Supporting Information
Azuma-Mukai et al. 10.1073/pnas.0800334105

Fig. S1. Amino acid sequence alignment of the N-terminal regions of hAgo1, hAgo2, hAgo3, and hAgo4. The regions were used as antigens for immunizing
mice. The pink boxes indicate amino acids perfectly identical among all Argonautes. Amino acids highly conserved are highlighted with light green boxes.

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Fig. S2. Monoclonal antibodies against individual hAgos. (A) N-terminal regions of hAgo1 and hAgo2 fused to GST produced in and purified from E. coli
(GST-hAgo1-N and GST-hAgo2-N). The N-terminal regions of hAgo3 and hAgo4 were fused to 6⫻ His-tag and produced in and purified from E. coli (His-hAgo3-Nand His-hAgo4-N). Red asterisks show the expected full-length fusion proteins of each hAgo. (B) One-tenth of purified proteins shown in A separated on
SDS/PAGE gel and, after transferring to a membrane, probed with anti-hAgo1 monoclonal antibody. Only GST-hAgo1-N (red asterisk) is detected, indicating thatanti-hAgo1 we raised is specific to hAgo1. (C) Blot prepared as in B and probed with anti-hAgo2 antibody. (D) Blot prepared as in B probed with anti-hAgo3
antibody. (E) Blot prepared as in B probed with anti-hAgo4 antibody. (F) Total lysates of Jurkat cells probed with anti-hAgo2 and -hAgo3 antibodies. The blot
indicated as ‘‘control’’ was probed only with secondary antibody.

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Fig. S3. Characterization of immunoprecipitates with anti-hAgo2, -hAgo3, and -hAgo4 antibodies from Jurkat cells. (A) Anti-hAgo2 immunoprecipitates (Fig.
1 A) analyzed by Western blot analysis with anti-hAgo1, anti-hAgo3, and anti-hAgo4. (B) Anti-hAgo3 immunoprecipitates (Fig. 1 A) analyzed by Western blot
analysis with anti-hAgo1, anti-hAgo2, and anti-hAgo4. (C) Anti-hAgo4 immunoprecipitates (Fig. 1 A) analyzed by Western blot analysis with anti-hAgo4. The
120-kDa band appearing in Fig. 1 A (double asterisks) reacted with anti-hAgo4 even in Western blot analysis.

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C
5'ACAGCUGUAAUUAGU CAGUUUUCUGUC U GU
3'UGUUGACAUUGAUCUGCCAAAAGACA CAC C
hsa-miR-1537

chr1: 234082923-234082983(-)

C
5' GGGAACAGCAGCAACAUGGGCCUCGCU U C
U
3' UCCUUGUCGUCGU G GCCCGG GCGG C C G
C GC

chr16: 68157212-68157272(-)

hsa-miR-1538

5'

C
C
G
GGC UCUG GGC UGCAGG GC G A

3'C

CCG AGAC CUG GCGUCC UG A AUCChsa-miR-1539

hsa-miR-1284
A
5' C UAUACAGAC CCU GGCUUUUCUUA AUU
UU
3' G UUAUGUUUG UAC CCGAAAGGUUA AUA
U

hsa-miR-103-1(as)
AUA
C
G
A
AU CCA
5'UC
GC CU UACA UGCUGCUU G
U
A
AG
CG
GA
AUGU
ACGACGGA
U
3'
A
G
C
AA A
C
CAAC
hsa-miR-103-2(as)CC
AU
C
5'UC AGC CUG UACAA UGCUGCUU G A UGA
AU
3'AG UCGAAGAA AUGUC ACGACGGA A
CAUCG

chr18: 45267744-45267790(+)

chr3:71673843-71673900(-)

chr5: 167920487-167920548(+)

chr20: 3846149-3846210(-)

Fig. S4. Our sequence data analysis found five novel microRNA candidates (hsa-miR-20071, hsa-miR-20072, hsa-miR-20073, hsa-miR-2004, and hsa-miR-2005;
temporal names). miR-20071 resides inside of lyst (lysosomal trafficking regulator isoform 1) intron, which is supported by a microRNA prediction (HS92) byBerezikov et al. (1). miR-20072 is inside of nfat5 (nuclear factor of activated T cells 5 isoform a) 5⬘ UTR, which is supported by a computational microRNA prediction(miRRim2837) by Terai et al. (2). miR-20073 is inside of rpl17 (putative NF␬B activating protein) intron, which is supported by a prediction HS276 (1). miR-20074
is located inside of foxp1 (forkhead box P1 isoform 1) and is supported by a computational prediction miRRim999 (2). miR-20075 has a distinct feature: it is mapped
onto known microRNA (miR-103-1) but its strand is opposite. miR-20075 precursors could be mapped onto chromosome 5 and chromosome 20, as indicated.

1. Berezikov E, et al. (2006) Diversity of microRNAs in human and chimpanzee brain. Nat Genet 38:1275–1277.
2. Terai G, Komori T, Asai K, Kin T (2007) miRRim: A novel system to find conserved miRNAs with high sensitivity and specificity. RNA 13:2081–2090.

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hAgo2-top1

miR-150

5P-------------UGUCUCCCAACCCUUGUACCAGU-------------------------------------------------------------GUCUCCCAACCCUUGUACCAGUG------------------------------------------------------------aUCUCCCAACCCUUGUACCAGUG-------------------------------------------------------------UCUCCCAACCCUcGUACCAGU--------------------------------------------------------------aCUCCCAACCCUUGUACCAGUG-------------------------------------------------------------UCUCCCAACCCUcGUACCAGUG-------------------------------------------------------------gCUCCCAACCCUUGUACCAGUG-------------------------------------------------------------UCUCCCAACCCUUGcACCAGUG-------------------------------------------------------------UCUCCCAACCCUUGUACCAGUa-------------------------------------------------------------UCUCCCAACCCUUGUACCAGUu-------------------------------------------------------------UCUCCCAAuCCUUGUACCAGUG-------------------------------------------------------------UCUCCCAACCCUUuUACCAGUG-------------------------------------------------------------UCUCCCAACCCUUGUACCAGUGC------------------------------------------------------------UCUCCCAACCCUUGUACCAGUGa------------------------------------------------------------UCUCCCAACCCUUGUACCAGU--------------------------------------------------------------UCUCCCAACCCUUGUACCAGUGu------------------------------------------------------------UCUCCCAACCCUUGUACCAGUG--------------------------------------------------------------CUCCCAACCCUUGUACCAGUGu-------------------------------------------------------------CUCCCAACCCUUGUACCAGUG---------------------------------------------------------------UCCCAACCCUUGUACCAGUG-----------------------------------------------

11
13
64
10
11
12
13
18
23
31
35
47
141
362
381
530
4478
14
167
11

CUCCCCAUGGCCCUGUCUCCCAACCCUUGUACCAGUGCUGGGCUCAGACCCUGGUACAGGCCUGGGGGACAGGGACCUGGGGAC.((((((.((((((((((((((..(((.(((((((.((((....))).).))))))))))..)))))))))))).)))))))).
Fig. S5. miRNA sequence variations found in the hAgo2 and hAgo3 small RNA libraries. Variants of the top 30 miRNAs in both libraries are shown. For individual
miRNAs, only variants that appeared a total of 10 times or more in the hAgo2 and hAgo3 small RNA libraries are listed. Nucleotide sequences shown on the bottom
in each case are precursor sequences of the miRNAs, in which authentic mature miRNA sequences registered in databases are highlighted in yellow. Sequencesin red (upper) are authentic mature miRNA sequences registered in databases. Additional nucleotides found in the variants of the 5⬘ and 3⬘ ends compared withthe authentic miRNAs are shown in colors. Letters with green boxes indicate nucleotides most likely added to mature miRNAs after miRNA maturation processing(nontemplated extensions), whereas letters with purple boxes indicate nucleotides most likely originating from precursors together with authentic mature
miRNAs (alternative ends), because those nucleotides completely match to the precursor sequences. Numbers shown on the right show total appearances of eachvariant in the libraries.

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hAgo2-top2

miR-99b

5P-----aCACCCGUAGAACCGACCUUGCG-----------------------------------------------CACCCGUAGAACCGACCUUGuG-----------------------------------------------CACCCGUAGAACCGACCUUGCu-----------------------------------------------CACCCGUAGAAuCGACCUUGCG-----------------------------------------------CACCCGUAGAACCGAuCUUGCG-----------------------------------------------CACCCGUAuAACCGACCUUGCG-----------------------------------------------CACCCuUAGAACCGACCUUGCG-----------------------------------------------CACCCGUAGAACCGACuUUGCG-----------------------------------------------CACCCGcAGAACCGACCUUGCG-----------------------------------------------CACCCGUAGAACCuACCUUGCG-----------------------------------------------CACCCGUAGAACCGACCUUGC------------------------------------------------CACCCGUAGAACuGACCUUGCG-----------------------------------------------CACCCGUAGAACCGACCUUGCGa----------------------------------------------CACCCGUAGAACCGACCUUGCGu----------------------------------------------CACCCGUAGAACCGACCUUGCG------------------------------------------

85
12
12
12
13
15
17
17
24
27
28
30
155
293
4330

GGCACCCACCCGUAGAACCGACCUUGCGGGGCCUUCGCCGCACACAAGCUCGUGUCUGUGGGUCCGUGUC(((((..(((((((((..(((.((((.(((((....))).).).)))).)))..)))))))))..)))))

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hAgo2-top3
hAgo2-top8

miR-142-3p
miR-142-5p

3P
5P-------------CCCAUAAAGUAGAAAGCACUACU---------------------------------------------------------------CCCAUAAAGUAGAAAGCACUACa---------------------------------------------------------------CCCAUAAAGUAGAAAGCACUACc---------------------------------------------------------------CCCAUAAAGUAGAAAGCACUA-----------------------------------------------------------------CCCAUAAAGUAGAAAGCACUAC-----------------------------------------------------------------CCAUAAAGUAGAAAGCACUAC------------------------------------------------------------------CAUAAAGUAGAAAGCACUACUA----------------------------------------------------------------CAUAAAGUAGAAAGCACUAC------------------------------------------------------------------CAUAAAGUAGAAAGCACUACU-----------------------------------------------------------------------------------------------------UGUAGUGUUUCCUACUUUAUGGAa--------------------------------------------------------------UGUAGUGUUUCCUACUUUAU------------------------------------------------------------------aGUAGUGUUUCCUACUUUAUGGA---------------------------------------------------------------UGUAGUGUUUCCUACUUUAUGG----------------------------------------------------------------UGUAGUGUUUCCUACUUUAUGGA----------------------------------------------------------------GUAGUGUUUCCUACUUUAUGGAU---------------------------------------------------------------GcAGUGUUUCCUACUUUAUGGA----------------------------------------------------------------GUAGUGcUUCCUACUUUAUGGA----------------------------------------------------------------GUAGUGUUUCCUACUUUAUGGu----------------------------------------------------------------GUAGUGUUUCCUACUUUAUG------------------------------------------------------------------uUAGUGUUUCCUACUUUAUGGA----------------------------------------------------------------GUAGUGUUUCCUACUUUAUGGAa---------------------------------------------------------------GUAGUGUUUCCUACUUUAUGG-----------------------------------------------------------------GUAGUGUUUCCUACUUUAUGGA-----------------------------------------------------------------cAGUGUUUCCUACUUUAUGGA-----------------------------------------------------------------UAGUGUUUCCUACUUUAUGGAU----------------------------------------------------------------UAGUGUUUCCUACUUUAUGGAa----------------------------------------------------------------UAGUGUUUCCUACUUUAUGGA------------------------------------------------------------------AGUGUUUCCUACUUUAUGGAU------------

13
17
34
120
1831
23
12
14
40
12
17
24
42
373
10
11
13
17
22
34
65
260
2952
19
27
42
104
16

GACAGUGCAGUCACCCAUAAAGUAGAAAGCACUACUAACAGCACUGGAGGGUGUAGUGUUUCCUACUUUAUGGAUGAGUGUACUGUG.((((((((.(((.(((((((((((.(((((((((...(..(....)..)..))))))))).))))))))))).))).)))))))).

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hAgo2-top4

miR-21

5P------aUAGCUUAUCAGACUGAUGUUGA-------------------------------------------------UAGCUUAUCAGACUGAUGUUGACU-----------------------------------------------cAGCUUAUCAGACUGAUGUUGA-------------------------------------------------UAGCUUAcCAGACUGAUGUUGA-------------------------------------------------UAGCUUAUCAGACUGAUuUUGA-------------------------------------------------UAGCUUAUCAGACUGAUuUUGAC------------------------------------------------UAGCUUAUCAGACUGAUGUUGAa------------------------------------------------UAGCUUAUCAGACUGAUGUUGACa-----------------------------------------------UAGCUUAUCAGACUGAUGUUG--------------------------------------------------UAGCUUAUCAGACUGAUGUUGAC------------------------------------------------UAGCUUAUCAGACUGAUGUUGA--------------------------------------------------AGCUUAUCAGACUGAUGUUGAC-------------------------------------------------AGCUUAUCAGACUGAUGUUGA---------------------------------------------------GCUUAUCAGACUGAUGUUGAC--------------------------------------------------GCUUAUCAGACUGAUGUUGA----------------------------------------------------CUUAUCAGACUGAUGUUGA-------------------------------------------

10
12
13
14
16
18
19
58
59
1544
2053
29
30
10
14
16

UGUCGGGUAGCUUAUCAGACUGAUGUUGACUGUUGAAUCUCAUGGCAACACCAGUCGAUGGGCUGUCUGACA.(((((..((((((((.(((((.(((((.((((.(.....)))))))))).)))))))))))))..))))).

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hAgo2-top5

miR-92a-1

3P----------------------------------------------GUAUUGCACUUGUCCCGGCCUGU-------------------------------------------------------UAUUGCACUUGUCCCGcCCUG--------------------------------------------------------UAUUGuACUUGUCCCGGCCUGU-------------------------------------------------------UAUUGCACUUGUCCCGGCCUGUU------------------------------------------------------UAUUuCACUUGUCCCGGCCUGU-------------------------------------------------------UAUUGCACUUGUCCCGGCCUa--------------------------------------------------------UAUUGCACUUGUCCCGGCCUcU-------------------------------------------------------UAUUGCACUUGUCCCGGCCUaU-------------------------------------------------------UAUUGCACUUGUCCCGuCCUGU-------------------------------------------------------UAUUGCACUUuUCCCGGCCUGU-------------------------------------------------------UAUUGCACUUGUCCCGGCCU---------------------------------------------------------UAUUGCACUcGUCCCGGCCU---------------------------------------------------------UAUUGCACcUGUCCCGGCCUGU-------------------------------------------------------UAUUGCAuUUGUCCCGGCCUGU-------------------------------------------------------UAUUGCACUUGUCCCGGCCUGUa------------------------------------------------------UAUUGCACUUGUCCCGGCCUGa-------------------------------------------------------UAUUGCACUUGUCCCGGCCUuU-------------------------------------------------------UAUUGCACUUGUCCCGGCCUG--------------------------------------------------------UAUUGCACUUGUCCCGGCCUGU--------------------------------------------------------AUUGCACUUGUCCCGGCCUG---------------------------------------------------------AUUGCACUUGUCCCGGCCUGU---------------------------------------------------------UUGCACUUGUCCCGGCCUGU----------------------------------------------------------UGCACUUGUCCCGGCCUGU---------

13
10
11
12
12
14
17
18
24
26
26
27
27
36
37
42
72
160
1855
14
151
29
14

CUUUCUACACAGGUUGGGAUCGGUUGCAAUGCUGUGUUUCUGUAUGGUAUUGCACUUGUCCCGGCCUGUUGAGUUUGG..(((...((((((((((((.(((.(((((((((((......)))))))))))))).)))))))))))).))).....

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hAgo2-top5

miR-92a-2

3P----------------------------------------------GUAUUGCACUUGUCCCGGCCUGU----------------------------------------------------UAUUGCACUUGUCCCGcCCUG-----------------------------------------------------UAUUGuACUUGUCCCGGCCUGU----------------------------------------------------UAUUGCACUUGUCCCGGCCUGUu---------------------------------------------------UAUUuCACUUGUCCCGGCCUGU----------------------------------------------------UAUUGCACUUGUCCCGGCCUa-----------------------------------------------------UAUUGCACUUGUCCCGGCCUcU----------------------------------------------------UAUUGCACUUGUCCCGGCCUaU----------------------------------------------------UAUUGCACUUGUCCCGuCCUGU----------------------------------------------------UAUUGCACUUuUCCCGGCCUGU----------------------------------------------------UAUUGCACUUGUCCCGGCCU------------------------------------------------------UAUUGCACUcGUCCCGGCCU------------------------------------------------------UAUUGCACcUGUCCCGGCCUGU----------------------------------------------------UAUUGCAuUUGUCCCGGCCUGU----------------------------------------------------UAUUGCACUUGUCCCGGCCUGUa---------------------------------------------------UAUUGCACUUGUCCCGGCCUGa----------------------------------------------------UAUUGCACUUGUCCCGGCCUuU----------------------------------------------------UAUUGCACUUGUCCCGGCCUG-----------------------------------------------------UAUUGCACUUGUCCCGGCCUGU-----------------------------------------------------AUUGCACUUGUCCCGGCCUG------------------------------------------------------AUUGCACUUGUCCCGGCCUGU------------------------------------------------------UUGCACUUGUCCCGGCCUGU-------------------------------------------------------UGCACUUGUCCCGGCCUGU------

13
10
11
12
12
14
17
18
24
26
26
27
27
36
37
42
72
160
1855
14
151
29
14

UCAUCCCUGGGUGGGGAUUUGUUGCAUUACUUGUGUUCUAUAUAAAGUAUUGCACUUGUCCCGGCCUGUGGAAGA((.(((.(((((.(((((..((.(((.(((((((((...)))).))))).)))))..))))).))))).))).))

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hAgo2-top6

miR-342-3p

3P-----------------------------------------------------------aUCUCACACAGAAAUCGCACCCGU---------------------------------------------------------------------------UCUCACACAGAAAUCGCACCCGUu--------------------------------------------------------------------------UCUCACACAGAAAUCGCACCCGc---------------------------------------------------------------------------UCUCACACAGAAAUCGCACCCGUa--------------------------------------------------------------------------UCUCACACAGAAAUCGCACCCaU---------------------------------------------------------------------------UCUCACACAGAAAUCGCACCCG----------------------------------------------------------------------------UCUCACACAGAAAUCGCACCCGUC--------------------------------------------------------------------------UCUCACACAGAAAUCGCACCCGU----------------------------------------------------------------------------CUCACACAGAAAUCGCACCCGUC---------------------------------------------------------------------------CUCACACAGAAAUCGCACCCGU-----------------------------------------------------------------------------UCACACAGAAAUCGCACCCGUCA---------------------------------------------------------------------------UCACACAGAAAUCGCACCCGUC---------------

11
10
11
17
50
62
669
1273
26
35
42
56

GAAACUGGGCUCAAGGUGAGGGGUGCUAUCUGUGAUUGAGGGACAUGGUUAAUGGAAUUGUCUCACACAGAAAUCGCACCCGUCACCUUGGCCUACUUA.....(((((.((((((((.((((((..((((((.....((((((...((....))..)))))).))))))....)))))).)))))))))))))....

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hAgo2-top7

miR-484

5P------aUCAGGCUCAGUCCCCUCCCGAU--------------------------------------------------------UCAGGCUCAGUCCCCUCCCGAUA-------------------------------------------------------UCAGGCUCAGUCCCCUCCCGAaA-------------------------------------------------------UCAGGCUCAuUCCCCUCCCGAU--------------------------------------------------------UCAGGCUCAGUCCuCUCCCGAU--------------------------------------------------------UCAGGCUCAGUCCCCUCCCGAa--------------------------------------------------------UCAGGCUCAGUCCCCUCCCGuU--------------------------------------------------------UCAGGCUCAGUCCCCUCCCGA---------------------------------------------------------UCAGGCUCAGUCCCCUCCCGAUu-------------------------------------------------------UCAGGCUCAGUCCCCUCCCGAU---------------------------------------------------------CAGGCUCAGUCCCCUCCCGAUu--------------------------------------------------------CAGGCUCAGUCCCCUCCCGAU----------------------------------------------------------AGGCUCAGUCCCCUCCCGAU--------------------------------------------------

22
14
18
20
22
28
34
73
137
1573
14
121
13

AGCCUCGUCAGGCUCAGUCCCCUCCCGAUAAACCCCUAAAUAGGGACUUUCCCGGGGGGUGACCCUGGCUUUUUUGGCG.(((..((((((....(((.(((((((..(((.((((....))))..)))..))))))).)))))))))......))).

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hAgo2-top9

miR-425

5P------------aAAUGACACGAUCACUCCCGUUGA---------------------------------------------------------------AAUGACACGAUCACUCCCGUUGu---------------------------------------------------------------AAUGACACGAUCACUCCCuUUGA---------------------------------------------------------------AAUGACACGAUCACUCCCGUUGAG--------------------------------------------------------------AAUGACACGAUCACUCCCGUU-----------------------------------------------------------------AAUGACACuAUCACUCCCGUUGA---------------------------------------------------------------AAUGACACGAUCACUCCCGUUG----------------------------------------------------------------AAUGACACGAUCACUCCCGUUGA----------------------------------------------------------------AUGACACGAUCACUCCCGUUGAG---------------------------------------------------------------AUGACACGAUCACUCCCGUUGAGU--------------------------------------------------------------AUGACACGAUCACUCCCGUUG-----------------------------------------------------------------AUGACACGAUCACUCCCGUUGA-----------------------------------------------------------------UGACACGAUCACUCCCGUUGA---------------------------------------------------

10
10
20
25
26
38
386
714
13
18
25
83
11

GAAAGCGCUUUGGAAUGACACGAUCACUCCCGUUGAGUGGGCACCCGAGAAGCCAUCGGGAAUGUCGUGUCCGCCCAGUGCUCUUUC(((((.((.((((...(((((((((((((.....)))))....(((((.......)))))...))))))))...)))).)).)))))

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hAgo2-top10

miR-532-5p

5P-------------------CAUGCCUUGAGUGUAuGACCGU--------------------------------------------------------------------CAUGCCUUGAGUuUAGGACCGU--------------------------------------------------------------------CAUGCCUUGAGUGUAGGACC----------------------------------------------------------------------CAUGCCUUGAGUGUAGGACCGUU-------------------------------------------------------------------CAUGCCUUGAGUGUAGuACCGU--------------------------------------------------------------------CAUGCCUUGAGUGUAGGACCGa--------------------------------------------------------------------CAUGCCUUGAGUGUAGGACCGUa-------------------------------------------------------------------CAUGCCUUGAGUGUAGGACCG---------------------------------------------------------------------CAUGCCUUGAGUGUAGGACCGU---------------------------------------------------------------------AUGCCUUGAGUGUAGGACCGU----------------------------------------------------------------------UGCCUUGAGUGUAGGACCGU--------------------------------------------------

11
11
12
14
15
17
22
32
1036
65
14

CGACUUGCUUUCUCUCCUCCAUGCCUUGAGUGUAGGACCGUUGGCAUCUUAAUUACCCUCCCACACCCAAGGCUUGCAAAAAAGCGAGCCU.(.((((((((........((.((((((.((((.(((..((.............))..))).)))).)))))).))....)))))))).).

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hAgo2-top11

miR-17*

3P-------------------------------------------------aACUGCAGUGAAGGCACUUGUAGC------------------------------------------------------------ACUGCAGUcAAGGCACUUGUAG-------------------------------------------------------------ACUGCAGUGAAGGCACUUGUAG-------------------------------------------------------------ACUGCAGUGAAGGCACUUGUAGC-------------------------------------------------------------CUGCAGUGAAGGCACUUGUAG--------------------------------------------------------------CUGCAGUGAAGGCACUUGUAGC--------------------------------------------------------------UGCAGUGAAGGCACUUGUAG---------------------------------------------------------------UGCAGUGAAGGCACUUGUAGC-----------

10
10
232
423
86
139
12
64

GUCAGAAUAAUGUCAAAGUGCUUACAGUGCAGGUAGUGAUAUGUGCAUCUACUGCAGUGAAGGCACUUGUAGCAUUAUGGUGAC((((..(((((((..((((((((.((.(((((.(((((.......)).)))))))).)).))))))))...)))))))..))))

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hAgo2-top12

miR-210

3P-----------------------------------------------------------------CUGUGCGUGUGACAGCGaCUGA---------------------------------------------------------------------------------------CUGUGCGUGUGACAuCGGCUGA---------------------------------------------------------------------------------------CUGUGCGUGUGACAGCGGCUGu---------------------------------------------------------------------------------------CUGUGCGUGUGACAGCGGCUGAa--------------------------------------------------------------------------------------CUGUGCGUGUGACAGCGGCUGAU--------------------------------------------------------------------------------------CUGUGCGUGUGACAGCGGCUGA----------------------------------------------------------------------------------------UGUGCGUGUGACAGCGGCUGA-----------------------------------------------------------------------------------------GUGCGUGUGACAGCGGCUGA----------------------ACCCGGCAGUGCCUCCAGGCGCAGGGCAGCCCCUGCCCACCGCACACUGCGCUGCCCCAGACCCACUGUGCGUGUGACAGCGGCUGAUCUGUGCCUGGGCAGCGCGACCC....((..((((.(((((((((((..(((((.(((..(((((((((.((.((((...))).).)).)))))).))).))).)))))..)))))))))))..))))..)).

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11
13
14
15
22
721
15
13

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hAgo2-top13

miR-16-1

5P-------------UAGCAGCACGUAAAUAUUGGCGU-----------------------------------------------------------------UAGCAGCACGUAAAUAUUGGCG------------------------------------------------------

51
641

GUCAGCAGUGCCUUAGCAGCACGUAAAUAUUGGCGUUAAGAUUCUAAAAUUAUCUCCAGUAUUAACUGUGCUGCUGAAGUAAGGUUGAC((((((..(((.((((((((((((.((((((((.(.(((..........))).).)))))))).)).)))))))))).)))..))))))

hAgo2-top13

miR-16-2

5P---------UAGCAGCACGUAAAUAUUGGCGU---------------------------------------------------------UAGCAGCACGUAAAUAUUGGCG--------------------------------------------------

51641

GUUCCACUCUAGCAGCACGUAAAUAUUGGCGUAGUGAAAUAUAUAUUAAACACCAAUAUUACUGUGCUGCUUUAGUGUGAC((..((((..(((((((((..((((((((.((..(((........))).)).))))))))..)))))))))..))))..))

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hAgo2-top14

miR-25

3P---------------------------------------------------CAUUGCACUUGUCUCGGUCUGAa------------------------------------------------------------uAUUGCACUUGUCUCGGUCUGA-------------------------------------------------------------CAUUGCACUUGUCUCGGUCUGA-----------

11
20
605

GGCCAGUGUUGAGAGGCGGAGACUUGGGCAAUUGCUGGACGCUGCCCUGGGCAUUGCACUUGUCUCGGUCUGACAGUGCCGGCC((((.(..(((..((((.(((((..(.(((((.(((((.......))..)))))))).)..))))).))))..)))..).))))

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hAgo2-top15

miR-423-3p

3P---------------------------------------------------AAGCUCGGUCUGAGGCCCCUCAGU----------------------------------------------------------------------AGCUCGGUCUGAGGCCCCUCAuU----------------------------------------------------------------------AGCUCGGUCUGAuGCCCCUCAGU----------------------------------------------------------------------AGCUCGGUCUGAGGCCCCUCAGUu---------------------------------------------------------------------AGCUCGGUCUGAGGCCCCUCAG-----------------------------------------------------------------------AGCUCGGUCUGAGGCCCCUCAGU-----------------------------------------------------------------------GCUCGGUCUGAGGCCCCUCAGU------------------------------------------------------------------------CUCGGUCUGAGGCCCCUCAGU-------------------

24
10
10
10
54
439
31
41

AUAAAGGAAGUUAGGCUGAGGGGCAGAGAGCGAGACUUUUCUAUUUUCCAAAAGCUCGGUCUGAGGCCCCUCAGUCUUGCUUCCUAACCCGCGC....(((((((.((((((((((((..(((.((((.(((((.........))))))))).)))...)))))))))))).))))))).........

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hAgo2-top16

miR-92b

3P------------------------------------------------------------UAUUGCACUCGUCCCGGCCUCCu------------------------------------------------------------------------UAUUGCACUCGUCCCGGCCUu--------------------------------------------------------------------------UAUUGCACUCGUCCCGGCCUCu-------------------------------------------------------------------------UAUUGCACUuGUCCCGGCCU---------------------------------------------------------------------------UAUUGCACUCGUCCCGGCCU---------------------------------------------------------------------------UAUUGCACUCGUCCCGGCCUCa-------------------------------------------------------------------------UAUUGCACUCGUCCCGGCCUC--------------------------------------------------------------------------UAUUGCACUCGUCCCGGCCUCC--------------

10
13
16
26
27
29
140
314

CGGGCCCCGGGCGGGCGGGAGGGACGGGACGCGGUGCAGUGUUGUUUUUUCCCCCGCCAAUAUUGCACUCGUCCCGGCCUCCGGCCCCCCCGGCCC.(((((..(((.((((.(((((..(((((((.((((((((((((.............))))))))))))))))))).))))).))))))).)))))

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hAgo2-top17

let-7d*3P-------------------------------------------------------------CUAUACGACCUGCUGCCUUUCa----------------------------------------------------------------CUAUACGACCUGCUGCCUUUC-----------------------------------------------------------------CUAUACGACCUGCUGCCUUUCU-----------------------------------------------------------------UAUACGACCUGCUGCCUUUCU------------------------------------------------------------------AUACGACCUGCUGCCUUUCU----

14
50
396
96
21

CCUAGGAAGAGGUAGUAGGUUGCAUAGUUUUAGGGCAGGGAUUUUGCCCACAAGGAGGUAACUAUACGACCUGCUGCCUUUCUUAGG(((((((.((((((((((((((.((((((...((((((.....)))))).(.....)..)))))).)))))))))))))))))))))

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hAgo2-top18

miR-1915P---------------CAACGuAAUCCCAAAAGCAGCUG--------------------------------------------------------------------CAACGGAAUCCCAAAAGCAGC----------------------------------------------------------------------CAACGGAAUCCCAAAAGCAGCUGU-------------------------------------------------------------------CAACGGAAUCCCAAAAGCAGCUG--------------------------------------------------------------------CAACGGAAUCCCAAAAGCAGCU-------------------------------------------------------

11
22
26
172
264

CGGCUGGACAGCGGGCAACGGAAUCCCAAAAGCAGCUGUUGUCUCCAGAGCAUUCCAGCUGCGCUUGGAUUUCGUCCCCUGCUCUCCUGCCU.(((.(((.((((((..(((((((((.((..(((((((..((((....)).))..)))))))..)))))))))))..)))))).))).))).

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hAgo2-top19

miR-128-1

3P-------------------------------------------------UCACAGUGAACCGGUCUCUUUa-----------------------------------------------------------UCACAGUGAACCGGUCUCUUUU-----------------------------------------------------------UCACAGUGAACCGGUCUCUUU------------

10
61
442

UGAGCUGUUGGAUUCGGGGCCGUAGCACUGUCUGAGAGGUUUACAUUUCUCACAGUGAACCGGUCUCUUUUUCAGCUGCUUC.((((.((((((...(((((((...((((((..(((((((....)))))))))))))...)))))))...)))))).)))).

hAgo2-top19

miR-128-2

3P---------------------------------------------------UCACAGUGAACCGGUCUCUUUa-------------------------------------------------------------UCACAGUGAACCGGUCUCUUUu-------------------------------------------------------------UCACAGUGAACCGGUCUCUUU------------

10
61
442

UGUGCAGUGGGAAGGGGGGCCGAUACACUGUACGAGAGUGAGUAGCAGGUCUCACAGUGAACCGGUCUCUUUCCCUACUGUGUC.(..(((((((.((((((((((...((((((..((((.(........).))))))))))...)))))))))).)))))))..).

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hAgo2-top20

miR-28-3p

3P-----------------------------------------------------CACUAGAUUGUGAGCUCCUGGAa--------------------------------------------------------------CACUAGAUUGUGAGCUCCUGG----------------------------------------------------------------CACUAGAUUGUGAGCUCCUGGA-----------

25
62
311

GGUCCUUGCCCUCAAGGAGCUCACAGUCUAUUGAGUUACCUUUCUGACUUUCCCACUAGAUUGUGAGCUCCUGGAGGGCAGGCACU(((.(((((((((.((((((((((((((((.(((((((......)))))....)).)))))))))))))))).))))))))).)))

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hAgo2-top21

miR-221

3P----------------------------------------------------------------AGCUACAUUGUCUGCUGGGUU----------------------------------------------------------------------------------------AGCUACAUUGUCUGCUGGGUUUu--------------------------------------------------------------------------------------AGCUACAUUGUCUGCUGGGUUUC--------------------------------------------------------------------------------------AGCUACAUUGUCUGCUGGGUUU----------------------------------------------------------------------------------------GCUACAUUGUCUGCUGGGUUU------------------------

15
15
91
254
15

UGAACAUCCAGGUCUGGGGCAUGAACCUGGCAUACAAUGUAGAUUUCUGUGUUCGUUAGGCAACAGCUACAUUGUCUGCUGGGUUUCAGGCUACCUGGAAACAUGUUCUC.((((((((((((....(((.((((((..(((.(((((((((....(((((((.....))).))))))))))))).)))..)).)))).))))))))))....)))))..

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hAgo2-top22

miR-192

5P-----------------------CUGACCUAUGAAUUGACAGCCAG--------------------------------------------------------------------------------------CUGACCUAUGAAUUGACAGCCAu--------------------------------------------------------------------------------------CUGACCUAUGAAUUGACAGCCA---------------------------------------------------------------------------------------CUGACCUAUGAAUUGACAGCC-----------------------------------------------------------------------------------------UGACCUAUGAAUUGACAGCCAGU--------------------------------------------------------------------------------------UGACCUAUGAAUUGACAGCCA----------------------------------------------------------------------------------------UGACCUAUGAAUUGACAGCCAG----------------------------------------------------------------

11
14
71
110
15
21
104

GCCGAGACCGAGUGCACAGGGCUCUGACCUAUGAAUUGACAGCCAGUGCUCUCGUCUCCCCUCUGGCUGCCAAUUCCAUAGGUCACAGGUAUGUUCGCCUCAAUGCCAGC.........(((.(((((.(.((.((((((((((((((.(((((((................))))))).))))).))))))))).)).).)))..))))).........

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hAgo2-top23

miR-15a5P-------------UAGCAGCACAUAAUGGUUUGUGa-----------------------------------------------------------UAGCAGCACAUAAUGGUUUGU-------------------------------------------------------------UAGCAGCACAUAAUGGUUUGUG------------------------------------------------

15
91
233

CCUUGGAGUAAAGUAGCAGCACAUAAUGGUUUGUGGAUUUUGAAAAGGUGCAGGCCAUAUUGUGCUGCCUCAAAAAUACAAGG(((((.......(..((((((((..((((((((((..((.....))..))))))))))..))))))))..).......)))))

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hAgo2-top24

miR-941-1

3P------------------------------------------------------CACCCGGCUGUGUGCACAUGUG------------------------------------------------------------------CACCCGGCUGUGUGCACAUGUGC------------31309

CCCGGCUGUGUGGACAUGUGCCCAGGGCCCGGGACAGCGCCACGGAAGAGGACGCACCCGGCUGUGUGCACAUGUGCCCAGGGCCCGGG(((((((.((.((((((((((.((.((((.(((...(((((.(....).)).))).))))))).)).)))))))).)))).)).)))))

hAgo2-top24

miR-941-2

3P------------------------------------------------------CACCCGGCUGUGUGCACAUGUG------------------------------------------------------------------CACCCGGCUGUGUGCACAUGUGC------------31309

CCCGGCUGUGUGCACAUGUGCCCAGGGCCCGGGACAGCGCCACGGAAGAGGACGCACCCGGCUGUGUGCACAUGUGCCCAGGGCCCGGG(((((((.((.((((((((((.((.((((.(((...(((((.(....).)).))).))))))).)).)))))))))).)).)).)))))

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hAgo2-top25

miR-197

3P-----------------------------------------------UUCACCACCUUCUCCACCCAG-----------------------------------------------------UUCACCACCUUCUCCACCCAGCA---------------------------------------------------UUCACCACCUUCUCCACCCAGu----------------------------------------------------UUCACCACCUUCUCCACCCAGC------

11
18
34
243

GGCUGUGCCGGGUAGAGAGGGCAGUGGGAGGUAAGAGCUCUUCACCCUUCACCACCUUCUCCACCCAGCAUGGCC((((((((.((((.(((((((..((((..((((((....))).)))..))))..))))))).)))).))))))))

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hAgo2-top26

miR-29c3P-----------------------------------------------------UAGCACCAUUUGAAAUCGGUU------------------------------------------------------------------UAGCACCAUcUGAAAUCGGUUA-----------------------------------------------------------------UAGCACCAUUUGAAAUCGGUUA-------------

18
74
301

AUCUCUUACACAGGCUGACCGAUUUCUCCUGGUGUUCAGAGUCUGUUUUUGUCUAGCACCAUUUGAAAUCGGUUAUGAUGUAGGGGGA.(((((((((((...(((((((((((...((((((((((((.....)))))...)))))))...))))))))))))).))))))))).

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hAgo2-top27

miR-766

3P----------------------------------------------------------------ACUCCAGCCCCACAGCCUCAGa----------------------------------------------------------------------------------------ACUCCAGCCCCACAGCCUCAG-----------------------------------------------------------------------------------------ACUCCAGCCCCACAGCCUCAGu----------------------------------------------------------------------------------------ACUCCAGCCCCACAGCCUCAGC-------------------------

16
41
73
173

GCAUCCUCAGGACCUGGGCUUGGGUGGUAGGAGGAAUUGGUGCUGGUCUUUCAUUUUGGAUUUGACUCCAGCCCCACAGCCUCAGCCACCCCAGCCAAUUGUCAUAGGAGC((.((((...(((...((((.(((((((..((((...(((.(((((....(((.........)))..))))).)))...)))).))))))).))))....)))..))))))

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hAgo2-top28

miR-345

5P-----------------GCUGACUCCUAGUCCAGGGCUCu--------------------------------------------------------------------------GCUGACUCCUAGUCCAGGGCUu---------------------------------------------------------------------------GCUGACUCCUAGUCCAGGGCU----------------------------------------------------------------------------GCUGACUCCUAGUCCAGGGCUC----------------------------------------------------------------------------CUGACUCCUAGUCCAGGGCUC-----------------------------------------------------------

13
31
72
134
24

ACCCAAACCCUAGGUCUGCUGACUCCUAGUCCAGGGCUCGUGAUGGCUGGUGGGCCCUGAACGAGGGGUCUGGAGGCCUGGGUUUGAAUAUCGACAGC...(((((((.((((((.(.(((((((.((.(((((((((..(....)..))))))))).)).))))))).).)))))))))))))............

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hAgo2-top29

miR-93*

3P-------------------------------------------------ACUGCUGAGCUAGCACUUCCCGu--------------------------------------------------------ACUGCUGAGCUAGCACUUCCCG---------------------------------------------------------ACUGCUGAGCUAGCACUUCCCGA--------

33
106
122

CUGGGGGCUCCAAAGUGCUGUUCGUGCAGGUAGUGUGAUUACCCAACCUACUGCUGAGCUAGCACUUCCCGAGCCCCCGG((((((((((..((((((((((((.((((.(((..((......))..)))))))))))).)))))))...))))))))))

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hAgo2-top30

miR-501-3p

3P--------------------------------------------------AAUGCACCCGGGCAAuGAUUCU-------------------------------------------------------------AAUGCACCCGGGCAAGGAUUCU--------------------------------------------------------------AUGCACCCGGGCAAGGAUUCU------------

10
199
19

GCUCUUCCUCUCUAAUCCUUUGUCCCUGGGUGAGAGUGCUUUCUGAAUGCAAUGCACCCGGGCAAGGAUUCUGAGAGGGUGAGC((((.(((((((.(((((((.(.(((.(((((....(((.........)))...))))))))))))))))..))))))).))))

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hAgo3-top1
hAgo3-top16

miR-142-3p
miR-142-5p

3P
5P-------------CCCAUAAAGUAGAAAGCACUACc---------------------------------------------------------------CCCAUAAAGUAGAAAGCACUA-----------------------------------------------------------------CCCAUAAAGUAGAAAGCACUAC------------------------------------------------------------------CAUAAAGUAGAAAGCACUACU-----------------------------------------------------------------CAUAAAGUAGAAAGCACUACUA----------------------------------------------------------------------------------------------------UGUAGUGUUUCCUACUUUAUGGA---------------------------------------------------------------UGUAGUGUUUCCUACUUUAUGG-----------------------------------------------------------------GUAGUGUUUCCUACUUUAUGGu----------------------------------------------------------------uUAGUGUUUCCUACUUUAUGGA----------------------------------------------------------------GUAGUGUUUCCUACUUUAUGGAa---------------------------------------------------------------GUAGUGUUUCCUACUUUAUG------------------------------------------------------------------GUAGUGUUUCCUACUUUAUGGA----------------------------------------------------------------GUAGUGUUUCCUACUUUAUGG------------------------------------------------------------------UAGUGUUUCCUACUUUAUGGA-------------

18
46
181
37
227
386
651
19
37
42
86
3685
12636
20

GACAGUGCAGUCACCCAUAAAGUAGAAAGCACUACUAACAGCACUGGAGGGUGUAGUGUUUCCUACUUUAUGGAUGAGUGUACUGUG.((((((((.(((.(((((((((((.(((((((((...(..(....)..)..))))))))).))))))))))).))).)))))))).

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hAgo3-top2

miR-150

5P-------------UGUCUCCCAACCCUUGUACCAGU-------------------------------------------------------------aUCUCCCAACCCUUGUACCAGUG-------------------------------------------------------------UCUCCCAACCCUUGUACCAGUu-------------------------------------------------------------UCUCCCAACCCUUGcACCAGUG-------------------------------------------------------------UCUCCCAACCCUcGUACCAGUG-------------------------------------------------------------UCUCCCAACCCUUuUACCAGUG-------------------------------------------------------------UCUCCCAACCCUUGUACCAGUa-------------------------------------------------------------UCUCCCAACCCUUGUACCAGUGC------------------------------------------------------------UCUCCCAACCCUUGUACCAGU--------------------------------------------------------------UCUCCCAACCCUUGUACCAGUGa------------------------------------------------------------UCUCCCAACCCUUGUACCAGUGu------------------------------------------------------------UCUCCCAACCCUUGUACCAGUG--------------------------------------------------------------CUCCCAACCCUUGUACCAGUGu-------------------------------------------------------------CUCCCAACCCUUGUACCAGUG-----------------------------------------------

10
14
13
16
23
30
71
263
286
663
755
9951
13
52

CUCCCCAUGGCCCUGUCUCCCAACCCUUGUACCAGUGCUGGGCUCAGACCCUGGUACAGGCCUGGGGGACAGGGACCUGGGGAC.((((((.((((((((((((((..(((.(((((((.((((....))).).))))))))))..)))))))))))).)))))))).

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hAgo3-top3

miR-92b

3P------------------------------------------------------------UAUUGCACUCGUCCCGGCCU---------------------------------------------------------------------------UAUUGCACUuGUCCCGGCCU---------------------------------------------------------------------------UAUUGCACUCGUCCCGGCCUu--------------------------------------------------------------------------UAUUGCACUCGUCCCGGCCUCu-------------------------------------------------------------------------UAUUGCACUCGUCCCGGCCUCCu------------------------------------------------------------------------UAUUGCACUCGUCCCGGCCUCa-------------------------------------------------------------------------UAUUGCACUCGUCCCGGCCUC--------------------------------------------------------------------------UAUUGCACUCGUCCCGGCCUCC--------------

16
20
45
211
335
439
1292
4835

CGGGCCCCGGGCGGGCGGGAGGGACGGGACGCGGUGCAGUGUUGUUUUUUCCCCCGCCAAUAUUGCACUCGUCCCGGCCUCCGGCCCCCCCGGCCC.(((((..(((.((((.(((((..(((((((.((((((((((((.............))))))))))))))))))).))))).))))))).)))))

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hAgo3-top4

miR-425

5P------------aAAUGACACGAUCACUCCCGUUGA---------------------------------------------------------------AAUGACACGAUCACUCCCuUUGA---------------------------------------------------------------AAUGACACGAUCACUCCCGUUG----------------------------------------------------------------AAUGACACuAUCACUCCCGUUGA---------------------------------------------------------------AAUGACACGAUCACUCCCGUUGA----------------------------------------------------------------AUGACACGAUCACUCCCGUUGA-----------------------------------------------------------------UGACACGAUCACUCCCGUUGA---------------------------------------------------

33
11
97
105
4070
180
10

GAAAGCGCUUUGGAAUGACACGAUCACUCCCGUUGAGUGGGCACCCGAGAAGCCAUCGGGAAUGUCGUGUCCGCCCAGUGCUCUUUC(((((.((.((((...(((((((((((((.....)))))....(((((.......)))))...))))))))...)))).)).)))))

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hAgo3-top5

miR-532-5p

5P-------------------CAUGCCUUGAGUGUAGGACCGUU-------------------------------------------------------------------CAUGCCUUGAGUGUAGuACCGU--------------------------------------------------------------------CAUGCCUUGAGUGUAuGACCGU--------------------------------------------------------------------CAUGCCUUGAGUGUAGGACCGa--------------------------------------------------------------------CAUGCCUUGAGUGUAGGACC----------------------------------------------------------------------CAUGCCUUGAGUGUAGGACCGUa-------------------------------------------------------------------CAUGCCUUGAGUGUAGGACCG---------------------------------------------------------------------CAUGCCUUGAGUGUAGGACCGU---------------------------------------------------------------------AUGCCUUGAGUGUAGGACCGU--------------------------------------------------

14
17
17
32
66
92
395
3653
177

CGACUUGCUUUCUCUCCUCCAUGCCUUGAGUGUAGGACCGUUGGCAUCUUAAUUACCCUCCCACACCCAAGGCUUGCAAAAAAGCGAGCCU.(.((((((((........((.((((((.((((.(((..((.............))..))).)))).)))))).))....)))))))).).

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hAgo3-top6

miR-92a-1

3P-----------------------------------------------UAUUGCACUUGUCCCGGCCUa--------------------------------------------------------UAUUGCACUcGUCCCGGCCU---------------------------------------------------------UAUUGCACUUGUCCCGGCCUcU-------------------------------------------------------UAUUGCACUUGUCCCGGCCU---------------------------------------------------------UAUUGCAuUUGUCCCGGCCUGU-------------------------------------------------------UAUUGCACUUuUCCCGGCCUGU-------------------------------------------------------UAUUGCACUUGUCCCGGCCUuU-------------------------------------------------------UAUUGCACUUGUCCCGGCCUGUU------------------------------------------------------UAUUGCACUUGUCCCGGCCUGUa------------------------------------------------------UAUUGCACUUGUCCCGGCCUGa-------------------------------------------------------UAUUGCACUUGUCCCGGCCUG--------------------------------------------------------UAUUGCACUUGUCCCGGCCUGU--------------------------------------------------------AUUGCACUUGUCCCGGCCUGU---------

15
16
16
20
24
30
35
47
65
67
280
2171
53

CUUUCUACACAGGUUGGGAUCGGUUGCAAUGCUGUGUUUCUGUAUGGUAUUGCACUUGUCCCGGCCUGUUGAGUUUGG..(((...((((((((((((.(((.(((((((((((......)))))))))))))).)))))))))))).))).....

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hAgo3-top6

miR-92a-2

3P-----------------------------------------------UAUUGCACUUGUCCCGGCCUa-----------------------------------------------------UAUUGCACUcGUCCCGGCCU------------------------------------------------------UAUUGCACUUGUCCCGGCCUcU----------------------------------------------------UAUUGCACUUGUCCCGGCCU------------------------------------------------------UAUUGCAuUUGUCCCGGCCUGU----------------------------------------------------UAUUGCACUUuUCCCGGCCUGU----------------------------------------------------UAUUGCACUUGUCCCGGCCUuU----------------------------------------------------UAUUGCACUUGUCCCGGCCUGUu---------------------------------------------------UAUUGCACUUGUCCCGGCCUGUa---------------------------------------------------UAUUGCACUUGUCCCGGCCUGa----------------------------------------------------UAUUGCACUUGUCCCGGCCUG-----------------------------------------------------UAUUGCACUUGUCCCGGCCUGU-----------------------------------------------------AUUGCACUUGUCCCGGCCUGU------

15
16
16
20
24
30
35
47
65
67
280
2171
53

UCAUCCCUGGGUGGGGAUUUGUUGCAUUACUUGUGUUCUAUAUAAAGUAUUGCACUUGUCCCGGCCUGUGGAAGA((.(((.(((((.(((((..((.(((.(((((((((...)))).))))).)))))..))))).))))).))).))

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hAgo3-top7

miR-629*

3P------------------------------------------------------------aUUCUCCCAACGUAAGCCCAGC--------------------------------------------------------------------------GUUCUCCCAACGUAAGCCCAGC---------------------------------------------------------------------------UUCUCCCAACGUAAGCCCAGC---------------

358
1318
51

UCCCUUUCCCAGGGGAGGGGCUGGGUUUACGUUGGGAGAACUUUUACGGUGAACCAGGAGGUUCUCCCAACGUAAGCCCAGCCCCUCCCCUCUGCCU..........(((((((((((((((((((((((((((((((((((..((....)).)))))))))))))))))))))))))))))))))))......

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hAgo3-top8

miR-210

3P-----------------------------------------------------------------CUGUGCGUGUGACAGCGGCUGu---------------------------------------------------------------------------------------CUGUGCGUGUGACAuCGGCUGA---------------------------------------------------------------------------------------CUGUGCGUGUGACAGCGGCUGAa--------------------------------------------------------------------------------------CUGUGCGUGUGACAGCGGCUGAU--------------------------------------------------------------------------------------CUGUGCGUGUGACAGCGGCUGA-----------------------

17
19
31
37
2561

ACCCGGCAGUGCCUCCAGGCGCAGGGCAGCCCCUGCCCACCGCACACUGCGCUGCCCCAGACCCACUGUGCGUGUGACAGCGGCUGAUCUGUGCCUGGGCAGCGCGACCC....((..((((.(((((((((((..(((((.(((..(((((((((.((.((((...))).).)).)))))).))).))).)))))..)))))))))))..))))..)).

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hAgo3-top9

miR-484

5P-------UCAGGCUCAGUCCCCUCCCGA---------------------------------------------------------UCAGGCUCAGUCCCCUCCCGuU--------------------------------------------------------UCAGGCUCAGUCCCCUCCCGAaA-------------------------------------------------------UCAGGCUCAGUCCCCUCCCGAa--------------------------------------------------------UCAGGCUCAGUCCCCUCCCGAUA-------------------------------------------------------UCAGGCUCAGUCCCCUCCCGAUu-------------------------------------------------------UCAGGCUCAGUCCCCUCCCGAU--------------------------------------------------

57
59
72
81
142
605
813

AGCCUCGUCAGGCUCAGUCCCCUCCCGAUAAACCCCUAAAUAGGGACUUUCCCGGGGGGUGACCCUGGCUUUUUUGGCG.(((..((((((....(((.(((((((..(((.((((....))))..)))..))))))).)))))))))......))).

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hAgo3-top10

miR-1915P---------------CAACGGAAUCCCAAAAGCAGC----------------------------------------------------------------------CAACGGAAUCCCAAAAGCAGCUG--------------------------------------------------------------------CAACGGAAUCCCAAAAGCAGCU-------------------------------------------------------

132
145
1273

CGGCUGGACAGCGGGCAACGGAAUCCCAAAAGCAGCUGUUGUCUCCAGAGCAUUCCAGCUGCGCUUGGAUUUCGUCCCCUGCUCUCCUGCCU.(((.(((.((((((..(((((((((.((..(((((((..((((....)).))..)))))))..)))))))))))..)))))).))).))).

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hAgo3-top11

miR-21

5P-------UAGCUUAUCAGACUGAUGUUGAa------------------------------------------------UAGCUUAUCAGACUGAUGUUG--------------------------------------------------UAGCUUAUCAGACUGAUGUUGAC------------------------------------------------UAGCUUAUCAGACUGAUGUUGA-------------------------------------------

31
55
62
1191

UGUCGGGUAGCUUAUCAGACUGAUGUUGACUGUUGAAUCUCAUGGCAACACCAGUCGAUGGGCUGUCUGACA.(((((..((((((((.(((((.(((((.((((.(.....)))))))))).)))))))))))))..))))).

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hAgo3-top12

miR-99b

5P------CACCCGUAGAACCGACCUUGC------------------------------------------------CACCCGUAGAACCGACCUUGCGa----------------------------------------------CACCCGUAGAACCGACCUUGCG------------------------------------------

17
20
717

GGCACCCACCCGUAGAACCGACCUUGCGGGGCCUUCGCCGCACACAAGCUCGUGUCUGUGGGUCCGUGUC(((((..(((((((((..(((.((((.(((((....))).).).)))).)))..)))))))))..)))))

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hAgo3-top13

miR-93*

3P-------------------------------------------------ACUGCUGAGCUAGCACUUCCCGu--------------------------------------------------------ACUGCUGAGCUAGCACUUCCCG---------------------------------------------------------ACUGCUGAGCUAGCACUUCCCGA--------

35
265
367

CUGGGGGCUCCAAAGUGCUGUUCGUGCAGGUAGUGUGAUUACCCAACCUACUGCUGAGCUAGCACUUCCCGAGCCCCCGG((((((((((..((((((((((((.((((.(((..((......))..)))))))))))).)))))))...))))))))))

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hAgo3-top14

miR-222

3P--------------------------------------------------------------------AGCUACAUCUGGCUACUGGGUCUC-------------------------------------------------------------------------------------AGCUACAUCUGGCUACUGGGUCU--------------------------------------------------------------------------------------AGCUACAUCUGGCUACUGGGUC----------------------------------------------------------------------------------------GCUACAUCUGGCUACUGGGUCU---------------------------------------------------------------------------------------GCUACAUCUGGCUACUGGGUC--------------------

52
237
332
15
17

GCUGCUGGAAGGUGUAGGUACCCUCAAUGGCUCAGUAGCCAGUGUAGAUCCUGUCUUUCGUAAUCAGCAGCUACAUCUGGCUACUGGGUCUCUGAUGGCAUCUUCUAGCU...(((((((((((......((.(((..(((((((((((((((((((...(((...........)))...))))).))))))))))))))..))).))))))))))))).

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hAgo3-top15
hAgo3-top28

let-7d*let-7d

3P
5P-------AGAGGUAGUAGGUUGCAUAGU-----------------------------------------------------------------AGAGGUAGUAGGUUGCAUAGUU----------------------------------------------------------------------------------------------------------------------CUAUACGACCUGCUGCCUUUCa----------------------------------------------------------------CUAUACGACCUGCUGCCUUUC-----------------------------------------------------------------CUAUACGACCUGCUGCCUUUCU----

15
166
14
42
539

CCUAGGAAGAGGUAGUAGGUUGCAUAGUUUUAGGGCAGGGAUUUUGCCCACAAGGAGGUAACUAUACGACCUGCUGCCUUUCUUAGG(((((((.((((((((((((((.((((((...((((((.....)))))).(.....)..)))))).)))))))))))))))))))))

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hAgo3-top17

miR-25

3P---------------------------------------------------CAUUGCACUUGUCUCGGUCUGA-----------

429

GGCCAGUGUUGAGAGGCGGAGACUUGGGCAAUUGCUGGACGCUGCCCUGGGCAUUGCACUUGUCUCGGUCUGACAGUGCCGGCC((((.(..(((..((((.(((((..(.(((((.(((((.......))..)))))))).)..))))).))))..)))..).))))

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hAgo3-top18

miR-26a5P---------UUCAAGUAAUCCAGGAUAGG--------------------------------------------------------UUCAAGUAAUCCAGGAUAGGC-------------------------------------------------------UUCAAGUAAUCCAGGAUAGGCU----------------------------------------------

48
107
222

GUGGCCUCGUUCAAGUAAUCCAGGAUAGGCUGUGCAGGUCCCAAUGGGCCUAUUCUUGGUUACUUGCACGGGGACGC(((.((((((.(((((((((.((((((((((.((.......))...)))))))))).))))))))).)))))).)))

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hAgo3-top19

miR-324-5p

5P---------------CGCAUCCCCUAGGGCAUUGGUGU---------------------------------------------

14

CUGACUAUGCCUCCCCGCAUCCCCUAGGGCAUUGGUGUAAAGCUGGAGACCCACUGCCCCAGGUGCUGCUGGGGGUUGUAGUC..((((((..(((((.(((.(.(((.(((((.(((.((..........))))).))))).))).).))).)))))..))))))

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hAgo3-top20

miR-23a3P--------------------------------------------AUCACAUUGCCAGGGAUUUCC---------------------------------------------------AUCACAUUGCCAGGGAUUaCCA--------------------------------------------------AUCACAUUGCCAGGGAUUUCCAA-------------------------------------------------AUCACAUUGCCAGGGAUUUCCA-------

13
29
72
265

GGCCGGCUGGGGUUCCUGGGGAUGGGAUUUGCUUCCUGUCACAAAUCACAUUGCCAGGGAUUUCCAACCGACC((.(((.((((((((((((.((((.((((((..........)))))).)))).))))))).))))).))).))

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hAgo3-top21

miR-378

3P------------------------------------------ACUGGACUUGGAGUCAGAAGG--------------------------------------------ACUGGACUUGGAGUCAGAAGGC--------------------------------------------CUGGACUUGGAGUCAGAAGGC--

16
270
27

AGGGCUCCUGACUCCAGGUCCUGUGUGUUACCUAGAAAUAGCACUGGACUUGGAGUCAGAAGGCCU(((.((.((((((((((((((...((((((........)))))).)))))))))))))).)).)))

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hAgo3-top22

miR-1865P--------------CAAAGAAUUCUCCUUUUGGGCUU--------------------------------------------------------------CAAAGAAUUCUCCUUUUGGGCU--------------------------------------------------------------CCAAAGAAUUCUCCUUUUGGGCU--------------------------------------------------

109
110
72

UGCUUGUAACUUUCCAAAGAAUUCUCCUUUUGGGCUUUCUGGUUUUAUUUUAAGCCCAAAGGUGAAUUUUUUGGGAAGUUUGAGCU.(((((.(((((((((((((((((.(((((.((((((..............))))))))))).)))))))))))))))))))))).

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hAgo3-top23

miR-29c3P-----------------------------------------------------UAGCACCAUUUGAAAUCGGUU------------------------------------------------------------------UAGCACCAUcUGAAAUCGGUUA-----------------------------------------------------------------UAGCACCAUUUGAAAUCGGUUA-----------------------------------------------------------------UAGCACCAUUUGAAAUCGGUUAA------------

23
78
243
12

AUCUCUUACACAGGCUGACCGAUUUCUCCUGGUGUUCAGAGUCUGUUUUUGUCUAGCACCAUUUGAAAUCGGUUAUGAUGUAGGGGGA.(((((((((((...(((((((((((...((((((((((((.....)))))...)))))))...))))))))))))).))))))))).

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hAgo3-top24

miR-423-3P

3P---------------------------------------------------AAGCUCGGUCUGAGGCCCCUCA------------------------------------------------------------------------AGCUCGGUCUGAGGCCCCUCAGU----------------------------------------------------------------------AGCUCGGUCUGAGGCCCCUCA------------------------------------------------------------------------AGCUCGGUCUGAGGCCCCUC--------------------------------------------------------------------------GCUCGGUCUGAGGCCCCUCA---------------------

15
33
169
20
10

AUAAAGGAAGUUAGGCUGAGGGGCAGAGAGCGAGACUUUUCUAUUUUCCAAAAGCUCGGUCUGAGGCCCCUCAGUCUUGCUUCCUAACCCGCGC....(((((((.((((((((((((..(((.((((.(((((.........))))))))).)))...)))))))))))).))))))).........

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hAgo3-top25

miR-130b*

5P------------ACUCUUUCCCUGUUGCACUACU------------------------------------------------252

GGCCUGCCCGACACUCUUUCCCUGUUGCACUACUAUAGGCCGCUGGGAAGCAGUGCAAUGAUGAAAGGGCAUCGGUCAGGUC((((((.((((..(((((((..(((((((((.((.(((....)))...)).)))))))))..)))))))..)))).))))))

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hAgo3-top26

miR-151-5p

5P---------CUCGAGGAGCUCACAGUCUAGU--------------------------------------------------------------------UCGAGGAGCUCACAGUCUAGU--------------------------------------------------------------------UCGAGGAGCUCACAGUCUAGUA------------------------------------------------------------------CUCGAGGAGCUCACAGUCUAGUA----------------------------------------------------------

84
72
78
17

UUUCCUGCCCUCGAGGAGCUCACAGUCUAGUAUGUCUCAUCCCCUACUAGACUGAAGCUCCUUGAGGACAGGGAUGGUCAUACUCACCUC.((((((.((((((((((((..((((((((((.(........).)))))))))).)))))))))))).)))))).(((.......)))..

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hAgo3-top27

miR-501-3p

3P--------------------------------------------------AAUGCACCCGGGCAAGGAUUCU-------------------------------------------------------------AAUGCACCCGGGCAAGGAUUCUG-----------

214
14

GCUCUUCCUCUCUAAUCCUUUGUCCCUGGGUGAGAGUGCUUUCUGAAUGCAAUGCACCCGGGCAAGGAUUCUGAGAGGGUGAGC((((.(((((((.(((((((.(.(((.(((((....(((.........)))...))))))))))))))))..))))))).))))

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hAgo3-top29

miR-221

3P----------------------------------------------------------------AGCUACAUUGUCUGCUGGGUU----------------------------------------------------------------------------------------AGCUACAUUGUCUGCUGGGUUUC--------------------------------------------------------------------------------------AGCUACAUUGUCUGCUGGGUUU------------------------

10
25
131

UGAACAUCCAGGUCUGGGGCAUGAACCUGGCAUACAAUGUAGAUUUCUGUGUUCGUUAGGCAACAGCUACAUUGUCUGCUGGGUUUCAGGCUACCUGGAAACAUGUUCUC.((((((((((((....(((.((((((..(((.(((((((((....(((((((.....))).))))))))))))).)))..)).)))).))))))))))....)))))..

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hAgo3-top21

miR-378

3P------------------------------------------ACUGGACUUGGAGUCAGAAGG--------------------------------------------ACUGGACUUGGAGUCAGAAGGC--------------------------------------------CUGGACUUGGAGUCAGAAGGC--

16
270
27

AGGGCUCCUGACUCCAGGUCCUGUGUGUUACCUAGAAAUAGCACUGGACUUGGAGUCAGAAGGCCU(((.((.((((((((((((((...((((((........)))))).)))))))))))))).)).)))

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Fig. S6. Distributions of nontemplated bases found in the top 30 miRNAs in the hAgo2 and hAgo3 libraries. Only variants that appeared a total of 10 times
or more were considered as subjects for this statistical study. Apparent preference to adenine and uracil is shown, while nontemplated guanine is extremely rare.

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Fig. S7. (A) Immunoprecipitates from Jurkat cells using anti-hAgo2 and -hAgo3 stained with SYPRO Ruby (Molecular Probes). hAgo3 was immunopurified fromthree volumes of the Jurkat cell lysate that was used for hAgo2 immunopurification, where the amount of hAgo3 immunopurified was ⬇80% of that of hAgo2.
(B) RNAs purified from the immunoprecipitates prepared as in A confirmed with a DNA oligo recognizing miR-150. Total RNAs from Jurkat cells, and DNA oligos
with sequences of miR-150 (sense) and miR-150* (antisense) were used as controls.

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targetmiR-142-5p

miR-142-5p-Me- target23 nt -

13 nt 11 nt -miR-142-5P target

*
**

*
**

13nt
5'-AGUAGUGCUUUCUACUUUAUGGG-3'3'-UCAUCACGAAAGAUGAAAUACCC-5' miR-142-5P+CC
11nt
5'-AGUAGUGCUUUCUACUUUAUGGG-3'3'-UCAUCACGAAAGAUGAAAUAC-5'
miR-142-5P
cleavage inhibitedMe

miR-142-5P-Me target

5'-AGUAGUGCUUUCUACUUUAUGGG-3'3'-UCAUCACGAAAGAUGAAAUACCC-5' miR-142-5P+CC

11nt
Me
5'-AGUAGUGCUUUCUACUUUAUGGG-3'3'-UCAUCACGAAAGAUGAAAUAC-5'
miR-142-5P
Fig. S8. In vitro target RNA cleavage assays using miR-142-5p target. (Upper Left) Target RNA harboring a sequence completely complementary to miR-142-5p(miR-142-5p target; 23 nt; the sequence is shown below) performed by using hAgo2 immunoprecipitated complexes from Jurkat cells. The hAgo2 complex
predominantly cleaved the target at two major sites; one corresponding to the site of the cleavage directed by registered miR-142-5p (double asterisks; 11 nt),
and the other corresponding to the site of the cleavage directed by one very abundant miR-142-5p variant that has two additional nucleotides (mostly CC) at
the 5⬘ end (miR-142-5p⫹CC) (asterisk; 13 nt). (Upper Right) miR-142-5p target modified with a methyl group at the expected cleavage site by miR-142-5p⫹CC[miR-142-5p-Me target; nucleotide 13 (U, highlighted in blue) was methylated; see Lower] showed strong resistance to miR-142-5p⫹CC but not to registered
miR-142-5 cleavage.

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Table S1. Top 30 miRNAs appearing in the hAgo2 and hAgo3 libraries
miRNA

pre-miRNA arm

Most abundant sequence (mas)Count of mas/total countmiRBase sequence ⫽ mas (Y, yes; N, no)hAgo2
miR-150
miR-99b
miR-142-3p
miR-21
miR-92amiR-342-3p
miR-484
miR-142-5p
miR-425
miR-532-5p
miR-17*miR-210
miR-16miR-25
miR-423-3p
miR-92b
let-7d*miR-191
miR-128-1/-2
miR-28-3p
miR-221
miR-192
miR-15amiR-941
miR-197
miR-29c
miR-766
miR-345
miR-93*miR-501-3p

5p
5p
3p
5p
3p
3p
5p
5p
5p
5p
3p
3p
5p
3p
3p
3p
3p
5p
3p
3p
3p
5p
5p
3p
3p
3p
3p
5p
3p
3p

UCUCCCAACCCUUGUACCAGUG
CACCCGUAGAACCGACCUGCGGUAGUGUUUCCUACUUUAUGGAUAGCUUAUCAGACUGAUGUUGAUAUUGCACUUGUCCCGGCCUGUUCUCACACAGAAAUCGCACCCGUUCAGGCUCAGUCCCCUCCCGAU
CCCAUAAAGUAGAAAGCACUAC
AAUGACACGAUCAGUCCCGUUGACAUGCCUUGAGUGUAGGACCGUACUGCAGUGAAGGCACUUGUAGC
CUGUGCGUGUGACAGCGGCUGAUAGCAGCACGUAAAUAUUGGCG
CAUUGCACUUGUCUCGGUCUGAAGCUCGGUCUGAGGCCCCUCAGUUAUUGCACUCGUCCCGGCCUCCCUAUACGACCUGCUGCCUUUCUCAACGGAAUCCCAAAAGCAGCUUCACAGUGAACCGGUCUCUUUCACUAGAUUGUGAGCUCCUGGAAGCUACAUUGUCUGCUGGGUUUCUGACCUAUGAAUUGACAGCCUAGCAGCACAUAAUGGUUUGUG
CACCCGGCUGUGUGCACAUGUGCUUCACCACCUUCUCCACCCAGC
UAGCACCAUUUGAAAUCGGUUAACUCCAGCCCCACAGCCUCAGCGCUGACUCCUAGUCCAGGGCUCACUGCUGAGCUAGCACUUCCCGAAAUGCACCCGGGCAAGGAUUCU
hAgo3

miR-142-3p
miR-150
miR-92bmiR-425
miR-532-5p
miR-92amiR-629*
miR-210
miR-484
miR-191
miR-21
miR-99b
miR-93*miR-222
let-7d*miR-142-5p
miR-25miR-26amiR-324-5p
miR-23amiR-378
miR-186
miR-29c
miR-423-3p
miR-130b*
miR-151-5p
miR-501-3p
let-7dmiR-221
miR-28-3p

3p
5p
3p
5p
5p
3p
3p
3p
5p
5p
5p
5p
3p
3p
3p
5p
3p
5p
5p
3p
3p
5p
3p
3p
5p
5p
3p
5p
3p
3p

GUAGUGUUUCCUACUUUAUGG
UCUCCCAACCCUUGUACCAGUG
UAUUGCACUCGUCCCGGCCUCC
AAUGACACGAUCAGUCCCGUUGACAUGCCUUGAGUGUAGGACCGUUAUUGCACUUGUCCCGGCCUGUGUUCUCCCAACGUAAGCCCAGC
CUGUGCGUGUGACAGCGGCUGAUCAGGCUCAGUCCCCUCCCGAU
CAACGGAAUCCCAAAAGCAGCUUAGCUUAUCAGACUGAUGUUGACACCCGUAGAACCGACCUGCG
ACUGCUGAGCUAGCACUUCCCGAAGCUACAUCUGGCUACUGGGUCCUAUACGACCUGCUGCCUUUCUCAUAAAGUAGAAAGCACUACTACAUUGCACUUGUCUCGGUCUGAUUCAAGUAAUCCAGGAUAGGCUCGCAUCCCCUAGGGCAUUGGUAUCACAUUGCCAGGGAUUUCCAACUGGACUUGGAGUCAGAAGGCCAAAGAAUUCUCCUUUUGGGCUUAGCACCAUUUGAAAUCGGUUAAGCUCGGUCUGAGGCCCCUCAACUCUUUCCCUGUUGCACUACUCUCGAGGAGCUCACAGUCUAGUAAUGCACCCGGGCAAGGAUUCUAGAGGUAGUAGGUUGCAUAGUU
AGCUACAUUGUCUGCUGGGUUUCACUAGAUUGUGAGCUCCUGGA

4,478/6,713
4,330/5,285
2,952/4,359
2,053/4,237
1,855/2,918
1,273/2,533
1,573/2,401
1,831/2,220714/1,563
1,036/1,372
423/1,077
721/935
641/779605/731439/698314/682396/667
264/644442/495
311/484
254/444
110/418
233/390309/386
243/381301/363
173/348134/327
122/302
199/299

Y
Y
N
Y
Y
Y
Y
N
Y
Y
N
Y
Y
Y
Y
Y
Y
N
Y
Y
N
Y
Y
Y
Y
Y
Y
Y
N
Y

12,636/18,409
9,951/12,732
4,835/8,324
4,070/5,022
3,653/4,753
2,171/3,131
1,318/3,122
2,561/2,893
813/2,207
1,273/1,847
1,191/1,412
717/830
367/767
332/720539/656
227/619429/477
222/432
360/426265/388
270/364
110/320243/302169/294
252/291
84/288214/265
166/227
131/194
151/193

Y
Y
Y
Y
Y
Y
Y
Y
Y
N
Y
Y
N
N
Y
N
Y
Y
N
N
N
N
Y
N
N
N
Y
Y
N
Y

Shown are all kinds of miRNAs identified in the hAgo2 and hAgo3 small RNA libraries.

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Other Supporting Information Files
Dataset S1
Dataset S2

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