the anti - mili monoclonal antibody 17.8 was generated by immunizing mice with recombinant gst - mili .
other antibodies used were anti - flag ( sigma ) , anti - sdma ( sym11 ; millipore ) , anti - adma ( asym24 ; millipore ) , anti - tmg ( santa cruz biotech ) , anti--tubulin ( developmental studies hybridoma bank ) ; anti - hnrnpc ( 4f4 ) , anti - smn ( 2b1 ) , anti - gemin3 ( 12h12 ) , anti - gemin5 ( 10g11 ) , y12 were gifts from g. dreyfuss and antibodies against drosophila ago1 , aub , piwi and ago3 were gifts from mc .
csul flies ( csul : w;csul / cyo ) , were a gift from j. anne and a deletion that uncovers csul ( w n ; df(2r)jp7 , was obtained from bloomington drosophila stock center ( indiana university ) .
directional ligation of adapters and cdna generation was performed using the small rna sample prep kit ( illumina ) .
recombinant flag - miwi and flag - mili and gst - mili were produced in baculovirus infected sf9 cells .
the cdnas for flag - miwi and flag - mili production and flag - miwi truncations mutants ( bc for 68 - 862aa of miwi and np for 1 - 212aa ) were gifts from s. kuramochi - miyagawa and t. nakano .
arginine residues were changed to lysine (  wt : 10a - r - g - r - g - r - g - r - k18  changed to  mutant : 10a - k - g - k - g - k - g - k - k18  ) , by site - directed mutagenesis ( quickchange xl , stratagene ) using the following primers ; forward : 5-caccaaaccctgtaattgctaaaggaaaaggtaaaggaaaaaagcccaataatgtagaggc-3 and reverse : 5-gcctctacattattgggcttttttcctttaccttttcctttagcaattacagggtttggtg-3. wild - type and mutant prh - flag - aub expression vectors were co - transfected with pcoblast ( invitrogen ) at a ratio of 5:1 using cellfectin at 27 c .
twenty - four hours post - transfection , cells were grown in complete schneider cell medium containing 15 g / ml blasticidin .
recombinant aub expression was induced by growing s2 cells seeded at 2  10 cells / ml in complete schneider cell medium containing 500 m cuso4 for 48 hours , and the protein was purified by immunoprecipitation using anti - flag m2 agarose ( sigma ) .
defolliculated oocytes were fixed with 10% neutral buffered formalin , paraffin embedded and sectioned at 5m ; in situ hybridization was performed with a locked nucleic acid ( lna)-modified probe against xl - pir-3 ( 5- uaaguagaagagcaccaaugucaugucc ) , an abundant pirna sequenced from x. laevis oocytes .
the sequence of the lna probe was : 5- ggacatgacattggtgctcttctactta -3-dig ( capital letters : lna - modified nucleotides ; dig : digoxigenin ) . for northern
blotting , total rna was isolated from x. laevis mixed stage oocytes and from liver and probed with 5-end radiolabeled dna probes antisense to xl - pir-3 ( probe : 5- ggacatgacattggtgctcttctactta ) and antisense to mir-16 ( probe : 5- caccaatatttacgtgctgcta ) .
a synthetic pirna containing a photo - reactive residue , 4-thio - uridine ( su ) , at position 1 ( 5-(su)gacaugaacacaggugcucagauagcuuu-3 ) was 5-end labeled using [ -p ] atp and t4 polynucleotide kinase ( t4 pnk , new england biolabs ) .
the p - labeled rnas ( 20,000 cpm ) were incubated with wild - type or mutant flag - aub beads at 28 c for 60 min in a buffer containing 20 mm tris - hcl ph 7.5 , 200 mm nacl , 2.5 mm mgcl2 , 0.05 % np-40 and complete edta - free protease inhibitors ( roche ) .
crosslinking was performed on ice by irradiation for 30 min with a 365 nm hand - held lamp ( el series uv lamp , uvp ) .
cross - linked proteins were separated by nupage and detected by storage - phosphor autoradiography .
data and analysis for x. laevis pirnas are found in the supplement and in : http://cbcsrv.watson.ibm.com / piwi_modification/.
the anti - mili monoclonal antibody 17.8 was generated by immunizing mice with recombinant gst - mili .
other antibodies used were anti - flag ( sigma ) , anti - sdma ( sym11 ; millipore ) , anti - adma ( asym24 ; millipore ) , anti - tmg ( santa cruz biotech ) , anti--tubulin ( developmental studies hybridoma bank ) ; anti - hnrnpc ( 4f4 ) , anti - smn ( 2b1 ) , anti - gemin3 ( 12h12 ) , anti - gemin5 ( 10g11 ) , y12 were gifts from g. dreyfuss and antibodies against drosophila ago1 , aub , piwi and ago3 were gifts from mc .
csul flies ( csul : w;csul / cyo ) , were a gift from j. anne and a deletion that uncovers csul ( w n ; df(2r)jp7 , was obtained from bloomington drosophila stock center ( indiana university ) .
testis and liver tissues were procured from euthanized animals . y12-immunopurified x. laevis pirnas from testis and oocytes were 5-end labeled and gel purified .
directional ligation of adapters and cdna generation was performed using the small rna sample prep kit ( illumina ) .
recombinant flag - miwi and flag - mili and gst - mili were produced in baculovirus infected sf9 cells .
the cdnas for flag - miwi and flag - mili production and flag - miwi truncations mutants ( bc for 68 - 862aa of miwi and np for 1 - 212aa ) were gifts from s. kuramochi - miyagawa and t. nakano .
were changed to lysine (  wt : 10a - r - g - r - g - r - g - r - k18  changed to  mutant : 10a - k - g - k - g - k - g - k - k18  ) , by site - directed mutagenesis ( quickchange xl , stratagene ) using the following primers ; forward : 5-caccaaaccctgtaattgctaaaggaaaaggtaaaggaaaaaagcccaataatgtagaggc-3 and reverse : 5-gcctctacattattgggcttttttcctttaccttttcctttagcaattacagggtttggtg-3. wild - type and mutant prh - flag - aub expression vectors were co - transfected with pcoblast ( invitrogen ) at a ratio of 5:1 using cellfectin at 27 c .
twenty - four hours post - transfection , cells were grown in complete schneider cell medium containing 15 g / ml blasticidin .
recombinant aub expression was induced by growing s2 cells seeded at 2  10 cells / ml in complete schneider cell medium containing 500 m cuso4 for 48 hours , and the protein was purified by immunoprecipitation using anti - flag m2 agarose ( sigma ) .
defolliculated oocytes were fixed with 10% neutral buffered formalin , paraffin embedded and sectioned at 5m ; in situ hybridization was performed with a locked nucleic acid ( lna)-modified probe against xl - pir-3 ( 5- uaaguagaagagcaccaaugucaugucc ) , an abundant pirna sequenced from x. laevis oocytes .
the sequence of the lna probe was : 5- ggacatgacattggtgctcttctactta -3-dig ( capital letters : lna - modified nucleotides ; dig : digoxigenin ) . for northern
blotting , total rna was isolated from x. laevis mixed stage oocytes and from liver and probed with 5-end radiolabeled dna probes antisense to xl - pir-3 ( probe : 5- ggacatgacattggtgctcttctactta ) and antisense to mir-16 ( probe : 5- caccaatatttacgtgctgcta ) .
a synthetic pirna containing a photo - reactive residue , 4-thio - uridine ( su ) , at position 1 ( 5-(su)gacaugaacacaggugcucagauagcuuu-3 ) was 5-end labeled using [ -p ] atp and t4 polynucleotide kinase ( t4 pnk , new england biolabs ) . the p - labeled rnas ( 20,000 cpm ) were incubated with wild - type or mutant flag - aub beads at 28 c for 60 min in a buffer containing 20 mm tris - hcl ph 7.5 , 200 mm nacl , 2.5 mm mgcl2 , 0.05 % np-40 and complete edta - free protease inhibitors ( roche ) .
crosslinking was performed on ice by irradiation for 30 min with a 365 nm hand - held lamp ( el series uv lamp , uvp ) .
cross - linked proteins were separated by nupage and detected by storage - phosphor autoradiography .
data and analysis for x. laevis pirnas are found in the supplement and in : http://cbcsrv.watson.ibm.com / piwi_modification/.
 piwi family proteins are essential for germline development and bind piwi - interacting rnas ( pirnas 1 
 2 
 3 ) . 
 the grandchildless gene aub of drosophila melanogaster encodes the pirna - binding protein aub that is essential for formation of primordial germ cells ( pgcs ) 4 . here 
 we report that mouse , xenopus laevis and drosophila melanogaster piwi family proteins contain symmetrical dimethylarginines ( sdmas ) . 
 we find that piwi proteins are expressed in x. laevis oocytes and we identify numerous x. laevis pirnas . 
 we report that the drosophila homolog of protein methyltransferase 5 ( dprmt5 , csul / dart5 ) , which is also the product of a grandchildless gene 5 , 6 , is required for arginine methylation of drosophila piwi , ago3 and aub proteins , in vivo . 
 loss of dprmt5 activity leads to reduction of pirnas and in particular of ago3 and aub protein levels and accumulation of retrotransposons in the drosophila ovary . 
 our studies explain the relationship between aub and dprmt5 ( csul / dart5 ) genes by demonstrating that dprmt5 is the enzyme that methylates aub . 
 our findings underscore the significance of sdma modification of piwi proteins in the germline and suggest an interacting pathway of genes that are required for pirna function and pgc specification . 
Methods
Antibodies
Drosophila stocks
Xenopus laevis and piRNA sequencing
Recombinant proteins and cDNA constructs
In situ hybridization and Northern blotting
UV crosslinking
Supplementary Material