Nature Structural & Molecular Biology: doi:10.1038/nsmb€¦ · aligning to each genomic feature. The reads corresponding to sense rRNA sequences were excluded from the analysis

Nature Structural & Molecular Biology: doi:10.1038/nsmb.3376

Supplementary Figure 1

Antisense ribosomal siRNAs were enriched in susi-1 mutant.

(A) Deep sequencing of small RNAs in wild-type N2 and susi-1(R457H) mutant animals. The relative

abundance of rRNA-related sequences is indicated. (B) Size distribution and 5'-end nucleotide preference of

sense rRNA reads in wild-type N2 and susi-1(R457H) animals. (C) Pie charts display the proportion of reads

aligning to each genomic feature. The reads corresponding to sense rRNA sequences were excluded from the

analysis. (D) Relative abundance of endogenous smalls from wild-type N2 and susi-1(R457H) animals. Source

data (for panels B-C) are available on-line.




risiRNA belongs to 22G-RNAs.

(A) Relative abundance of Argonaute-associated risiRNAs from published data sets. (B) risiRNA was pre-

treated with or without calf intestinal alkaline phosphatase (CIAP), followed by p32 labeling. Uncropped gel

image is shown in Supplementary Data Set 2. (C) risiRNA was pre-treated with guanylyl transferase followed

by p32 labeling. Uncropped gel image is shown in Supplementary Data Set 2. (D) risiRNA was pre-labeled with

p32 followed by -elimination reactions. Uncropped gel image is shown in Supplementary Data Set 2. (E)

Relative abundance of Argonaute-associated small RNAs from published data sets. The blue dashed lines

represent risiRNA. Source data (for panels A, E) are available on-line.



risiRNA silences pre-rRNA through the nuclear RNAi pathway.

(A) Images of C. elegans' embryos expressing risiRNA sensor after feeding exogenous dsRNA targeting gfp

and 26S rRNA. (B) Brood size of indicated animals at 20°C. (C) NRDE-3-associated RNAs in the indicated

animals were immunoprecipitated and quantified by qRT-PCR. Ratios are presented as +/- exogenous dsRNA.

mean ± s.d. n=3 independent animals. Source data (for panels B-C) are available on-line.



Low temperature upregulates risiRNA expression.

(A) Brood size of the indicated animals at different temperatures. (B, C) Total RNA samples were collected

from bleached embryos of the indicated genotypes. The abundance of risiRNA was quantified by Taqman qRT-

PCR and is shown relative to levels of wild-type animals at 20°C. mean ± s.d. n=3 independent animals.

*p<0.05, **p<0.01, ***p<0.001, NS, not significant. two-tailed student t-test. (D) Images of representative

seam cells of the indicated animals expressing GFP::NRDE-3. The percentage of nuclear localized NRDE-3 is

quantified at the right panel. Source data (for panels A-D) are available on-line.




Lowering temperature triggers risiRNA generation.

(A) Relative abundance of small RNAs in wild-type N2 animals at different temperatures. The blue dashed lines

represent risiRNA. (B) Relative abundance of NRDE-3-associated small RNAs at different temperatures.

NRDE-3-associated small RNAs at 20°C have been deep sequenced previously 1. The blue dashed lines

represent risiRNA. (C) Relative abundance of NRDE-3-associated small RNAs at different temperatures in eri-

1(mg366);gfp::nrde-3 animals. The small RNA deep sequencing data of NRDE-3 immunoprecipitation in eri-

1(mg366);dpy-13(e458);dpy-13(RNAi); gfp::nrde-3 animals 1 were re-analyzed here. The blue dashed lines

represent risiRNA. Source data (for panels A-C) are available on-line.



SUSI-1 localized to the cytoplasm and was required for fertility.


(A) Images of representative seam cells of indicated animals. susi-1p::mCherry::SUSI-1 rescued the nuclear

localization of NRDE-3. (B) Images of representative seam cells of indicated animals expressing GFP::SUSI-1

and its variants. (C) Synergistic fertility defects in eri-1(mg366);susi-1 double mutants. (D) Synergistic

embryonic lethality in eri-1(mg366);susi-1 double mutants. The number of counted embryos are shown above

each column. Source data (for panels C-D) are available on-line.



TAIL-seq analysis identified nontemplated addition of single nucleotide at the 3' ends of 26S rRNA.

(A, B) Tail-seq of 26S rRNA and the comparison of 3'-end untemplated addition of single nucleotide. Total

RNA of indicated animals were isolated from bleached embryos and subjected to Tail-seq assay. The sense 26S

rRNA reads were compared to annotated 26S rRNA sequences of WS250 transcriptome assembly. Source data

(for panels A-B) are available on-line.




UV irradiation elicits the translocation of NRDE-3 to the nucleus and stimulates risiRNA generation.

(A) Bleached embryos of eri-1(mg366);gfp::nrde-3 animals were exposed to 50 mJ/cm2 UV and the percentage

of nuclear localized NRDE-3 was scored at indicated times post irradiation. (B) Bleached embryos of eri-

1(mg366);gfp::nrde-3;mCherry::fib-1 animals were exposed to 50 mJ/cm2 UV irradiation and the subcellular

localization of GFP::NRDE-3 and mCherry::FIB-1 were visualized. white arrows, nucleoli; red triangle,

nucleus. (C) NRDE-3-associated small RNAs were immunoprecipitated and risiRNAs were quantified by

Taqman qRT-PCR. mean ± s.d. n=3 independent animals. 18S and 26S, risiRNA sequences; 21UR-1 and

21UR-5045, piRNA sequences; e01g4.5 #1 and #2, endo-siRNA sequences. Source data (for panels C-D) are

available on-line. (D) Total RNAs were isolated from bleached embryos after UV irradiation and the abundance

of risiRNAs was quantified by qRT-PCR. mean ± s.d. n=3 independent animals.


Table S1: SUSI-1 plays marginal, if any, direct roles in feeding RNAi. pos-1

(lethality) lir-1

(lethality*) unc-15

(paralysis) dpy-13

(dumpy*) lin-15b

(multivulva*) N2 ++++ - ++++ + - eri-1(mg366) ++++ ++++ ++++ ++++ ++++ eri-1(mg366);nrde-3(gg66) ++++ - +++ + - susi-1(ust1) ++++ - ++++ + - eri-1(mg366);susi-1(ust1) ++++ ++++ ++++ ++ - eri-1(mg366):gfp::nrde-3 N/A N/A N/A ++++ N/A eri-1(mg366);susi-1(ust1);gfp::nrde-3

N/A N/A N/A ++ N/A


Table S2. Manual cloning and sequencing of NRDE-3-associated small RNAs.

number of reads sequences mapped to genome4 GAGATAGAGATGCCTCCCGA F31C3

4 GAGATAGAGATGCCTCCCGACA F31C3

2 GATCGAACTTGAATATTTGCTA F31C3

2 GCTATCCAGAACGGGGCTCG F31C3

2 GACAACGCACATGTCGCAACGG F31C3

2 GGACATGGAAGAATTTCAACGGT F31C3

1 GGAAACAACGTCCCCATCTCTG F31C3

1 GTCCGAAGACTCCCACCTATGCT F31C3

2 GCACACACCAGAAAGCGTCCGC F31C3

1 GCACACACCAGAAAGCGTCCGCA F31C3

1 GCATTCACGTTGACAGTTCACT F31C3

3 GATAGAGATGCCTCCCGACACC F31C3

2 GATAGAGATGCCTCCCGACAC F31C3

2 GCAACCGCCACGACCTTCCTAC F31C3

5 GCAACCGCCACGACCTTCCTA F31C3

2 GAAGTTACGGATCTAGTTTGCC F31C3

5 GCATAAGCTCTCGTGTTGCCAC F31C3

2 GCATAAGCTCTCGTGTTGCCA F31C3

1 ACTGCTCCCCACTACTAAATTTT F52E10

2 GCCAAGGCCATACATAGGGCATA F31C3

1 GCACGTCAGAACCGATACGGAC F31C3

1 GGAAGAGCCGACATCGAAGGA F31C3

1 GCTCATAGCCTTCAACGCAACC F31C3

1 GGGACACCTACGTTACGATTTG F31C3

1 GGACACCTACGTTACGATTTGA F31C3

2 GGGACACCTACGTTACGATTTGA F31C3

1 GAACCCGCTGTTCCTCTCGTA F31C3

1 GCAATGATAGGAAGAGCCGA F31C3

1 GGATACCGACTTCCATGGCCAC F31C3

2 GGATACCGACTTCCATGGCCA F31C3

2 GCCTTAGGACACCTACGTTACGA F31C3

2 GATCGACTGACCCACATCCAAC F31C3

2 CTGTGGTGTCTCGTGCTCTTTGA F31C3

1 GGGGACGTTGTTTCCGAAAAGCACTGCGGT F31C3

1 CGATACGAATGCCCCCGGTTTG F31C3

1 GCCAACCGTTCCAACCACTGGA F31C3

1 GGCCAACCGTTCCAACCACTGGA F31C3

1 GAAAGTATCAAGCAGACAGAA F31C3

1 GAAAGCGTCCGCAAGCAAGCAC F31C3

1 CCACCTATGCTACACCTCTCAT F31C3

1 GAAGATTCGCCAACTATTTAG F31C3

1 CCACCTCCACCGCCACCAGCTC wip-1

1 GAGGTCCAAGCACACACCAGA F31C3

1 GACGGAAGCTCCAGCCGAGCCTA F31C3

1 GATAAAAATGGTTTCCGCAA F31C3


1 GGCAAATGCTTTCGCTGTTGGG F31C3

1 GGCAAATGCTTTCGCTGTTGGG F31C3

1 GACAGACAGAACTGCGCCGGAC F31C3

1 GCACTGACGGAAGCTCCAGCC F31C3

1 GCACTGACGGAAGCTCCAGC F31C3

1 GTACCGGTTCCAGTACGGTTGT F31C3

1 GCAAGCTTTTCGCCACCGATGA F31C3

1 GGCCACCGTCCTGCTGTCAATAT F31C3

1 GGCCACCGTCCTGCTGTCA F31C3

1 GGCCACCGTCCTGCTGTCAATA F31C3

1 GTAGCACGCGTGCAGCCCCGGA F31C3

2 GCCACGACCTTCCTACTCGTTAC F31C3

1 GCCCCGACATTGAACCAGAG F31C3

1 GCACAGGGCCGTGGTGCGCT F31C3

1 GACGCCAAGGCCATACATAGGG F31C3

1 GGAAGAATTTCAACGGTTTAC F31C3

1 GGGCCGTTCAAAGAGCACGAGA F31C3

1 GATCAGGATACTAGAACCGAG F31C3

1 GCATAGGAAACGACACCCGGCAA F31C3

1 GACGACCTTGACCTTGGTTCCGG rpl-32

1 GGCAGGTGAGTTGTTACACACT F31C3

1 GACATCGAAGGATCAAAAAGCA F31C3

1 GACATCGAAGGATCAAAAAG F31C3

1 GTCGGTGATGAACCCACGGTGG hsp-2ps

1 GCGGATATCGGTACGATTGAG F31C3

1 GCAAGAAATTTTCGCCTTGACAA F31C3

1 ACAAGAGTCGATGAAAGTGGTGG F31C3

1 GACCTTGACGTTGTTAGCGGC rpl-22

1 GCTACACCTCTCATGACTCTTC F31C3

1 GCCATCCATTTTCAGGGCTAG F31C3

1 GCCATCCATTTTCAGGGCTAGT F31C3

1 GCTTCGAAAAAAGGCCGTGAA F31C3

1 GCCACGAACCCACTACCGCCAG F31C3

1 GAACCAAATGTTTGAACCCGCT F31C3

1 GTACTAAGCAGGATTACTTTCGC F31C3

1 GAACCCGAAGATTCGCCAACTAT F31C3

1 GCAAGCACAATCACTAGTCCGC F31C3

1 GCCCTGCCGAGGAGCTATCCG F31C3

1 GGAGACCTAATGCGGATATCGG F31C3

1 ACCATTACGATCTGCACTGAC F31C3

1 GATGAATCAAAGCATGCATGGC F31C3

1 GCTTTTCGCCACCGATGATACAA F31C3

1 GGGCATAAGCTCTCGTGTTGC F31C3

1 GTAATCTCTCAAATGGTAGCTT F31C3

1 GGTTCCAGTACGGTTGTTTAGA F31C3

1 CACGAACCCACTACCGCCAGC F31C3

1 GCCAACTATTTAGCAGGCTAGA F31C3

1 GATCAAGTCTATGAACCAAATG F31C3


1 GTAAAACTAACCTGTCTCACGA F31C3

1 GCATCAACCAACTGGCCAACCGTA F31C3

2 GTCAGTCCTAATGGTGTCCGG F31C3

1 GCAACCGCCACGACCTCCCTAC F31C3

1 GCGAACTGCGGCACGTAACCTA F31C3

1 GGGAAGCAAGAAATTTTCGCCT F31C3

1 GACTGACCCACATCCAACTACT F31C3

1 GGCATAGGAAAAACCCGCTGAC F31C3

1 GCAAGTGATTTAGCGCCCCGA F31C3

1 GAACACAATCTTTGCATCTCAGT F31C3

number of reads sequences not mapped to genome1 CCGCGTCCCCCCCTCGCCGTCTCC

1 GGCAAATGGTAGCAATGGTA

1 CGGGGCTCAAACTGTGCA

1 ACGGGTGCTACTGGCGCTAC

1 CAGCGCCGAATCCCCGCCCCGC

1 GGGGGTGCGAGGCAGGGGA

2 TACGAAGGTGCGCTCAGCGTG

2 CGGCGTAGGGTGAGTCGGCCCC

1 CGGTGGAGCATGTGGTTTAATTC

1 CCGCGTGTAGCAGTGAAATGC

1 TAGGTGGGAGGCTTTGAA

1 CCGGGGGGACGCCTCGCCGCCG

1 CCGGTCAGCCTCCCCCCGGCCCC

1 CCCCTGGGCCGATACTGACGC


Table S3: Mutant alleles of susi-1 that had been tested for their ability to elicit the

subcellular redistribution of NRDE-3.

susi-1 alleles Percentage of nuclear localization of GFP::NRDE-3

H654Y(ust1) 100 Y46N(gk174720) 0 P253Q(gk174722) 0 R457H(gk835603) 95 T489M(gk523266) 0 M636I(gk632674) 0 E697K(gk638682) 25 L710F(gk174725) 0 V739I(gk174726) 0 2869bp deletion(ust39) 100


Table S4: Strains used in this study.

YY178: eri-1(mg366);FLAG::GFP::NRDE-3(ggIS1)

YY208: rrf-3(pk1426); FLAG::GFP::NRDE-3(ggIS1)

SHG11: eri-1(mg366); FLAG::GFP::NRDE-3(ggIS1);susi-1(ust1)

SHG12: eri-1(mg366);susi-1(ust1)

SHG83: susi-1(ust1)

SHG84: eri-1(mg366); FLAG::GFP::NRDE-3(ggIS1);susi-1(gk174722)








SHG101: eri-1(mg366); FLAG::GFP::NRDE-3(ggIS1);susi-1(ust1);ergo-1(gg98)

SHG110: eri-1(mg366); FLAG::GFP::NRDE-3(ggIS1); rde-1(ne219)

SHG114: eri-1(mg366);susi-1(gk638682)

SHG115: susi-1(gk638682)

SHG116: eri-1(mg366);FLAG::GFP::NRDE-3(PAZ*)

SHG120: eri-1(mg366);susi-1(gk835603)

SHG121: susi-1(gk835603)

SHG122: eri-1(mg366); FLAG::GFP::NRDE-3(ggIS1);susi-1(ust1);rrf-1(pk1417)


SHG123: eri-1(mg366); FLAG::GFP::NRDE-3(ggIS1);susi-1(ust1);rrf-2(ok210)

SHG124: eri-1(mg366); FLAG::GFP::NRDE-3(ggIS1);susi-1(ust1);rrf-3(pk1426)

SHG125: eri-1(mg366); FLAG::GFP::NRDE-3(ggIS1);susi-1(ust1);rde-4(ne301)

SHG126: eri-1(mg366); FLAG::GFP::NRDE-3(ggIS1);susi-1(ust1);rde-1(ne219)

SHG135: eri-1(mg366); FLAG::GFP::NRDE-3(ggIS1);rrf-1(pk1417)

SHG168: rrf-3(pk1426); FLAG::GFP::NRDE-3(ggIS1):susi-1(ust1)

SHG169: eri-1(mg366); FLAG::GFP::NRDE-3(ggIS1); rrf-3(pk1426)

SHG248: control_sensor(ustIS38)

SHG278: risiRNA_sensor(ustIS37)

SHG285: risiRNA_sensor(ustIS37);susi-1(ust1)

SHG287: risiRNA_sensor(ustIS37);susi-1(ust1);eri-1(mg366)

SHG297: risiRNA_sensor(ustIS37);eri-1(mg366)

SHG360: eri-1(mg366); FLAG::GFP::NRDE-3(ggIS1); ergo-1(gg98)

SHG362: eri-1(mg366); FLAG::GFP::NRDE-3(ggIS1); rde-12(ust17)

SHG388: mCherry::FIB-1(ustIS36)

SHG390: eri-1(mg366); FLAG::GFP::NRDE-3(ggIS1); mCherry::FIB-1(ustIS36)

SHG391: eri-1(mg366); FLAG::GFP::NRDE-3(ggIS1);susi-1(ust1);

mCherry::FIB-1(ustIS36)


Table S5: Primers used for quantitative real-time PCR analysis.

eft-3 mRNA: (ACTTGATCTACAAGTGCGGAGGA) and

(AAAGATCCCTTACCCATCTCCTG)

eft-3 pre-mRNA: (ACTTGATCTACAAGTGCGGAGGA) and

(CGGGTGAGAAAATCTTTCAAACTA)

lin-15b mRNA: (GCCCTCCGTCCACACATTATTGC) and

(TTCTTCAATTCCGCTGGCGTTTC)

pre-rRNA #1: (CTGTGTTTACACCCGAATGATTCTAG) and

(CTAATCGTGAGATGGGACACTCATACA)

pre-rRNA #2: (CGCAGACATATAGTCTAGCGAG) and

(GATCCATAGATATTGCTGATGATTC)

pre-rRNA #3: (AAAGTCGTAACAAGGTAG) and (ATCTTAAGGTTTGTGGAT)

pre-rRNA #4: (GATTTGTTGTTGAGATTTG) and (CAATTCGTGGTAAGTAAC)

pre-rRNA #5: (AACGCATAGCACCAACTG) and (TCCGAAGAGAAGCCTAAG)

pre-rRNA #6: (AATACTGGGATTCGTCTA) and (GAGTTCAGGTTGAGATTAG)

pre-rRNA #7: (CCTTTTCCTACACTCATGTCTTTGCAG) and

(GTTCTAAGTTTTTCTAAAGCAAGCACATTG)

risiRNA sensor mRNA: (GAGGCATCTGAAGCATACCTCGTC) and

(GATTCTAACTTAGAGGCGTTCAG)

21UR-1 RT:

GTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCAACGCACGG


21UR-5045 RT:

GTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCAACGCCGTT

e01g4.5 siRNA #1 RT:

GTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCAACTCCGAG

e01g4.5 siRNA #2 RT:

GTTGGCTCTGGTGCAGGGTCCGAGGTATTCGCACCAGAGCCAACCTTCTT

21UR-1: (GATGGTGGTACGTACGTTAA) and (GTGCAGGGTCCGAGGTATT)

21UR-5045: (GATGGGTGCAGGGTCCGA) and

(TGCCAAACTCCATTTAACGGC)

e01g4.5 siRNA #1: (GATGGGTTTGGCCATCTTCAA) and

(GTGCAGGGTCCGAGGTATT)

e01g4.5 siRNA #2: (GGCGGGTAGTGACCATCATAA) and

(GTGCAGGGTCCGAGGTATT)


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Nature Structural & Molecular Biology: doi:10.1038/nsmb€¦ · aligning to each genomic feature. The reads corresponding to sense rRNA sequences were excluded from the analysis