genetics.org/cgi/content/full/genetics.109.110171/DC1


http://www.wormbase.org/db/gene/gene?name=CSR-1;class=Gene


http://www.wormbase.org/db/gene/gene?name=EGO-1;class=Gene

http://www.wormbase.org/db/gene/gene?name=DRH-3;class=Gene

http://www.wormbase.org/db/gene/gene?name=PGL-1;class=Gene


http://www.wormbase.org/db/gene/gene?name=GLH-1;class=Gene


http://www.wormbase.org/db/gene/gene?name=glh-1;class=Gene



http://www.wormbase.org/db/gene/gene?name=DEPS-1;class=Gene




http://www.wormbase.org/db/gene/gene?name=IMB-2;class=Gene


http://www.wormbase.org/db/gene/gene?name=IMA-3;class=Gene

http://www.wormbase.org/db/gene/gene?name=MEX-1;class=Gene




and Priess 1997); many of the Sm spliceosome compo-nents (Barbee et al. 2002); the eIF-5A homolog IFF-1(Hanazawa et al. 2004); the germ-line enriched proteinMEG-1 (Leacock and Reinke 2008); and the oocytematuration factors OMA-1 and OMA-2 (Shimada et al.2006). Of these, MEX-1, MEG-1, OMA-1, and the Smproteins are also P-granule components. This led us topredict that additional genes that affect the compositionand behavior of P granules could be identified through agenomewide RNAi screen for PGL-1 accumulation andlocalization defects. Unlike the forward mutagenesis thatidentified deps-1, an RNAi screen would not requiremutants to be viable and fertile. In addition, the variableexpressivity of RNAi phenotypes could be used todiscover P-granule defects accompanying the incompleteloss of essential genes.

In this article we report the identification of 173 genesrequired for the normal assembly and localization ofPGL-1. Many of these components fall into specific geneclasses with shared P-granule phenotypes, allowing us tobetter understand how cellular processes converge on P-granule assembly and function. We looked closely at thestriking phenotype, accumulation of enlarged P gran-ules, caused by RNAi depletion of CSR-1, an Argonautethat was previously shown to target its mRNA slicingactivity through secondary siRNAs (Yigit et al. 2006;Aoki et al. 2007). Loss of other predicted members of asecondary siRNA pathway, the RNA-dependent RNApolymerase EGO-1 and the DExH helicase DRH-3,causes a similar phenotype. We propose that CSR-1(1), EGO-1(1), and DRH-3(1) antagonize thegrowth and accumulation of P granules through anendogenous siRNA pathway that functions in the germline.

MATERIALS AND METHODS

Strains and culture: C. elegans strains were maintained asdescribed by Brenner (1974). Strains used for this studyinclude N2(Bristol) as wild type (WT), SS747 bnIs1(pie-1TGFPTPGL-1; unc-119(1)) (Cheeks et al. 2004), ZT3 csr-1(fj54)IV/nT1[qIs51](IV:V), FX892 csr-1(tm892)/unc-24 IV,NL2098 rrf-1(pk1417)I, NL2099 rrf-3(pk1426)II, EL500 ego-1(om84)I/hT2G(I:III), and SS889 rde-3(r459)I/hT2G(I:III).

GFPTPGL-1 RNAi screen: SS747 worms carrying aGFPTPGL-1 transgene inserted into LGI were maintained at24�. The following screen protocol was followed for 10 weeks:

Day 1. Worms were washed off of 8 recently starved plates into100 ml of S medium with 2 g of HT115 bacteria and grown at24� with shaking (150 rpm) (Lewis and Fleming 1995).Three liters of NGM medium 1 60 mg/ml Carbenicillin 11 mm IPTG (Kamath et al. 2001) were poured into 8024-well plates (1 ml/well).

Day 2. Five 384-well plates from the Ahringer RNAi library(Kamath et al. 2003) were replicated onto Nunc platescontaining LB agar 1 50 mg/ml tetracycline 1 50 mg/mlampicillin and incubated overnight at 37�.

Day 3. The 5 spotted Nunc plates were used to inoculate LBmedium 1 60 mg/ml Carbenicillin cultures in 5 384-wellplates (50 ml/well), which were grown overnight at 37�. glh-1

RNAi feeding bacteria were inoculated into 4 empty wells ofthe 384-well plates each week as a positive control.

Day 4. Worms were collected from the liquid culture (startedon day 1) by centrifugation. Embryos, prepared by bleachtreatment of adults, were transferred to 3 NGM plateswithout food and allowed to hatch overnight at 24�. RNAibacteria from the 50-ml cultures were seeded onto the 8024-well plates and incubated overnight at 37�.

Day 5. The 80 seeded 24-well plates were placed at roomtemperature. Hatched L1s were washed off the unseededplates into 100 ml of S media 1 2 g of bacteria and incubatedat 24� with shaking.

Day 6. After 30–33 hr of growth, the synchronized worms(mainly L3/L4 larvae) were washed and distributed onto the80 24-well plates using a COPAS Biosort (Union Biometrica)to deliver 12–15 worms/well. Worms were incubated on RNAiplates at 24�.

Day 7. Approximately 28 hr after sorting, worms were placed at15� to slow growth. All observable P0 adults and F1 embryosin each well were screened for GFPTPGL-1 phenotypesusing a Zeiss SV11 fluorescence dissecting microscope fittedwith a stereo and 103 compound objective. Secondary andtertiary screens were performed using a Leica MZ16Ffluorescence dissecting microscope fitted with a stereoand 53 compound objective.

Immunocytochemistry: Embryos and worms were fixedusing methanol/acetone (Strome and Wood 1983). Anti-body dilutions were 1:30,000 rabbit anti-PGL-1 (Kawasaki

et al. 1998), 1:5000 mouse anti-NPC MAb414 (Covance)(Blobel 1985), 1:5000 rat anti-PGL-3 (Kawasaki et al.2004), 1:1000 rabbit anti-GLH-1 (Gruidl et al. 1996; Kawasaki

et al. 2004), 1:400 Alexa Fluor 488 goat anti-rabbit IgG, 1:400Alexa Fluor 594 goat anti-mouse IgG, and 1:400 Alexa Fluor 594goat anti-rat IgG (Molecular Probes, Eugene, OR). Imageswere acquired with a Volocity spinning disk confocal system(Perkin-Elmer/Improvision, Norwalk, CT) fitted on a NikonEclipse TE2000-E inverted microscope.

Quantitative RT–PCR: Four biological replicates of N2worms fed empty vector or csr-1 RNAi bacteria at 24� for30 hr were washed and frozen prior to RNA isolation usingTrizol (Invitrogen, Carlsbad, CA). cDNA was synthesized usingSuperscript III First Strand synthesis (Invitrogen). Quantita-tive PCR was performed using a 23 SYBR green mix (Roche,Indianapolis) on a Roche LightCycler 480. pgl-1, pgl-3, and glh-1 amplicons were normalized to act-2. Primers were as follows:pgl-1, f-tgttgttggagtcgcgaag; pgl-1, r-tccgcaatggctcgtctt; pgl-3,f-ctcgagcagtgcttttctca; pgl-3, r-tttcgttgttcaactcgcttt; glh-1,f-actctggttttggggaagga; glh-1, r-gtcactcgatcgatgtcctg; act-2,f-cgtcatcaaggagtcatggtc; and act-2, r-catgtcgtcccagttggtaa.

SYTO14 staining of RNA: Gonads from N2 worms 3 daysposthatching were dissected in 118 mm NaCl, 48 mm KCl, and5 um SYTO14 (Schisa et al. 2001), incubated for 10 min, andthen imaged with a confocal microscope.

RNAi conditions: RNAi depletion of csr-1, ego-1, and drh-3was performed by feeding bacteria from the Ahringer library(Kamath et al. 2003) seeded on agar containing 50 mg/mlcarbenicillin and 1 mm IPTG. Worms at the L3/L4 stage werefed RNAi bacteria for 30 hr before imaging or fixation.Embryos or worms labeled as wild type were fed HT115bacteria containing the L4440 empty vector as a control. Toenhance gene product depletion, worms used for SYTO14staining were fed either control or RNAi bacteria starting atthe L2/L3 stage for 45 hr before dissection. On the basis ofsequence data from the Ahringer lab (J. Ahringer, personalcommunication), we resequenced RNAi clones that had beenflagged as targeting the wrong sequence. Correct gene targetsare as follows: JA:B0464.7 targets atx-2, JA:Y55F3A_739.a

1398 D. L. Updike and S. Strome

http://www.wormbase.org/db/gene/gene?name=IFF-1;class=Gene

http://www.wormbase.org/db/gene/gene?name=MEG-1;class=Gene

http://www.wormbase.org/db/gene/gene?name=OMA-1;class=Gene


http://www.wormbase.org/db/gene/gene?name=MEX-1;class=Gene

http://www.wormbase.org/db/gene/gene?name=MEG-1;class=Gene



http://www.wormbase.org/db/gene/gene?name=deps-1;class=Gene









http://www.wormbase.org/db/gene/strain?name=N2;class=Strain

http://www.wormbase.org/db/gene/strain?name=SS747;class=Strain

http://www.wormbase.org/db/gene/transgene?name=bnIs1;class=Transgene

http://www.wormbase.org/db/gene/gene?name=pie-1;class=Gene



http://www.wormbase.org/db/gene/gene?name=unc-119;class=Gene

http://www.wormbase.org/db/gene/strain?name=ZT3;class=Strain

http://www.wormbase.org/db/gene/gene?name=csr-1;class=Gene


http://www.wormbase.org/db/gene/variation?name=fj54

http://www.wormbase.org/db/gene/rearrange?name=nT1;class=Rearrangement

http://www.wormbase.org/db/gene/transgene?name=qIs51;class=Transgene


http://www.wormbase.org/db/gene/variation?name=tm892

http://www.wormbase.org/db/gene/gene?name=unc-24;class=Gene

http://www.wormbase.org/db/gene/strain?name=NL2098;class=Strain

http://www.wormbase.org/db/gene/gene?name=rrf-1;class=Gene

http://www.wormbase.org/db/gene/variation?name=pk1417

http://www.wormbase.org/db/gene/strain?name=NL2099;class=Strain


http://www.wormbase.org/db/gene/variation?name=pk1426

http://www.wormbase.org/db/gene/gene?name=ego-1;class=Gene


http://www.wormbase.org/db/gene/variation?name=om84

http://www.wormbase.org/db/gene/variation?name=r459

http://www.wormbase.org/db/gene/strain?name=SS747;class=Strain


http://www.wormbase.org/db/gene/strain?name=HT115;class=Strain








http://www.wormbase.org/db/gene/gene?name=pgl-1;class=Gene




http://www.wormbase.org/db/gene/gene?name=act-2;class=Gene












http://www.wormbase.org/db/gene/gene?name=drh-3;class=Gene

http://www.wormbase.org/db/gene/strain?name=HT115;class=Strain

http://www.wormbase.org/db/gene/gene?name=B0464.7;class=Gene

http://www.wormbase.org/db/gene/gene?name=atx-2;class=Gene

http://www.wormbase.org/db/seq/clone?name=Y55F3A;class=Clone

targets cct-8, JA:Y65B4B_13.b targets imb-5, JA:ZK1058.2 targetsrpn-1, JA:Y47D9A.d targets rps-3, JA:C24H12.6 targets an AT-rich area with multiple targets, JA:T05H10.3 targets F15E11.5,and JA:ZK675.1 targets F59E12.11.

Western blot analysis: L3 larvae were fed control or csr-1(RNAi) bacteria for 30 hr. Forty adult worms boiled in SDS–PAGE sample buffer were loaded in each lane. Blots wereprobed with 1:50,000 rabbit anti-PGL-1 and 1:1000 mouse anti-tubulin [Sigma (St. Louis) DM 1a] for a loading control.Normalized ratios of PGL-1 were compared between sixsamples of control and csr-1(RNAi) lysates.

RESULTS

Multiple components are required for the properorganization and localization of PGL-1: To identifyeffectors of P-granule assembly, stability, and function,

we used the Ahringer RNAi feeding library (Kamath

et al. 2003), which targets 16,757 genes (87% of the C.elegans genome), to screen for abnormal accumulationand/or localization of GFP-tagged PGL-1 in adult germlines and their embryonic progeny (Figure 1A). Histor-ically, PGL-1 antibody and GFPTPGL-1 have been usedto identify and characterize P granules, so often theterms P granules and PGL-1 granules are used inter-changeably; however, the phenotypes observed in ourscreen rely solely on PGL-1 localization unless otherwisestated. Our visual GFP screen allowed us to observepatterns that deviated from normal GFPTPGL-1 pat-terns, including differences in GFP intensity, subcellu-lar and organismal distribution, and granule shapethroughout adult germ-line and embryonic develop-ment (Table 1; Figure 1, B and C). We initially identified

Figure 1.—Genomewide GFPTPGL-1 screen. (A) GFPTPGL-1 screen strategy. L3/L4 stage worms were placed on bacteriafrom the Ahringer RNAi feeding library for 30 hr at 24�. GFPTPGL-1 phenotypes were examined in the adult (P0) germ lineof living animals and their embryonic progeny (F1) under a fluorescence dissecting microscope. (B) RNAi depletion of 173 dif-ferent genes caused GFPTPGL-1 phenotypes in F1 embryos and/or phenotypes in P0 germ lines. The range of phenotypes in-cluded diffuse, high, low, and undetectable (und.) GFPTPGL-1 accumulation. ‘‘Diffuse’’ GFPTPGL-1 included both dispersedsmall granules and more homogeneously distributed GFPTPGL-1. Diffuse GFPTPGL-1 in embryos was either throughout theembryo (see glh-1) or restricted to germ-line progenitor (P) cells. (C) GFPTPGL-1 accumulation in the P0 germ line (top)and F1 embryos (bottom) of wild-type, glh-1, and csr-1 RNAi worms showing wild-type, diffuse, and high-accumulation phenotypes,respectively. Wild-type and control embryos display small GFPTPGL-1 granules in somatic cells from the �24- to �200-cell stage.Small somatic GFPTPGL-1 granules are much more prevalent in glh-1 and csr-1 RNAi embryos. csr-1 RNAi embryos also oftendisplay large perinuclear GFPTPGL-1 granules in multiple cells (also see Figure 7D). Bars, 25 mm.

C. elegans P-Granule Regulators 1399

http://www.wormbase.org/db/gene/gene?name=cct-8;class=Gene

http://www.wormbase.org/db/gene/gene?name=ZK1058.2;class=Gene

http://www.wormbase.org/db/gene/gene?name=rpn-1;class=Gene

http://www.wormbase.org/db/seq/clone?name=Y47D9A;class=Clone

http://www.wormbase.org/db/gene/gene?name=rps-3;class=Gene

http://www.wormbase.org/db/gene/gene?name=C24H12.6;class=Gene

http://www.wormbase.org/db/gene/gene?name=T05H10.3;class=Gene

http://www.wormbase.org/db/gene/gene?name=F15E11.5;class=Gene

http://www.wormbase.org/db/gene/gene?name=ZK675.1;class=Gene

http://www.wormbase.org/db/gene/gene?name=F59E12.11;class=Gene












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ecl

ass

cod

ecA

lter

edle

vel

Alt

ered

pat

tern

Alt

ered

leve

ldA

lter

edp

atte

rnSt

ain

edSa

me

asG

FP

eif-

3.F

D2

01

3.7

Eu

kary

oti

ctr

ansl

atio

nin

itia

tio

nfa

cto

r3

sub

un

it5

RL

ow

No

XX

elc-

1Y

82

E9

BR

.15

Elo

ngi

nC

UU

nd

etec

tab

leU

nd

.Ye

sX

Xem

b-9

K0

4H

4.1

Co

llag

enL

ow

No

Xfb

xa-8

1T

24

C2

.4F

-bo

xd

om

ain

UL

ow

No

Xfb

xb-5

1K

05

F6.3

F-b

ox

do

mai

nU

Hig

hH

igh

inP

cell

sL

arge

gran

ule

sN

oX

X

fbxb

-60

F55

C9

.7F

-bo

xd

om

ain

UH

igh

No

Xfl

p-1

3F3

3D

4.3

FM

RF

-like

amid

en

euro

tran

smit

ter

Hig

hH

igh

inP

cell

sD

iffu

sein

som

aN

oX

glb-

27

W0

1C

9.5

Un

kno

wn

Lo

wN

oX

gon

-1F2

5H

8.3

Secr

eted

met

allo

pro

teas

e(M

PT

)L

ow

Dif

fuse

No

XX

gsk-

3Y

18

D1

0A

.5G

lyco

gen

syn

thas

eki

nas

ere

qu

ired

for

po

lari

zati

on

of

EM

San

dP

2b

last

om

eres

CL

ow

Dif

fuse

inm

ult

iple

cell

sYe

s

his-

40

K0

3A

1.1

Sim

ilar

ity

toh

isto

nes

(pse

ud

oge

ne?

)D

iffu

sein

Pce

lls

Yes

his-

48

B0

03

5.8

H2B

his

ton

eL

ow

Yes

hsp-

6C

37

H5

.8H

SP70

-like

mit

och

on

dri

alch

aper

on

eL

ow

No

Xif

y-1

C2

7A

2.3

Lig

and

of

Fiz

zyp

rote

in.

Secu

rin

?C

Dif

fuse

Yes

imb-

4Z

K7

42

.1E

xpo

rtin

-1/

CR

M1

nu

clea

rex

po

rtin

ND

iffu

sein

Pce

lls

No

XX

imb-

5Y

48

G1

A.5

a,b

CSE

1/C

AS-

like

nu

clea

rim

po

rtin

ND

iffu

sein

Pce

lls

No

XX

inx-

8Z

K7

92

.2In

nex

in/G

AP

jun

ctio

nco

mp

on

ent

Lo

wN

oX

let-

70

M7

.1E

2u

biq

uit

in-c

on

juga

tin

gen

zym

eU

Lo

wD

iffu

seYe

sle

t-7

11

F57

B9

.2N

OT

1d

ead

enyl

atio

nco

mp

on

ent

Hig

hD

iffu

seH

igh

No

XX

let-

92

F38

H4

.9C

atal

ytic

sub

un

ito

fP

P2A

ph

osp

hat

ase

Dif

fuse

Yes

XX

let-

99

K0

8E

7.3

DE

Pd

om

ain

.Sp

ind

leo

rien

tati

on

CL

ow

No

XX

lin

-41

C1

2C

8.3

RIN

Gfi

nge

rB

bo

xco

iled

-co

ilp

rote

inL

ow

inso

me

No

X

lmn

-1D

Y3

.2N

ucl

ear

typ

eB

lam

inN

Dif

fuse

inP

cell

sN

oX

Xm

ag-1

R0

9B

3.5

Mag

on

ash

i.E

xon

–exo

nju

nct

ion

fact

or

SL

ow

inso

me

No

X

mbk

-2F4

9E

11

.1M

inib

rain

DYR

K2

kin

ase

CL

ow

No

XX

mcm

-2Y

17

G7

B.5

MC

Mli

cen

sin

gfa

cto

r2

ML

ow

Dif

fuse

inP

cell

sYe

sX

Xm

cm-4

Y3

9G

10

AR

.14

MC

Mli

cen

sin

gfa

cto

r4

ML

ow

Dif

fuse

inP

cell

s,p

un

cta

inso

ma

Yes

XX

mcm

-5R

10

E4

.4M

CM

lice

nsi

ng

fact

or

5M

Dif

fuse

inP

cell

sYe

sX

Xm

cm-6

ZK

63

2.1

MC

Mli

cen

sin

gfa

cto

r6

MD

iffu

sein

Pce

lls,

hig

hp

un

cta

inso

ma

Yes

XX

(con

tin

ued

)


TA

BL

E1

(Co

nti

nu

ed)

GF

PT

PG

L-1

inth

eP

0ge

rmli

ne

GF

PT

PG

L-1

inF

1em

bry

os

PG

L-1

ph

eno

typ

ep

rece

ded

by

gen

eral

emb

ryo

def

ects

?

En

do

gen

ou

sP

GL

-1

Gen

en

ame

Wo

rmb

ase

IDD

escr

ipti

on

Gen

ecl

ass

cod

ecA

lter

edle

vel

Alt

ered

pat

tern

Alt

ered

leve

ldA

lter

edp

atte

rnSt

ain

edSa

me

asG

FP

mcm

-7F3

2D

1.1

0M

CM

lice

nsi

ng

fact

or

7M

Dif

fuse

inP

cell

sYe

sX

Xm

dt-6

Y5

7E

12

AL

.5T

ran

scri

pti

oin

alm

edia

tor

Med

6-li

keL

ow

Dif

fuse

Yes

mel

-26

ZK

85

8.4

Ad

apto

ro

fth

eE

3u

biq

uit

inli

gase

UD

iffu

seYe

sm

ix-1

M1

06

.1SM

C2-

like

con

den

sin

gco

mp

lex

fact

or

Lo

wN

oX

nm

y-2

F20

G4

.3N

on

mu

scle

myo

sin

IIC

Lo

wD

iffu

seL

ow

Dif

fuse

Yes

XX

npp

-10

ZK

32

8.5

NU

P98

-like

nu

cleo

po

rin

ND

iffu

sein

Pce

lls

No

XX

npp

-20

Y7

7E

11

A.1

3N

ucl

eop

ori

nN

Lo

wD

iffu

seYe

sX

Xn

pp-6

F56

A3

.3N

UP

160-

like

nu

cleo

po

rin

ND

iffu

sein

Pce

lls

No

XX

npp

-7T

19

B4

.2N

ucl

eop

ori

nN

Dif

fuse

inP

cell

sN

oX

Xn

pp-9

F59

A2

.1N

ucl

eop

ori

nN

Dif

fuse

inP

cell

sN

oX

Xpa

a-1

F48

E8

.5R

egu

lato

rysu

bu

nit

of

PP

2Ap

ho

sph

atas

eD

iffu

seYe

sX

Xpa

r-1

H3

9E

23

.1Se

rin

e/th

reo

nin

eki

nas

eC

Lo

wo

ru

nd

.D

iffu

seN

oX

Xpa

r-2

F58

B6

.3C

3HC

4-ty

pe

RIN

G-fi

nge

rp

rote

inC

Lo

wo

ru

nd

.Ye

spa

r-5

M1

17

.214

-3-3

-co

nta

inin

gp

rote

inC

Lo

wD

iffu

seN

oX

Xpa

r-6

T2

6E

3.3

PD

Z-d

om

ain

-co

nta

inin

gp

rote

inC

Lo

wo

ru

nd

.D

iffu

seYe

spa

s-4

C3

6B

1.4

a4

20S

pro

teas

om

eco

resu

bu

nit

PL

ow

or

un

d.

Yes

XX

pas-

5F2

5H

2.9

a5

20S

pro

teas

om

eco

resu

bu

nit

PL

ow

or

un

d.

Yes

pbs-

1K

08

D1

2.1

b1

20S

pro

teas

om

eco

resu

bu

nit

PL

ow

Dif

fuse

Yes

XX

pbs-

3Y

38

A8

.2b

320

Sp

rote

aso

me

core

sub

un

itP

Un

d.

inso

me

Yes

pbs-

4T

20

F5.2

b4

20S

pro

teas

om

eco

resu

bu

nit

PD

iffu

seYe

spb

s-5

K0

5C

4.1

b5

20S

pro

teas

om

eco

resu

bu

nit

PD

iffu

seL

ow

Dif

fuse

Yes

XX

pbs-

6C

02

F5.9

b6

20S

pro

teas

om

eco

resu

bu

nit

PD

iffu

seYe

spb

s-7

F39

H1

1.5

b7

20S

pro

teas

om

eco

resu

bu

nit

PD

iffu

seYe

spg

l-1Z

K3

81

.4R

GG

bo

xm

oti

fs.

Un

d.

No

phi-

10

Y4

6G

5A

.4D

EA

D-b

ox

RN

Ah

elic

ase

BR

R2

SH

igh

Hig

hN

oX

Xph

i-1

2M

03

F8.3

HA

Tre

pea

t.m

RN

Asp

lici

ng

SL

ow

No

XX

phi-

21

T0

5H

4.6

Tra

nsl

atio

nre

leas

efa

cto

rL

ow

Dif

fuse

No

XX

phi-

6K

02

F2.3

snR

NP

-ass

oci

ated

fact

or

SH

igh

Hig

hin

Pce

lls

Dif

fuse

inso

ma

No

XX

phi-

62

F49

C1

2.1

2U

nkn

ow

nL

ow

No

?X

Xph

i-7

D1

08

1.8

mR

NA

spli

cin

gp

rote

inC

DC

5(M

yb)

SH

igh

Dif

fuse

Hig

hin

Pce

lls

No

XX

plk-

1C

14

B9

.4Se

rin

e/th

reo

nin

ep

olo

-like

kin

ase

CL

ow

Dif

fuse

Yes

XX

pri-

1F5

8A

4.4

DN

AP

ol

a-p

rim

ase

sub

un

itD

Lo

wN

oX

Xpr

p-8

C5

0C

3.6

U5

snR

NA

-ass

oci

ated

spli

cin

gfa

cto

rS

Hig

hD

iffu

seH

igh

Dif

fuse

No

XX

prs-

1T

20

H4

.3P

roli

ne

tRN

Ali

gase

Lo

wN

o?

XX

puf-

3Y

45

F10

A.2

PU

Ffa

mil

yR

NA

-bin

din

gp

rote

inD

iffu

seN

oX

X

(con

tin

ued

)


TA

BL

E1

(Co

nti

nu

ed)

GF

PT

PG

L-1

inth

eP

0ge

rmli

ne

GF

PT

PG

L-1

inF

1em

bry

os

PG

L-1

ph

eno

typ

ep

rece

ded

by

gen

eral

emb

ryo

def

ects

?

En

do

gen

ou

sP

GL

-1

Gen

en

ame

Wo

rmb

ase

IDD

escr

ipti

on

Gen

ecl

ass

cod

ecA

lter

edle

vel

Alt

ered

pat

tern

Alt

ered

leve

ldA

lter

edp

atte

rnSt

ain

edSa

me

asG

FP

ran

-1K

01

G5

.4R

AN

-ass

oci

ated

GT

P-b

ind

ing

pro

tein

NL

ow

Dif

fuse

No

XX

ran

-4R

05

D1

1.3

Nu

clea

rtr

ansp

ort

fact

or

2(N

TF

2)N

Lo

wD

iffu

sein

Pce

lls

No

XX

rnp-

4R

07

E5

.14

Exo

n–e

xon

jun

ctio

nco

mp

lex

fact

or

SD

iffu

sein

Pce

lls

Yes

rnr-

1T

23

G5

.1R

ibo

nu

cleo

tid

ere

du

ctas

eL

ow

or

un

d.

Dif

fuse

inP

cell

sN

oX

Xrn

r-2

C0

3C

10

.3R

ibo

nu

cleo

tid

ere

du

ctas

eD

iffu

sein

Pce

lls

No

XX

rpa-

1F1

8A

1.5

Lar

gesu

bu

nit

of

rep

lica

tio

np

rote

inA

Dif

fuse

inP

cell

s,h

igh

pu

nct

ain

som

a

Yes

rpb-

2C

26

E6

.4R

NA

Po

lII

b-s

ub

un

itD

iffu

sein

Pce

lls

Yes

XX

rpl-1

2JC

8.3

Rib

oso

mal

pro

tein

larg

esu

bu

nit

12R

Lo

wN

oX

rpl-1

8Y

45

F10

D.1

2R

ibo

som

alp

rote

inla

rge

sub

un

it18

RL

ow

Yes

XX

rpl-2

0E

04

A4

.8R

ibo

som

alp

rote

inla

rge

sub

un

it18

aR

Lo

wN

oX

Xrp

l-36

F37

C1

2.4

Rib

oso

mal

pro

tein

larg

esu

bu

nit

36R

Lo

wN

oX

Xrp

n-1

T2

2D

1.9

aP

rote

aso

me

regu

lato

ryp

arti

cle

PD

iffu

seYe

srp

n-1

1K

07

D4

.3P

rote

aso

me

regu

lato

ryp

arti

cle

PD

iffu

sein

Pce

lls

Yes

rpn

-3C

30

C1

1.2

Pro

teas

om

ere

gula

tory

par

ticl

eP

Dif

fuse

Yes

rps-

0B

03

93

.1R

ibo

som

alp

rote

insm

all

sub

un

it2A

RL

ow

No

XX

rps-

20

Y1

05

E8

A.1

6R

ibo

som

alp

rote

insm

all

sub

un

it20

RL

ow

Yes

rps-

22

F53

A3

.3R

ibo

som

alp

rote

insm

all

sub

un

it15

aR

Lo

wN

oX

Xrp

s-2

4T

07

A9

.11

Rib

oso

mal

pro

tein

smal

lsu

bu

nit

24R

Lo

wN

oX

rps-

3C

23

G1

0.3

aR

ibo

som

alp

rote

insm

all

sub

un

it3

RL

ow

No

Xrp

s-5

T0

5E

11

.1R

ibo

som

alp

rote

insm

all

sub

un

it5

RL

ow

No

Xrp

s-8

F42

C5

.8b

Rib

oso

mal

pro

tein

smal

lsu

bu

nit

8R

Lo

wD

iffu

seYe

srp

s-9

F40

F8.1

0R

ibo

som

alp

rote

insm

all

sub

un

it9

RL

ow

No

XX

rpt-

1C

52

E4

.4P

rote

aso

me

regu

lato

ryp

arti

cle

PL

ow

Dif

fuse

Yes

rpt-

3F2

3F1

2.6

Pro

teas

om

ere

gula

tory

par

ticl

eP

Dif

fuse

Yes

rpt-

5F5

6H

1.4

Pro

teas

om

ere

gula

tory

par

ticl

eP

Dif

fuse

Yes

rsp-

7D

20

89

.1SR

pro

tein

(sp

lici

ng

fact

or)

7S

Lo

wD

iffu

sein

Pce

lls

No

XX

ruvb

-1C

27

H6

.2R

uvB

-like

DN

Ah

elic

ase

Lo

wN

oX

ruvb

-2T

22

D1

.10

Ru

vB-li

keD

NA

hel

icas

eL

ow

No

Xsc

p-1

D2

01

3.8

WD

rep

eat,

sreb

clea

vage

pro

tein

GL

ow

inso

me

No

X

skp-

1T

27

F2.1

SKI-

bin

din

gp

rote

in(S

KIP

)o

rth

olo

gsS

Hig

hH

igh

Dif

fuse

inso

ma

No

XX

snr-

2W

08

E3

.1C

ore

spli

ceso

me

com

po

nen

tS

Lo

wYe

ssp

d-5

F56

A3

.4D

ynei

nh

eavy

chai

n1

CD

iffu

seYe

ssq

v-6

Y5

0D

4C

.4X

ylo

sylt

ran

sfer

ase

CL

ow

or

un

d.

Yes

srh-

81

W0

3F9

.7Se

rpen

tin

ere

cep

tor

clas

sH

Lo

wN

oX

sri-

25

C4

1G

6.1

0Se

rpen

tin

ere

cep

tor

clas

sI

Lo

wN

oX

(con

tin

ued

)


TA

BL

E1

(Co

nti

nu

ed)

GF

PT

PG

L-1

inth

eP

0ge

rmli

ne

GF

PT

PG

L-1

inF

1em

bry

os

PG

L-1

ph

eno

typ

ep

rece

ded

by

gen

eral

emb

ryo

def

ects

?

En

do

gen

ou

sP

GL

-1

Gen

en

ame

Wo

rmb

ase

IDD

escr

ipti

on

Gen

ecl

ass

cod

ecA

lter

edle

vel

Alt

ered

pat

tern

Alt

ered

leve

ldA

lter

edp

atte

rnSt

ain

edSa

me

asG

FP

srj-6

F31

F4.8

Serp

enti

ne

rece

pto

rcl

ass

JL

ow

No

Xsr

u-4

1Y

51

H7

BR

.6Se

rpen

tin

ere

cep

tor

clas

sU

Lo

wN

oX

srx-

38

C0

3A

7.5

Serp

enti

ne

rece

pto

rcl

ass

XL

ow

No

Xta

g-1

98

F09

G8

.2P

uta

tive

DN

ase

IIL

ow

No

Xta

g-4

9A

C7

.1G

-pro

tein

rece

pto

rL

ow

No

XX

tbb-

2C

36

E8

.5b

-Tu

bu

lin

Dif

fuse

Yes

XX

tbg-

1F5

8A

4.8

g-T

ub

uli

nC

Lo

wYe

stt

r-1

4T

05

A1

0.3

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234 genes whose depletion by RNAi caused GFPTPGL-1phenotypes. These initial positives were taken throughtwo additional rounds of screening, which narrowed ourlist of positives to 173 genes whose depletion consis-tently caused aberrant GFPTPGL-1 phenotypes (Table1). GFPTPGL-1 phenotypes observed under the fluores-cence dissecting scope were placed in broad classes ofhigh, low, or undetectable (und.) GFPTPGL-1 accumu-lation (Figure 1B, Table 1). GFPTPGL-1 dispersed in thecytoplasm instead of in perinuclear aggregates wasclassified as ‘‘diffuse’’; this category included dispersedsmall granules and PGL-1 signal homogeneously dis-tributed in the cytoplasm. Altered GFP distributionswere scored as throughout the embryo or restricted tothe germ-line blastomeres. Examples of wild-typeGFPTPGL-1 accumulation, diffuse GFPTPGL-1 inglh-1(RNAi), and high GFPTPGL-1 accumulation incsr-1(RNAi) are shown in Figure 1C.

We wanted to know if GFPTPGL-1 defects reflectdefects associated with endogenous PGL-1 or insteadare specific to the GFP-tagged transgenic protein. TheGFPTPGL-1 transgene was integrated into the genomethrough particle bombardment (Cheeks et al. 2004)and as a consequence should be present at low copynumber and shielded from transgene silencing effects.However, since the reporter uses the germ-line-specificpie-1 promoter (Cheeks et al. 2004), it is possible thatsome RNAi depletions specifically affect expression ofthe transgene and not endogenous PGL-1. It is alsopossible that the post-transcriptional processing, ex-pression, or stability of transgenic PGL-1 is affected bythe GFP tag. Therefore, we repeated RNAi against 126screen positives in wild-type (not transgenic) worms,stained with antibody against PGL-1, and examinedthe P0 germ line and F1 embryos using both widefieldfluorescence and spinning disk confocal microscopy(Table 1). In the majority of RNAi depletions that wetested (76 of 126), endogenous PGL-1 recapitulated theGFPTPGL-1 phenotypes. Furthermore, the improvedresolution of fixed and stained samples allowed us toexamine the PGL-1 phenotypes in greater detail. How-ever, subtle differences in PGL-1 levels were harder tosee in fixed and stained embryos than by GFP imaging.Fifty of 126 RNAi depletions that caused reduced levelsof GFPTPGL-1 did not cause noticeably reduced levelsof endogenous PGL-1. This set of 50 genes mayspecifically affect expression of the GFP transgene ormay result in changes in PGL-1 protein levels or distribu-tion that are more easily seen by GFP than by antibodystaining.

As expected, the list of screen positives is significantlyenriched for genes expressed in the germ line. Seventy-four of the 173 genes identified are in the set of 3144genes with germ-line-enriched expression defined byReinke et al. (2004), which is a 2.7-fold enrichment overthe number expected by chance. One hundred twenty-seven of the 173 genes identified are in the set of 4699

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genes expressed in dissected gonads as detected usingSAGE (Wang et al. 2009), which is a 3.5-fold enrichmentover the number expected by chance.

The majority of screen positives were grouped intolarge gene classes that display common phenotypes(Table 2). These classes are discussed below, followed bymore in-depth analysis of an argonaute gene identifiedin our screen.

Cell cycle/polarity/cell division: The largest group ofscreen positives can be classified by their role in the cellcycle, establishing cell polarity, and the segregation ofcellular components during early cell divisions. Thisgroup includes 28 genes of the 173 total screen positives(Table 2). In wild-type animals, P granules are evenlydistributed in oocytes and newly fertilized zygotes, butprior to each P-cell division the granules are segregatedto the cytoplasm destined for the next P-cell daughter(Strome and Wood 1982). P granules left behind insomatic daughters are disassembled or degraded (Hird

et al. 1996; Saffman and Lasko 1999; DeRenzo et al.2003; Spike and Strome 2003). RNAi depletions thatcause early cell cycle arrest or altered cell divisionpatterns and eventually embryonic lethality may leadto a diffuse or ‘‘missegregated’’ P-granule pattern. Infact, P granules have been used as a marker to study cellpolarity for .20 years, and some examples of P-granule

missegregation have been intensively studied (e.g.,Gonczy and Rose 2005; Cowan and Hyman 2007). Inour secondary and tertiary screens, we more closelyexamined whether a defective GFPTPGL-1 phenotypeappeared to be correlated with abnormal nuclearmorphology, embryo patterning defects, and/or earlyarrest (Table 1). PGL-1 patterning defects appeared toprecede general embryo defects for only 8 of the 28genes in this class (bir-1, cdc-25.1, cdc-42, let-99, mbk-2,par-1, par-5, and F22B5.10).

As shown in Figure 2, RNAi depletion of genes in thecell cycle/polarity/cell division class tended to showtwo types of phenotypes. As exemplified by par-1 andcdk-9, RNAi depletion caused PGL-1 to be present in allcells of early embryos and reduced or absent from allcells of later embryos. This pattern likely reflects bothmissegregation of P granules and failure to properlyseparate germ-line and somatic lineages, with theirdifferent abilities to retain (germ-line) vs. disassemble(somatic) P granules (Kemphues et al. 1988). Asexemplified by let-99, par-5, and cdc-25.1, RNAi de-pletion caused PGL-1 to be present in all cells of earlyembryos and to persist in numerous cells of laterembryos (Rose and Kemphues 1998; Morton et al.2002). We suspect that the extra PGL-1-containing cellsin later embryos are in most cases a consequence of the

TABLE 2

Most prominent PGL-1 phenotypes associated with each gene class

Gene class (Table 1 code) Genesb

Most common PGL-1phenotype in class

Cell cycle/polarity/cell division(C) air-1, bir-1, cdc-25.1, cdc-42, cdk-1, cdk-9, cyk-4, dhc-1,dnc-1, dom-6, egg-3, gsk-3, ify-1, let-99, mbk-2, nmy-2,par-1, par-2, par-5, par-6, plk-1, spd-5, sqv-6, tbb-2,tbg-1, zen-4, zyg-9, F22B5.10

Low PGL-1 level, likely caused bysegregation defects in earlyF1 embryos

Proteasome/ubiquitin(P/U) Proteasome: pas-4, pas-5, pbs-1, pbs-3,pbs-4, pbs-5, pbs-6, pbs-7, rpn-1, rpn-3, rpn-11,rpt-1, rpt-3, rpt-5. Ubiquitin: apc-2, elc-1, fbxa-81,fbxb-51, fbxb-60, let-70, mel-26, uba-1, ubl-1

Diffuse PGL-1 in F1 embryos

Ribosome(R) egl-45, eif-3.f, rpl-12, rpl-18, rpl-20, rpl-36, rps-0,rps-3, rps-5, rps-8c, rps-9, rps-20, rps-22, rps-24

Low PGL-1 in F1 embryos

Splicing(S) cpf-2, mag-1, phi-6, phi-7, phi-10, phi-12, prp-8,rnp-4, rsp-7, skp-1, snr-2, C06A5.1

High PGL-1 in P0 germ lines andF1 embryos

Nuclear pore/envelope/transport(N) dlc-1, imb-4, imb-5c, lmn-1, npp-6, npp-7, npp-9,npp-10, npp-20, ran-1, ran-4

PGL-1 granules not associated withthe nuclear envelope, and PGL-1diffuse in the cytoplasm of P cells

MCM licensing(M) mcm-2, mcm-4, mcm-5, mcm-6, mcm-7 Diffuse PGL-1 in the cytoplasm ofP cells; associated with severeembryonic defects

Vacuole(V) unc-32, vha-2, vha-13, vha-19, vps-11 Low PGL-1 in F1 embryosChaperonin containing TCP-1

(CCT)(T)cct-2, cct-3, cct-7, cct-8c Low and diffuse PGL-1 in

F1 embryosGolgi/ER(G) arl-1, scp-1, B0361.10, F59E10.3 Low PGL-1 in F1 embryosOthera 59 genesUnknown 8 genes

a Groups with fewer than three genes not shown.b Genes in boldface type exhibit the most common PGL-1 phenotype in their class (described in right column).c Genes represented multiple times in the screen.




http://www.wormbase.org/db/gene/gene?name=bir-1;class=Gene

http://www.wormbase.org/db/gene/gene?name=cdc-25.1;class=Gene

http://www.wormbase.org/db/gene/gene?name=cdc-42;class=Gene

http://www.wormbase.org/db/gene/gene?name=let-99;class=Gene

http://www.wormbase.org/db/gene/gene?name=mbk-2;class=Gene

http://www.wormbase.org/db/gene/gene?name=par-1;class=Gene


http://www.wormbase.org/db/gene/gene?name=F22B5.10;class=Gene


http://www.wormbase.org/db/gene/gene?name=cdk-9;class=Gene




http://www.wormbase.org/db/gene/gene?name=cdc-25.1;class=Gene



earlier segregation defects, but another interestingpossibility is that they arise by extra divisions of the P-cell lineage.

Proteasome/ubiquitin: The next largest group ofscreen positives contains 23 protein degradation com-ponents from the proteasome and ubiquitin pathways(Table 2). RNAi depletion of the majority of genes inthis class resulted in a characteristic GFPTPGL-1 phe-notype: the uterus was filled with early embryos thatexhibit a diffuse PGL-1 distributed throughout theembryo (Table 1, Figure 3A). Often the embryonicphenotype was accompanied by diffuse PGL-1 distribu-tion in the P0 germ line. The PGL-1 phenotypedisplayed by this class is likely a result of compromisedprotein degradation mechanisms, which may stabilizePGL-1, and by extension P-granule proteins, in both theP0 germ line and in embryos (DeRenzo et al. 2003; Spike

and Strome 2003). This phenotype is probably com-pounded by the early embryonic arrest associated with

the depletion of all but 4 of the 23 genes. These 4include ubl-1(RNAi), whose RNAi depletion causedPGL-1 granules to persist in multiple cells prior toembryonic arrest, and the F-box-containing genes fbxa-81, fbxb-51, and fbxb-60, whose depletion did not causeembryonic lethality under our experimental condi-tions. This class emphasizes the importance of proteindegradation in regulating P-granule patterns in germlines and embryos.

Ribosome: We obtained 14 genes that encode ribo-somal components in our screen (Table 2). In contrastto the groups mentioned above, most PGL-1 pheno-types in this class (11/14) preceded apparent cell/embryo death (egl-45, eif-3.f, rpl-12, rpl-20, rpl-36, rps-0,rps-3, rps-5, rps-9, rps-22, and rps-24) (Table 1). RNAidepletion of all 14 of these genes resulted in reducedPGL-1 signal and small PGL-1 aggregates (Figure 3B).While compromised translation of GFPTPGL-1 orendogenous PGL-1 may account for this phenotype, it

Figure 2.—Cell cycle/cell division/polarity class. Antibody-stained PGL-1 (grayscale, green in merge) and nuclear pores (red)and DAPI-stained DNA (blue) in fixed embryos at the 4-,�30-, and�150-cell stages of development are shown. PGL-1 segregationdefects are shown at the 4-cell stage of par-1, cdk-9, cdc-25.1, par-5, and let-99 RNAi embryos. In later embryos, the absence of germ-line progenitors (par-1) or improper P-granule segregation accompanied by general embryo defects (cdk-9) caused PGL-1 to below or undetectable. Embryos depleted of cdc-25.1, par-5, and let-99 have multiple P-granule-containing cells later in development.Bar, 10 mm.







http://www.wormbase.org/db/gene/gene?name=ubl-1;class=Gene


http://www.wormbase.org/db/gene/gene?name=fbxa-81;class=Gene

http://www.wormbase.org/db/gene/gene?name=fbxa-81;class=Gene

http://www.wormbase.org/db/gene/gene?name=fbxb-51;class=Gene

http://www.wormbase.org/db/gene/gene?name=fbxb-60;class=Gene


http://www.wormbase.org/db/gene/gene?name=egl-45;class=Gene

http://www.wormbase.org/db/gene/gene?name=rpl-12;class=Gene













is also possible that the accumulation of P granules isdependent upon active translation. In comparison tosomatic cell differentiation, which relies heavily ontranscriptional regulation, this ‘‘ribosome’’ class empha-sizes the crucial role of post-transcriptional regulationin the identity and development of the germ line.Indeed, recent transcript profiling from dissectedgerm lines revealed that a relatively large percentageof germ-line expressed mRNAs are involved in trans-lation and ribosome structure and biogenesis (Wang

et al. 2009). P-granule integrity and distribution maybe sensitive to the translational status of germ cells andearly embryos.

Splicing: Our screen identified 12 genes that encodeknown or putative splicing factors (Table 2). The PGL-1phenotypes of 9 of these genes appeared to precedecell/embryo death (cpf-2, mag-1, phi-6, phi-7, phi-10, phi-12, prp-8, rsp-7, and skp-1) (Table 1). Several PGL-1phenotypes were shared by multiple members of thesplicing class of genes (Figure 4). RNAi depletion of 5genes (phi-6, phi-7, phi-10, prp-8, and skp-1) resulted inincreased PGL-1 signal in the germ line of P0 adults andin F1 embryos. RNAi depletion of 9 genes (cpf-2, phi-6,phi-7, phi-10, phi-12, prp-8, rnp-4, rsp-7, and skp-1) resultedin some diffuse PGL-1 in the embryonic germ-line

blastomeres and PGL-1 granules that are dissociatedfrom the nuclear periphery. In wild-type embryos,between the 4- and 8-cell stages, P granules start tobecome perinuclear in the germ-line blastomere, whileembryos depleted for splicing factors often containedPGL-1 granules dispersed in the cytoplasm, whichpersisted until the embryos arrested around the 100-cell stage. This arrest was accompanied by failure of theprimordial germ cell (P4) to migrate internally.

Sm components of the splicesome have been shownto localize to germ plasm in multiple species, andprevious RNAi depletion of the C. elegans Sm proteinsdisrupted P-granule patterns in embryos (Moussa et al.1994; Barbee et al. 2002; Chuma et al. 2003; Bilinski

et al. 2004; Barbee and Evans 2006). However, previousRNAi depletion of other splicing components did notdisrupt P-granule patterns in embryos (Barbee et al.2002), suggesting that P-granule integrity and perinu-clear localization in embryonic cells do not depend onpre-mRNA splicing. Our screen demonstrated that atleast some pre-mRNA splicing factors are necessary forproper PGL-1 localization to granules at the nuclearperiphery in embryos. Since the embryonic germ-lineblastomeres are not actively engaged in transcription(Seydoux et al. 1996), the requirement for proper

Figure 3.—Proteasome/ubiquitin and ribo-some classes. GFPTPGL-1(green) in live worms (A)and PGL-1 (green), nu-clear pores (red), andDNA (blue) in fixed em-bryos (B) are shown.Dashed boxes indicate thelocation of zoomed imageson the right. (A) Protea-some/ubiquitin class. De-pletion of proteasome(e.g., pbs-5) and ubiquitincomponents often causesearly embryonic arrest withdiffuse PGL-1 throughoutthe embryo. Arrested em-bryos accumulate in theuterus (bracketed bar) af-ter proteasome or ubiqui-tin depletion, whichcreates a distinctiveGFPTPGL-1 phenotype.(B) Ribosome class. Four-cell embryos are shown.Depletion of core ribo-somal components such asrps-0, rpl-20, and rpl-36 re-sults in reduced PGL-1and smaller granules (ar-rowheads). Bars: (A) 50 mmand (B) 10 mm.



http://www.wormbase.org/db/gene/gene?name=cpf-2;class=Gene

http://www.wormbase.org/db/gene/gene?name=mag-1;class=Gene

http://www.wormbase.org/db/gene/gene?name=prp-8;class=Gene

http://www.wormbase.org/db/gene/gene?name=rsp-7;class=Gene

http://www.wormbase.org/db/gene/gene?name=skp-1;class=Gene





http://www.wormbase.org/db/gene/gene?name=cpf-2;class=Gene


http://www.wormbase.org/db/gene/gene?name=rnp-4;class=Gene

http://www.wormbase.org/db/gene/gene?name=rsp-7;class=Gene






splicing regulation likely reflects that maternally gener-ated mRNAs must be properly spliced for P granules tolocalize normally in embryos. Whether properly splicedmRNAs or the process of splicing affect P-granule pattern-ing directly or indirectly remains to be determined.

We noted that several splicing factors obtained in ourscreen represent one subunit of multisubunit splicingcomplexes. This finding raised two possibilities: theother subunits in the complex do not participate inregulating P-granule distributions or the other subunitswere missed in our single-pass RNAi screen. To distin-guish between these possibilities, we tested all subunitsof the SF3b complex, which is absolutely essential forpre-mRNA splicing (Will et al. 1999). This complexcontains seven proteins, of which six have clear homo-logs in worms, encoded by the genes W03F9.10, phf-5,

C46F11.4, sap-49, phi-11, and phi-6 (supporting informa-tion, Table S1). Only phi-6 was identified in our screen.Of the six worm genes, only phi-6, phi-11, sap-49, andC46F11.4 are targeted by the Ahringer RNAi library. Wedepleted these four genes by RNAi and found that inaddition to phi-6, loss of both phi-11 and sap-49 resultedin diffuse PGL-1 in the embryonic germ-line blasto-meres, PGL-1 granules that are dissociated from thenuclear periphery, and failure of P4 to migrate internally(Figure 4B, Table S1). Late embryonic arrest was used asa measure of RNAi effectiveness, and unlike phi-6, phi-11, and sap-49, depletion of C46F11.4 did not result inlate embryonic arrest, diffuse PGL-1, or a P4 migrationdefect. Interestingly, the Ahringer RNAi clone targetingC46F11.4 contains a short sequence that is present innumerous copies throughout the genome, potentially

Figure 4.—Splicing class. Antibody-stained PGL-1 (green), nuclear pores (red), and DAPI-stained DNA (blue) in fixed embryosare shown. Dashed boxes indicate the location of zoomed images on the right. (A) Approximately 16-cell (left) and �150-cell(right) embryos. In wild-type embryos, PGL-1 granules are attached to the periphery of the nucleus in the primordial germ cellP4 (left embryo, �16 cells) and its daughters Z2 and Z3 (right embryo, �150 cells). P4, Z2, and Z3 nuclei are marked with asterisks.phi-12, phi-10, prp-8, phi-6, and skp-1 RNAi embryos contain P granules that are detached from the periphery of the nucleus (arrow-heads). Higher levels of diffuse PGL-1 in the cytoplasm of Z2/Z3 cells of phi-12, phi-10, and prp-8 embryos can be observed in theareas circumscribed by dashed lines, and Z2/Z3 fail to migrate internally. (B) SF3b complex components phi-6, phi-11, and sap-49RNAi embryos contain PGL-1 granules that are detached from the nucleus, and Z2/Z3 fail to migrate internally. Bar, 10 mm.


http://www.wormbase.org/db/gene/gene?name=W03F9.10;class=Gene

http://www.wormbase.org/db/gene/gene?name=phf-5;class=Gene

http://www.wormbase.org/db/gene/gene?name=C46F11.4;class=Gene

http://www.wormbase.org/db/gene/gene?name=sap-49;class=Gene

http://www.genetics.org/cgi/data/genetics.109.110171/DC1/1













diluting the effectiveness of this RNAi clone. Takentogether, our results suggest that the SF3b complex andat least certain aspects of pre-mRNA splicing areessential for proper PGL-1 localization. These resultsexemplify the limitations of RNAi screens (incompletegene coverage, ineffective RNAi, and missing pheno-types in single-pass screens) but also illustrate how RNAiscreen positives can lay the foundation for more targetedapproaches.

Nuclear pore/envelope/transport: We identified 11nuclear pore or nuclear pore/envelope associatedfactors in our screen (Table 2). RNAi depletion of 9 ofthese resulted in a distinctive phenotype that appearedto precede cell death in early embryos (imb-4, imb-5,lmn-1, npp-6, npp-7, npp-9, npp-10, ran-1, and ran-4). Incontrast to the perinuclear association of PGL-1 in themore than four-cell stage wild-type embryos, most PGL-1-containing granules in embryos depleted for nuclearpore components remained detached from the nuclearenvelope (Figure 5A). The finding that .75% ofnuclear pores are overlaid by P granules in the adultgerm line (Pitt et al. 2000) predicted that loss ofnuclear pore components might compromise associa-tion of P granules with the nuclear periphery.

Other labs have previously demonstrated a require-ment for the nuclear transportins IMB-2, IMB-3, IMB-5,and IMA-3 to maintain P-granule integrity or perinu-clear localization ( J. Ahringer, personal communica-tion; Geles and Adam 2001). There are three imb-5RNAi clones in the Ahringer RNAi library (two correctlyand one incorrectly annotated); our screen identifiedall three clones as causing PGL-1 granules to be de-tached from the nuclear periphery. However, our screendid not identify imb-2, imb-3, or ima-3. In targeted retestsof those three RNAi clones, depletion of imb-2 and imb-3resulted in detachment of PGL-1 from the nuclearperiphery of embryos (Table S2), while depletion ofima-3 did not. We note that a stronger RNAi regime byGeles and Adam resulted in diffuse P granules in theadult germ line but, similar to our results, not inembryos (Geles and Adam 2001).

Our screen identified 5 of the 20 NPP proteins in C.elegans. To determine if only a subset of NPPs partic-ipates in tethering PGL-1 granules to the nuclearperiphery, or if some NPPs were missed by our screen,we more closely examined PGL-1 patterns after RNAidepleting individual NPPs. The results support bothscenarios. Thirteen of the 15 remaining NPPs arecovered in the Ahringer RNAi library. Of these 13, RNAidepletion of 4 (npp-1, npp-3, npp-8, and npp-19) causedPGL-1 to be detached from the nuclear periphery in ahigh enough proportion of embryos that they shouldhave emerged as screen positives (Table S2). Thus, ourscreen was successful in identifying 5 of 9 of the geneswhose depletion results in high penetrance detachmentof PGL-1 from the nuclear periphery. We missed 4genes. Interestingly, RNAi depletion of 3 genes (npp-14,

npp-15, and npp-16) caused late embryonic lethality,demonstrating the effectiveness of RNAi, but did notcause any apparent defects in PGL-1 patterns. Thesefindings suggest that the localization of PGL-1 granulesto the nuclear periphery depends on many but not allNPPs.

Our results contribute to the emerging view that thelocalization of P granules to the nuclear periphery reliesheavily upon underlying nuclear pores and nucleartransport. It will be intriguing to determine if thislocalization depends on a direct interaction betweenP-granule and nuclear pore components, mRNAtrafficking through nuclear pores, or both. We arecurrently investigating the former possibility and thehypothesis that the GLH components of P granulesinteract via their phenylalanine–glycine (FG) domainswith the FG domains of nuclear pore proteins (seediscussion).

MCM licensing: One class of screen positives containsall five MCM licensing factors represented in the RNAilibrary (Table 2). MCM licensing factors ensure thatDNA replication occurs only once per cell cycle, so thisgroup could have been included in the cell cycle class ofpositives. We chose to put the MCM licensing factors intheir own class because depletion of all five factorscaused a similar phenotype: PGL-1 granules were nolonger perinuclear in the embryonic P cells (Figure 5B).RNAi of these five MCM factors caused severe abnor-malities, so at this time we cannot deduce whether thePGL-1 phenotype is a secondary effect of embryonicdeath due to compromised DNA replication or whetherthe MCM licensing factors serve a DNA replication-independent function that facilitates P-granule attach-ment to the nuclear periphery. In yeast and mammaliancells, MCMs vastly outnumber replication origins andtheir function is not restricted to DNA synthesis (Blow

and Dutta 2005). In Xenopus, MCMs associate withRNA polymerase II holoenzyme (Yankulov et al. 1999)and were recently found to be required for RNA poly-merase II-mediated transcription (Snyder et al. 2009).Therefore, the PGL-1 phenotype in MCM RNAi em-bryos may be independent of replication and depen-dent on transcription or transcriptional regulation ofother specific factors. It is worth noting that RNAidepletion of subunits of DNA polymerase d and DNApolymerase a-primase can delay early cell divisions andcause missegregation of P granules (Encalada et al.2000). We found that RNAi depletion of another DNApolymerase a-primase subunit, PRI-1, the large subunitof replication protein A, RPA-1, or the catalytic subunitof DNA polymerase alpha, Y47D3A.29, resulted in adiffuse cytoplasmic distribution of PGL-1 in early germ-line blastomeres. It would be interesting to investigatewhether any treatment that slows the timing of celldivisions alters P-granule distribution and segregationor whether the factors discussed above have more directeffects on P granules.



http://www.wormbase.org/db/gene/gene?name=lmn-1;class=Gene

http://www.wormbase.org/db/gene/gene?name=npp-6;class=Gene




http://www.wormbase.org/db/gene/gene?name=ran-1;class=Gene

http://www.wormbase.org/db/gene/gene?name=ran-4;class=Gene






http://www.wormbase.org/db/gene/gene?name=IMA-3;class=Gene


http://www.wormbase.org/db/gene/gene?name=imb-2;class=Gene


http://www.wormbase.org/db/gene/gene?name=ima-3;class=Gene





http://www.wormbase.org/db/gene/gene?name=ima-3;class=Gene


















http://www.wormbase.org/db/gene/gene?name=PRI-1;class=Gene

http://www.wormbase.org/db/gene/gene?name=RPA-1;class=Gene

http://www.wormbase.org/db/gene/gene?name=Y47D3A.29;class=Gene


Other small classes of screen positives: Anothersmall class of screen positives includes the actin/microtubule chaperonins cct-2, cct-3, cct-7, and cct-8(Table 2). Depletion of these CCT proteins resulted inlow and diffuse levels of PGL-1 in embryos. Severeembryonic defects prevented further characterizationof the PGL-1 phenotype, but we predict that PGL-1mislocalization (data not shown) stems from compro-mised actin-based P-granule segregation in these mu-tants. Interestingly, it was recently demonstrated thatRNAi depletion of other components of this chaper-onin complex, cct-4 and cct-6, causes ectopic expressionof PGL-1 in the intestine and hypodermis of adultworms, which may contribute to their long-lived phe-notype (Curran et al. 2009).

Remaining screen positives can be placed into ahandful of smaller categories. Some genes encodecomponents of subcellular organelles including va-cuoles, Golgi, and ER. RNAi depletion typically resultedin decreased PGL-1 signal (data not shown). We wereunable to assign 59 screen positives to a class larger thanthree members, and the functions of 8 positives are notyet predictable (Table 2).

Accumulation of P granules mediated throughmRNA homeostasis: In embryos, clusters of poly(A)1

RNAs colocalize with P granules (Seydoux and Fire

1994). In adult germ lines and embryos, SL1 sequences,which are trans-spliced onto the 59 end of most mRNAs(Zorio et al. 1994), also colocalize with P granules (Pitt

et al. 2000; Schisa et al. 2001). These and other resultssuggest that P granules transiently retain developmen-tally regulated mRNAs as they exit the nucleus (Schisa

et al. 2001). P granules may regulate associated mRNAsin various ways, including control of mRNA stabilityand/or translation, and delivery of mRNAs to the germ-line blastomeres and eventually the primordial germcells in embryos (Strome 2005). In somatic cells, mRNAdeadenylation, decapping, and degradation occur incytoplasmic RNP aggregates called P bodies, whichshare many components with P granules. For example,C. elegans P granules have been shown to contain theCCR4/Not1 deadenylase component CCF-1, as well asmultiple decapping coactivators including PATR-1,DCAP-1, DCAP-2, and CGH-1 (Navarro et al. 2001;Lall et al. 2005; Squirrell et al. 2006; Gallo et al.2008). Our screen identified several mRNA degradation

Figure 5.—Nuclear pore/envelope/transport and MCM licensing classes. PGL-1 (green), nuclear pores (red), and DNA (blue)in fixed �30-cell embryos are shown. Dashed boxes indicate the location of zoomed images on the right. (A) Nuclear pore/en-velope/transport class. Depletion of nuclear pore and nuclear pore-associated components results in the dispersal of PGL-1 gran-ules from the nuclear periphery. (B) MCM licensing class. Depletion of MCM licensing factors also causes PGL-1 granules to bedistributed throughout the cytoplasm; this phenotype is accompanied by severe embryonic defects. Bars, 10 mm.













http://www.wormbase.org/db/gene/gene?name=CCF-1;class=Gene

http://www.wormbase.org/db/gene/gene?name=PATR-1;class=Gene

http://www.wormbase.org/db/gene/gene?name=DCAP-1;class=Gene

http://www.wormbase.org/db/gene/gene?name=DCAP-2;class=Gene

http://www.wormbase.org/db/gene/gene?name=CGH-1;class=Gene

components as being required for regulation of PGL-1accumulation. Interestingly, their depletion causeddifferent PGL-1 phenotypes (Figure 6). RNAi depletionof the Not1 component LET-711 resulted in increasedPGL-1 accumulation in the adult germ line (Figure 6A)and in progeny embryos, while RNAi depletion of thedecapping factors CGH-1 and ATX-2 and the predictedSKI2 exosome homolog F01G4.3 resulted in reducedPGL-1 levels (Table 1; Figure 6B). Depletion of CGH-1and ATX-2 also caused PGL-1 to persist in numerouscells of later embryos (Figure 6B). These findings extendthe list of mRNA degradation components that are likelyto be shared between P bodies and P granules andsupport the view that the accumulation of P granules isclosely connected to mRNA levels.

CSR-1 downregulates RNA and P-granule accumula-tion through an endo-siRNA pathway in the germ line:The most striking PGL-1 phenotype observed in the P0

germ line was caused by RNAi of csr-1. Depletion of csr-1resulted in very intense PGL-1 staining and large PGL-1aggregates distributed throughout the cytoplasm of theadult germ line and embryos (Figures 1C and 7A). Todetermine if the large PGL-1 aggregates in csr-1(RNAi)germ lines are bona fide P granules, we tested for whetherthey contain other constitutive components of P gran-ules, such as the PGL-1 paralog PGL-3 (Kawasaki et al.2004) and the VASA homolog GLH-1 (Gruidl et al.1996). PGL-3 and GLH-1 colocalize with the large PGL-1aggregates in csr-1(RNAi) adult germ lines (Figure 7C).Quantitative PCR of pgl-1, pgl-3, and glh-1 transcripts inadult hermaphrodites showed a modest increase aftercsr-1 RNAi (1.69-fold for pgl-1, P ¼ 0.0053; 1.12-fold forpgl-3, P ¼ 0.0390; 2.17-fold for glh-1, P ¼ 0.0010). WhileGFPTPGL-1 reporter and antibody-stained endogenousPGL-1 appear dramatically brighter in csr-1(RNAi) worms(Figures 1C and 7A), Western blot analysis revealed amodest 1.4-fold increase in PGL-1 protein in csr-1(RNAi)worms compared to wild type (Figure S1), consistentwith the �1.7-fold change in pgl-1 transcript notedabove. The brighter, larger P granules observed incsr-1(RNAi) embryos may reflect a combination ofmodestly elevated levels of P-granule proteins andenhanced aggregation of those proteins into granules.We noted an additional phenotype in csr-1(RNAi)embryos. In many RNAi embryos (40%, n ¼ 70), PGL-1and PGL-3 granules were found in somatic blastomeres(Figure 7D). Interestingly, GLH-1 did not colocalize withthe PGLs in these ectopic granules (Figure 7D). This isreminiscent of the partial P granules (containing PGL-1and PGL-3; lacking GLH-1) observed in mutant em-bryos defective in autophagy (Zhang et al. 2009),raising the possibility that CSR-1 participates in auto-phagocytic removal of P-granule proteins from somaticcells.

CSR-1 is an Argonaute protein with endonucleolyticmRNA cleavage (slicing) activity (Yigit et al. 2006; Aoki

et al. 2007). The slicing activity of Argonautes mediates

siRNA silencing of mRNAs, which are then degraded byexonucleases in the exosome and decapping pathways(Orban and Izaurralde 2005). To determine if theformation of large P granules in csr-1(RNAi) animals isaccompanied by an increase in RNA accumulation, wedissected hermaphrodite gonads in the presence ofSYTO14, a nucleic acid stain that is used to visualizenucleolar and cytoplasmic RNA (Schisa et al. 2001).Both control(RNAi) and csr-1(RNAi) worms showedcomparable SYTO14 staining in germ-line nucleoli.However, we observed a much higher level of RNAaccumulation in the central cytoplasmic ‘‘rachis’’ of csr-1(RNAi) animals than in control worms fed empty RNAivector (Figure 7B, gonads with high levels of RNAaccumulation: control, 1 of 16 gonad arms; csr-1, 12 of18 gonad arms). Our several attempts to concomitantlyimage cytoplasmic RNA accumulations and P granuleswere unsuccessful. Thus, we do not know if they coloc-alize. The different locations and morphologies ofcytoplasmic RNA accumulations and enlarged P gran-ules in csr-1(RNAi) suggest that P granules are not thesites of excessive RNA accumulation. Previous studies

Figure 6.—mRNA degradation components are requiredfor proper P-granule accumulation. Antibody-stained PGL-1(green) and nuclear pores (red) and DAPI-stained DNA(blue) in fixed germ lines and embryos are shown. (A) Inthe germ lines of animals depleted of the Not1 deadenylaseLET-711, PGL-1 granules are larger and more numerousand more dispersed than in wild type. (B) After depletionof decapping coactivators ATX-2 and CGH-1, low levels ofPGL-1 are observed in the adult germ line, and PGL-1 is oftenobserved in multiple cells of late embryos (arrows). Bars, 10 mm.




http://www.wormbase.org/db/gene/gene?name=LET-711;class=Gene



http://www.wormbase.org/db/gene/gene?name=ATX-2;class=Gene

http://www.wormbase.org/db/gene/gene?name=F01G4.3;class=Gene



http://www.wormbase.org/db/gene/gene?name=ATX-2;class=Gene














































have shown that depletion of sperm and arrestedovulation cause RNA to accumulate in the rachis; thisphenotype is regulated by the major sperm proteinpathway (Schisa et al. 2001; Jud et al. 2008). csr-1(RNAi)animals exhibited no obvious defects in fertilization orovulation, suggesting that RNA accumulation in csr-1(RNAi) animals is not caused by loss of the major spermprotein pathway. We hypothesize that the accumulationof cytoplasmic RNA and of enlarged P granules ob-

served after depletion of CSR-1 is due to reduced slicingand degradation of mRNAs.

If loss of CSR-1 slicing activity causes enlarged anddisorganized P granules, then loss of proteins thatfunction in the CSR-1 pathway should cause similardefects. CSR-1 preferentially binds to secondary siRNAs,siRNAs that are produced from primary siRNA-targetedmRNAs by RNA-dependent RNA polymerase (RdRP)(Aoki et al. 2007). We tested whether enlarged and

Figure 7.—PGL-1 and RNA accumulation inanimals depleted of the Argonaute CSR-1. (A)Endogenous PGL-1 staining (green) in wild-typeand csr-1(RNAi) germ lines. Depletion of CSR-1results in the accumulation of large PGL-1 aggre-gates. (B) RNA staining with SYTO14 shows thatRNA accumulates in the syncytial cytoplasm ofthe adult germ line after CSR-1 is depleted.(C) Endogenous PGL-3 (green) and GLH-1(red) colocalize in large P-granule aggregates af-ter CSR-1 is depleted. (D) In csr-1(RNAi) embryos(�150-cell stage shown), PGL-3 accumulates insomatic cells, whereas GLH-1 is restricted togerm-line blastomeres. Bars, 10 mm.









disorganized PGL-1 granules are seen in mutants lack-ing the somatic RdRP RRF-1 or either of the two RdRPsthat are known to be active in the germ line, EGO-1 andRRF-3. We observed an accumulation of large PGL-1granules in ego-1 mutant germ lines (as observed byVought et al. 2005) but not in rrf-1 or rrf-3 mutant germlines (Figure 8A). The PGL-1 phenotype in ego-1 and csr-1mutants was typically more severe than the phenotypecaused by RNAi; in mutant germ lines, the large PGL-1aggregates were less perinuclear than observed follow-ing RNAi. Mutant germ lines may have less EGO-1 orCSR-1 than can be achieved by RNAi, or alternativelymutant germ lines may have compromised health andshow more severe defects. Enlarged PGL-1 granuleswere not observed in RNAi-defective mutants such asdcr-1 (Vought et al. 2005) or rde-3 (our observations,Figure 8A), suggesting that the phenotype is not due toa general defect in RNAi. Next we examined GFPTPGL-1in drh-3(RNAi) germ lines. drh-3 encodes a DExH-boxhelicase that is required for RdRP activity and sharesseveral phenotypes with ego-1 and csr-1 (Duchaine et al.2006; Yigit et al. 2006; Aoki et al. 2007; Rocheleau et al.2008; She et al. 2009). Depletion of drh-3 phenocopiedthe PGL-1 phenotype observed in ego-1 and csr-1 RNAigerm lines (Figure 8B), providing further evidence thatDRH-3 functions in a pathway with EGO-1 and CSR-1.

We wanted to see if elevated RNA accumulationcorrelates with elevated levels of PGL-1 or if the effectis specific to the depletion of csr-1. We found that, likecsr-1, depletion of both ego-1 and drh-3 also resulted inincreased cytoplasmic RNA as detected by SYTO14

staining (Table 3). We also examined other factorswhose depletion resulted in elevated PGL-1 in the adultgerm line (eft-1, let-711, phi-6, phi-7, phi-11, prp-8, and skp-1).In general, increased PGL-1 levels correlated withincreased cytoplasmic RNA. Depletion of csr-1, ego-1,and the splicing factors phi-7, phi-11, and skp-1 hadparticularly strong effects on RNA patterns (Table 3). Atthis time we do not know if the accumulation ofcytoplasmic RNA results from loss of function ofCSR-1, EGO-1, DRH-3, and the handful of RNA-bindingproteins whose depletion results in elevated PGL-1 or ifthe accumulation of RNA results from displaced andelevated PGL-1 itself. A third possibility is that P-granuleproteins and cytoplasmic RNAs interact with andstabilize each other.

CSR-1, EGO-1, and DRH-3 are thought to worktogether in a germ-line-specific endogenous siRNApathway (Maine et al. 2005; Robert et al. 2005; Duchaine

et al. 2006; Yigit et al. 2006; Aoki et al. 2007; Rocheleau

et al. 2008; She et al. 2009). Argonautes, like CSR-1, havebeen identified as germ granule components fromworms to mammals (Kotaja et al. 2006; Batista et al.2008; Wang and Reinke 2008). In addition, an RGGdomain, like that found in PGL-1 and PGL-3, in the Nterminus of CSR-1 also suggests that CSR-1 is likely toassociate with P granules. To test that prediction, weattempted to generate good anti-CSR-1 antibodies andinformative GFPTCSR-1 transgenic worms. Althoughboth antibodies and transgenic worms showed P-granulelocalization, neither passed our tests for specificity.Nevertheless, on the basis of the specific PGL-1 pheno-

Figure 8.—PGL-1 accu-mulates in numerous largegranules in the germ lineafter an endogenous siRNApathway is compromised.(A) Endogenous PGL-1staining (green) resembleswild type in the germ linesof rde-1, rrf-1, and rrf-3 mu-tants. Large PGL-1 gran-ules accumulate in thegerm lines of ego-1 andcsr-1 mutants. Most granulesare dissociated from thenuclear periphery. (B)GFPTPGL-1 accumulationin control(RNAi), drh-3(RNAi), and ego-1(RNAi)germ lines. RNAi depletionof EGO-1 and DRH-3 pro-duces the same large PGL-1granule phenotype as deple-tion of CSR-1. Bars, 10 mm.



http://www.wormbase.org/db/gene/gene?name=RRF-1;class=Gene


http://www.wormbase.org/db/gene/gene?name=RRF-3;class=Gene












http://www.wormbase.org/db/gene/gene?name=dcr-1;class=Gene



















http://www.wormbase.org/db/gene/gene?name=eft-1;class=Gene
























type of CSR-1 pathway mutants and the predictedP-granule localization of CSR-1, we hypothesize thatP granules function as a center for endogenous siRNAsilencing in the germ line and that disruption of thisendo-siRNA pathway results in not only the accumulationin the germ-line syncytium of RNA transcripts that in wildtype are silenced and degraded, but also the accumula-tion of large P granules.

DISCUSSION

Our RNAi screen proved to be an effective method toidentify genes whose depletion causes aberrant PGL-1phenotypes. One advantage of performing an RNAiscreen over a forward mutagenesis screen is that mutantsare not required to be viable and fertile. This allowed usto identify multiple gene classes, including some essentialgenes that are required for PGL-1 assembly and localiza-tion. One disadvantage of RNAi screens is missing somegenes due to ineffective RNAi or incomplete RNAilibrary coverage. To expedite our screen, RNAi wasperformed for only 30 hr, and only P0 and F1 progenywere examined. Because of the maternal contribution ofmany P-granule proteins, some phenotypes, like thatcaused by DEPS-1 loss, are not observable until the F2

generation after RNAi is started. Our P0/F1 screen didnot identify any additional genes whose depletion causesthe PGL-1 phenotypes of deps-1 and glh-1 mutants wherePGL-1 is dispersed in all cells of normally developingembryos. We do not know if DEPS-1 and GLH-1 are theonly proteins whose loss causes that specific phenotypeor if an F2 generation screen could identify more geneswith that phenotype.

Despite the limitations of RNAi screens, this type ofgenomewide screen serves as an excellent starting pointfor identification and in-depth analysis of gene familiesin particular cellular processes. For example, the 5 NPPsidentified in our screen drew our attention to nuclearpores as being involved in P-granule localization. Tar-geted retests of the remaining 13 NPP genes representedin the RNAi library identified 4 NPP genes that ourscreen should have identified, 5 NPP genes that have low-penetrance RNAi phenotypes and therefore requireother analysis strategies, and 4 NPP genes that may notparticipate in regulating P-granule localization. Anotherexample is our identification of one subunit of the SF3bsplicing complex. Targeted retests identified two addi-tional subunits. Loss of the remaining three SF3bcomponents, which are not covered or inefficientlycovered in the RNAi library, is likely to result in PGL-1phenotypes as well. Finally, a recent report used a similarRNAi screen and identified a single autophagy compo-nent, lgg-1, which is required for clearing small P granulesfrom somatic cells. Targeted tests of other autophagycomponents, by injection of double-stranded RNA toachieve stronger RNAi, revealed their involvement inclearing P granules from somatic cells (Zhang et al.

2009). Certainly, the 173 genes identified in our screenare an underestimate. We expect more stringent RNAiprocedures, such as injecting or soaking or feeding forlonger times, will identify additional genes that affectP granules in C. elegans.

The majority of genes identified in our screen weregrouped into large gene classes. While some classes werelikely identified because of P-granule segregation ordegradation defects (cell cycle/polarity/cell divisionand proteasome/ubiquitin), most classes associate Pgranules with post-transcriptional regulation in the germline. For example, splicing, nuclear pore, and ribosomalclasses emphasize the importance of mRNA maturation,nuclear transport, and translational regulation in con-trolling P-granule size and location.

Nascent transcripts are processed in the nucleus byspliceosomes prior to their export as mature mRNAs.Sm proteins are core components of nuclear spliceo-somes and are also concentrated in the cytoplasmicgerm granules of multiple species (Moussa et al. 1994;Chuma et al. 2003; Barbee et al. 2002; Bilinski et al.2004). In C. elegans, Sm proteins are required for properP-granule localization to the nuclear periphery; it hasbeen proposed that this role is independent of pre-mRNA splicing (Barbee and Evans 2006). We, too,identified the Sm protein, SNR-2, as required for properPGL-1 granule assembly. We also identified othersplicing and mRNA maturation components. Theseinclude the cleavage and polyadenylation factor CPF-2and the putative integrator complex subunit C06A5.1,both of which are involved in maturation of 39 ends ofmRNA; the putative exon–exon junction factors MAG-1and RNP-4; possible snRNP associated factor PRP-8along with SF3b splicesome components PHI-6, PHI-11, and SAP-49; and other factors whose homologs arerequired for pre-mRNA splicing, including RSP-7,

TABLE 3

Correlation of elevated PGL-1 and elevated RNA inRNAi germ lines

Target

% penetrance ofelevated PGL-1

phenotype (n ¼ 20)

% penetrance ofelevated cytoplasmic

RNA (n ¼ 10)a

Control 0 0csr-1 100 80ego-1 100 70drh-3 80 40eft-1 90 20let-711 100 40phi-6 100 10phi-7 100 80phi-11 95 60prp-8 100 30skp-1 90 80

a Based on SYTO14 staining.














http://www.wormbase.org/db/gene/gene?name=lgg-1;class=Gene

http://www.wormbase.org/db/gene/gene?name=SNR-2;class=Gene


http://www.wormbase.org/db/gene/gene?name=CPF-2;class=Gene

http://www.wormbase.org/db/gene/gene?name=C06A5.1;class=Gene

http://www.wormbase.org/db/gene/gene?name=MAG-1;class=Gene

http://www.wormbase.org/db/gene/gene?name=RNP-4;class=Gene

http://www.wormbase.org/db/gene/gene?name=PRP-8;class=Gene

http://www.wormbase.org/db/gene/gene?name=SAP-49;class=Gene

http://www.wormbase.org/db/gene/gene?name=RSP-7;class=Gene

SKP-1, PHI-7, PHI-10, and PHI-12. While the role of Smproteins in germ granules may still be independent oftheir role in splicing, our results suggest that propermRNA maturation in the nucleus affects the assemblyand localization of P granules. It will be interesting todetermine whether properly assembled P granulesrequire specific features of spliced mRNAs, splicingfactors associated with mRNAs, or mRNA exportthrough nuclear pores.

The integrity of nuclear pores appears to be vital forthe perinuclear distribution of P granules. At least 14 of20 nuclear pore components are required for PGL-1’sperinuclear distribution, as are nuclear transport factorssuch as IMB-1, IMB-5, RAN-1, and RAN-4. Nuclear laminLMN-1 is also required, as is dynein light chain-1, DLC-1.DLC-1’s yeast homolog DYN2 is a nucleoporin (Stelter

et al. 2007), and interestingly C. elegans DLC-1 was foundto interact with PGL-3 in a high-throughput bindingassay (Li et al. 2004). Therefore, it is possible that somenucleoporins, like the putative nucleoporin DLC-1,provide a physical linkage between P granules andnuclear pores. On that note, we find it interesting thatthe P-granule components GLH-1, GLH-2, and GLH-4contain multiple Phe–Gly (FG) repeat domains, do-mains that are found in many nucleoporins (Rexach

and Blobel 1995). Some FG domains form cohesivehydrophobic interactions with other FG domains in thenuclear pore (Patel et al. 2007). The close associationbetween P granules and nuclear pores, as well as similar-ities between GLH proteins and nucleoporins, has led tothe prediction that P granules serve as an extension ofnuclear pores in the germ line (Kuznicki et al. 2000;Pitt et al. 2000; Schisa et al. 2001). We are currentlytesting whether the FG domains of GLH-1, GLH-2, andGLH-4 interact with each other and with other FG-containing nucleoporins and whether FG proteins inP granules do indeed serve to extend the nuclear poreenvironment.

As RNAs transcribed in germ nuclei exit throughnuclear pores, many transcripts will encounter P gran-ules. Poly(A)1 and developmentally regulated mRNAshave been shown to accumulate in P granules (Seydoux

and Fire 1994; Schisa et al. 2001). P granules may act asa scaffold for multiple RNA processing activities, possi-bly by providing an environment that facilitates germ-line-specific binding interactions and regulates thetranslation, transport, modification, storage, and/ordegradation of RNA. Our screen results support manyof these possibilities. For example, we found thatmultiple ribosomal components are required forproper P-granule accumulation and stability. This find-ing could reflect a need for synthesis of P-granuleproteins or a requirement for ongoing translationduring the assembly of P granules.

We observed PGL-1 phenotypes when RNA dead-enlyase, decapping, and degradation components weredepleted. Depletion of LET-711, the NOT1 ortholog of

the CCR4/NOT1 mRNA deadenylation complex, hadpreviously been reported to cause PGL-1 localizationoutside of the embryonic germ line (Gallo et al. 2008).We identified LET-711 primarily on the basis of theelevated accumulation of transgenic and endogenousPGL-1 in the adult germ line. This phenotype wasobserved within 30 hr after initiation of RNAi andbefore germ-line morphology became altered, suggest-ing that the PGL-1 phenotype was a direct result of LET-711 depletion. The PGL-1 aggregates in let-711(RNAi)germ lines are smaller than those observed in csr-1mutants, but as in the case of csr-1, the enhancedaccumulation may be correlated with RNA stabilization.The accumulation of P granules may be sensitive tocytoplasmic RNA levels, requiring RNA destabilizingfactors like LET-711 and CSR-1 to control RNA levelsthrough exo- or endonucleolytic activity.

One exciting gene that emerged from our screen wascsr-1. CSR-1 is an Argonaute that exhibits endonucleo-lytic or slicing activity on RNA targets, showing apreference for using secondary siRNAs generated byRdRP activity rather than primary siRNAs generated byDicer (Aoki et al. 2007). Thus far, C. elegans is uniqueamong animals in its large number of Argonaute-encoding genes: 27 in C. elegans compared to 5 inDrosophila and 8 in humans (Hutvagner and Simard

2008). The diversity of Argonautes in C. elegans hasproven advantageous for dissecting out the multipleroles of these proteins. C. elegans Argonautes ALG-1 andALG-2 are essential for miRNA-mediated gene silenc-ing, RDE-1 for exogenous RNAi, ERGO-1 for endoge-nous RNAi, and PRG-1 and PRG-2 for piRNA-mediatedgene silencing, and a handful of other Argonautes arethought to bind secondary siRNAs but lack the ability toslice RNA targets (Grishok et al. 2001; Yigit et al. 2006;Batista et al. 2008; Das et al. 2008; Wang and Reinke

2008). csr-1 was first obtained in a germ-line cosuppres-sion screen, which suggested that it may be involved insilencing repetitive transgene arrays in the germ line(Robert et al. 2005). csr-1 worms are also partiallydeficient in RNAi (Yigit et al. 2006). It was recentlydiscovered that csr-1 shares multiple phenotypes withego-1 and drh-3, which encode an RdRP and an RdRPinteracting factor required for small endogenous RNAiin the germ line (Aoki et al. 2007; Rocheleau et al. 2008;She et al. 2009).

We discovered that csr-1 worms accumulate large Pgranules in the adult germ line and ectopic granules insomatic cells of embryos. This phenotype is accompa-nied by a dramatic increase in RNA in the centralcytoplasm of the germ line. While it was not obtained inour original screen, our targeted experiments demon-strated that depletion of EGO-1 and DRH-3 phenocopythe very specific PGL-1 phenotype of csr-1, supporting arole for CSR-1 in a germ-line-specific endo-RNAi path-way. One model proposed previously to explain theirregular P granules in ego-1 mutants is that EGO-1 may


http://www.wormbase.org/db/gene/gene?name=SKP-1;class=Gene



http://www.wormbase.org/db/gene/gene?name=RAN-1;class=Gene

http://www.wormbase.org/db/gene/gene?name=RAN-4;class=Gene

http://www.wormbase.org/db/gene/gene?name=LMN-1;class=Gene

http://www.wormbase.org/db/gene/gene?name=DLC-1;class=Gene



























http://www.wormbase.org/db/gene/gene?name=ALG-1;class=Gene

http://www.wormbase.org/db/gene/gene?name=ALG-2;class=Gene

http://www.wormbase.org/db/gene/gene?name=RDE-1;class=Gene

http://www.wormbase.org/db/gene/gene?name=ERGO-1;class=Gene

http://www.wormbase.org/db/gene/gene?name=PRG-1;class=Gene

http://www.wormbase.org/db/gene/gene?name=PRG-2;class=Gene














promote a chromatin state that regulates nuclearpore distribution and that the clumping of nuclearpore material leads to perturbed P-granule assembly(Vought et al. 2005). This model is strengthened by theobservation that EGO-1 is found in nuclear fractionsand is required for unpaired chromosomal regions toaccumulate high levels of histone H3 lysine K9 dime-thylation, a mark associated with heterochromatinassembly and transcriptional silencing (Maine et al.2005; She et al. 2009). However, we observed a differentPGL-1 phenotype when nuclear pore components weredepleted (PGL-1 granules dissociated from the nuclearperiphery in germ-line blastomeres of developing em-bryos) than we did when CSR-1, EGO-1, and DRH-3were depleted (enhanced accumulation of large PGL-1granules in the adult germ line). Loss of the nuclearenvelope component LMN-1, which also results in theclustering of nuclear pores (Liu et al. 2000), causes a P-granule phenotype that is more similar to that caused bydepletion of core nuclear pore proteins than to thelarge P-granule phenotype caused by depletion of CSR-1, EGO-1, and DRH-3. Another simple model is thatgenes typically silenced by endo-RNAi in the germ lineare desilenced when csr-1 is depleted, allowing thesegenes’ transcripts to accumulate in the cytoplasm. Innormal germ lines, these transcripts could be silenced atthe level of chromatin and regulation of transcription orat the level of exiting the nucleus into P granules wherethey are sliced by CSR-1 and subsequently degraded. Asall known P-granule components are thought to func-tion in RNA metabolism, the accumulation of large Pgranules in csr-1 animals may be due to increased levelsof cytoplasmic RNA in csr-1 germ lines. Another possi-bility is that transcripts encoding P-granule proteins arenormally downregulated by endo-RNAi, and the loss ofthis downregulation in csr-1 animals leads to the accu-mulation of large P granules. Profiling germ-line tran-scripts in csr-1 mutants may reveal which, if any, of thesemodels are correct.

The P-granule components DEPS-1 and PGL-1 areboth required for a robust exogenous RNAi response(Robert et al. 2005; Spike et al. 2008b). While PGL-1 hasnot been implicated in a specific RNAi pathway, deps-1mutants are thought to resist the effects of RNAi due to a7- to 10-fold decrease in rde-4 expression (Spike et al.2008b). RDE-4 binds dsRNA and forms a complex withDicer to produce siRNAs, and rde-4 mutants are RNAidefective (Tabara et al. 2002). Another componentidentified in screens for RNAi-defective mutants, RDE-3, is thought to act downstream of RDE-4 and Dicer.Comparison of transcriptional profiles of rde-3 and deps-1 mutants showed that nearly 30% of genes upregulatedin deps-1 mutant germ lines are also upregulated in rde-3worms, suggesting that RDE-3 and DEPS-1 functiontogether to regulate expression of at least their sharedtarget genes (Spike et al. 2008b). As DEPS-1 is aconstitutive P-granule component, it likely participates

in regulation at a post-transcriptional level. Our resultswith CSR-1, EGO-1, and DRH-3 strengthen the emergingview that P granules and RNAi are intimately related: P-granule components participate in regulating RNAi, andRNAi factors influence the structure, localization, andperhaps function of P granules. Better defining the RNAi–P-granule relationship will shed light on mechanisms ofpost-transcriptional gene regulation used by germ cells.

We thank Colin Thacker for training provided during the screen,use of equipment including the Copas Biosort, and preparation ofRNAi colonies from the Ahringer RNAi library; Robb Cundick for thedevelopment and use of a C. elegans RNAi database; Susan Mango forthe use of lab space and equipment during the first pass of screening;Julie Ahringer for unpublished information on the imb-2, imb-3, andimb-5 PGL-1 phenotypes; Xingyu She and Eleanor Maine for providingego-1 strain EL500; Andreas Rechtsteiner for comparison of germ-linedata sets; and the National Bioresource Project at Tokyo Women’sMedical College for providing the FX892 strain. This work wassupported by Ruth Kirchstein National Research Service Awardpostdoctoral fellowship GM084673 (to D.U.) and National Institutesof Health (NIH) grant GM34059 (to S.S.). Some C. elegans strains wereprovided by the Caenorhabditis Genetics Center, which is funded bythe NIH National Center for Research Resources.

Note added in proof: Since the acceptance of this article forpublication, an additional study was published that shows abnormalP granules in csr-1, drh-3, and ego-1 mutants ( J. M. Claycomb, P. J.Batista, K. M. Pang, W. Gu, J. J. Vasale et al., 2009, The ArgonauteCSR-1 and its 22G-RNA cofactors are required for holocentricchromosome segregation. Cell 139: 123–134). Claycomb et al. alsoshow that CSR-1, DRH-3, and EGO-1 colocalize with P granules.

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Communicating editor: B. J. Meyer


Supporting Information http://www.genetics.org/cgi/content/full/genetics.109.110171/DC1

A Genomewide RNAi Screen for Genes That Affect the Stability, Distribution and Function of P Granules in Caenorhabditis elegans

Dustin L. Updike and Susan Strome

Copyright © 2009 by the Genetics Society of America DOI: 10.1534/genetics.109.110171


D. L. Updike and S. Strome 2 SI

PGL-1

Tubulin

supplemental !gure 1

WT csr-1

100

75

50

FIGURE S1.—Western analysis of PGL-1 accumulation in control and csr-1(RNA) worms shows a modest increase in PGL-1 levels.


TABLE S1

RNAi of SF3b Complex

Gene Worm Homolog RNAi Clone Embryonic Lethality n=50 PGL-1 Phenotype

control empty vector 6% no

SF3b5/10 no homolog

SF3b125 C46F11.4 JA:C46F11.4 8% no

SF3b3/130 phi-6 JA:K02F2.3 98% yes

SF3b2/145 W03F9.10

SF3b14b phf-5

SF3b1/155 phi-11 JA:T08A11.2 100% yes

Sf3b4/49 sap-49 JA:C08B11.5 100% yes


TABLE S2

RNAi of Nuclear Pore Components

Target Diffuse PGL-1 in

Embryonic

Germline Cells?

Penetrance of

Diffuse PGL-1

n=50

Embryonic

Lethality n=50

control no 0% 8%

npp-6 yes 68% 100%

npp-1 yes 56% 100%

npp-2 yes 6% 86%

npp-3 yes 84% 100%

npp-4 yes 8% 54%

npp-5 yes 10% 46%

npp-8a yes 86% 100%

npp-8b yes 84% 100%

npp-8d yes 90% 100%

npp-12 yes 10% 94%

npp-14 no 0% 100%

npp-15 no 0% 36%

npp-16a no 0% 36%

npp-16b no 0% 100%

npp-17 yes 10% 30%

npp-18 no 0% 8%

npp-19 yes 38% 96%

imb-2 yes 74% 100%

imb-3a yes 4% 98%

imb-3i no 0% 10%

ima-3 no 0% 8%

a genomewide rnai screen for genes that affect the stability, … · 2009-12-11 · rnai...

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