SUPPLEMENTAL MATERIAL AND DATA

Molecular characterization of human Argonaute-containing ribonucleoprotein complexes and their bound target mRNAs

Markus Landthaler, Dimos Gaidatzis, Andrea Rothballer, Po Yu Chen, Steven Soll, Lana

Dinic, Tolulope Ojo, Markus Hafner, Mihaela Zavolan, and Thomas Tuschl

Supplementary Materials and Methods

Plasmids

Plasmids pENTR4 EIF2C1,-2,-3,-4, TNRC6A/GW182, TNRC6B, and TNRC6C, IGF2BP1 and

3, MOV10, DDX17, YBX1, QKI, PABPC4, and ELAVL1 were generated by PCR amplification of the respective coding sequences (CDS) followed by restriction digest and ligation into pENTR4 (Invitrogen). Primer sequences are listed under DNA oligonucleotides in Suppl.

Experimental Procedures. pENTR4 DICER was generated by cloning the CDS from pDEST10

DICER (Zhang et al., 2002) into pENTR4. pENTR4 constructs were recombined into pFRT/TO/FLAG/HA-DEST destination vector using GATEWAY LR recombinase according to manufacturer’s protocol (Invitrogen), which allow for inducible expression of stably transfected

FLAG/HA-tagged protein in Flp-In T-REx HEK293 cells from the TO/CMV promoter. Plasmids pENTR4 EIF2C1,-2,-3,-4 and pENTR QKI were recombined into pFRT/FLAG/HA-DEST, which only allows constitutive expression in Flp-In T-REx HEK293 cells. Myc-tagged constructs were generated by recombination of pENTR4 with plasmid pDESTmyc (Meister et al., 2004). psiCHECK reporter plasmids for miRNA target validation were generated by PCR amplification of genomic DNA (primer sequences listed in Supplemental Experimental Procedures). PCR products were XhoI and NotI digested and ligated into XhoI and NotI site of psiCHECK-2

(Promega).

1 Suppl.Figure 1

kDa C EIF2C1 EIF2C2 EIF2C3 EIF2C4 200 150

100 85

60 50

30

20 Suppl.Figure 2

Lysate SupernatantSupernatant FASupernatant IP FDSupernatant IP HDIP IP FA mADIP FDIP IP HA IP mAD FLAG/HA-EIF2C1

FLAG/HA-EIF2C2

FLAG/HA-EIF2C3

FLAG/HA-EIF2C4

FLAG/HA-DICER

FLAG/HA-TNRC6A

FLAG/HA-TNRC6B

FLAG/HA-TNRC6C

FLAG/HA-IGF2BP1

FLAG/HA-QKI Suppl.Figure 2

fmol 10 4 HEK QKI EIF2C1EIF2C2EIF2C2 (FA) EIF2C2 (FD)EIF2C2 (HD)QKI (mAD) (mAD)EIF2C3EIF2C4DICERTNRC6ATNRC6BTNRC6CIGF2BP1PABPC4 Suppl.Figure 3

EIF2C1(FA/1) EIF2C1(FA/2) EIF2C2(FA/1) EIF2C2(FA/2) EIF2C2(FD) EIF2C2(HD) EIF2C2(mAD) QKI(mAD) EIF2C3(FA) EIF2C4(FA) TNRC6A(FA/1) TNRC6A(FA/2) TNRC6B(FA) TNRC6B(FD) TNRC6C(FA/1) TNRC6C(FD) TNRC6C(FA/2) DICER1(FA/1) DICER1(FA/2) QKI(FA/1) QKI(FA/2) IGF2BP1(FA/1) IGF2BP1(FA/2) PABPC4(FA/1) PABPC4(FA/2)

log2 fold change Suppl.Figure 4

r=0.880 r=0.240 r=0.129 8 4 10

6 2 5 4 0

2 -2 0

0 DICER IP -4 PABPC4 IP PABPC4 -5 TNRC6A,-B,-C IP -2 -6

-4 -8 -10 -5 0 5 10 -5 0 5 10 -5 0 5 10 EIF2C1,-2,-3,-4 IP EIF2C1,-2,-3,-4 IP EIF2C1,-2,-3,-4 IP Antibodies and beads

Monoclonal anti-HA.11 (clone 16B12, Covance), monoclonal anti-myc (clone 9E10, Milipore), polyclonal anti-Ago2/EIF2C2 (Milipore), anti-YBX1, anti-RPS6, and anti-RPL7 were used as primary antibodies and HRP-conjugated anti-rabbit Ig and anti-mouse Ig were used as secondary antibodies for Western analysis. Polyclonal anti-DICER was generated by injection of GST-

DICER1 peptide. Dynabeads Protein G (Invitrogen) were used with anti-FLAG M2 (SIGMA), high affinity anti-HA (Roche) antibodies and monoclonal anti-Ago2/EIF2C2 (clone 9E8.2,

Milipore) as described by the manufacturer.

miRNA cloning and sequencing

RNA was extracted from suspension culture cells stably expressing FLAG/HA-TNRC6B and size-fractionated by a denaturing PAGE. miRNA cloning was performed as described previously

(Hafner et al., 2008; Landgraf et al., 2007). The cDNA library was sequenced by 454 sequencing. miRNA sequences were annotated as described (Berninger et al., 2008; Landgraf et al., 2007).

DNA oligonucleotides

Restriction sites are underlined. pENTR4 constructs

EIF2C1: 5’-ACCATGGAAGCGGGACCCTCGGGAG;

GCGAATTCCGTCAAGCGAAGTACATGGTGCGC,

EIF2C2: ACGCGTCGTCGACATGTACTCGGGAGCCGG;

ATAAGAATGCGGCCGCTCAAGCAAAGTACATGGTGC,

EIF2C3: ACGCGTCGTCGACATGGAAATCGGCTCCGCAGG;

CGGAATTCATTTAAGCGAAGTACATTGTGCGTAA,

EIF2C4: TACCATGGAGGCGCTGGGACCCG;

ATAAGAATGCGGCCGCGAATTCTCTCAGGCAAAATACATCG,

2 TNRC6A: ATGGATGCTGATTCTGCCTCCAGTTC;

TNRC6C: TTCAGTCGACATGGCTACAGGGAGTGCCCAGGGCAAC;

ATAAGAATGCGGCCGCCTACAGGGACTCCCCGCTGAGCAGG,

IGF2BP1: ACGCGTCGACATGAACAAGCTTTACATCGGCAACCTC;

ATAAGAATGCGGCCGCTCACTTCCTCCGTGCCTGGGCCTG,

IGF2BP3: ACGCGTCGACATGAACAAACTGTATATCGGAAACCTCAG; 5’

ATAAGAATGCGGCCGCTTACTTCCGTCTTGACTGAGGTGGTC,

DDX17: TTCAGTCGACATGCCCACCGGCTTTGTAGCCCCGATTC;

ATAAGAATGCGGCCGCTCATTTACGTGAAGGAGGAGGAGG,

PABPC4: TTCAGTCGACATGAACGCTGCGGCCAGCAGCTACC;

ATAAGAATGCGGCCGCCTAAGAGGTAGCAGCAGCAACAGC,

YBX1: ATGAGCAGCGAGGCCGAG,

ATAAGAATGCGGCCGCTTACTCAGCCCCGCCCTGCTCAG,

ELAVL1: ACGCGTCGACATGTCTAATGGTTATGAAGACCACATG;

ATAAGAATGCGGCCGCTTATTTGTGGGACTTGTTGGTTTTG

psiCHECK constructs

ENPP5: CCGCTCGAGAGATATGCATGCTGAAATAGCTCAACC,

ATAAGAATGCGGCCGCACCTGGTAGGTGGGTATTCACATATTTC;

SCN1B: CCGCTCGAGCTCAAGGAAGAGCCAGCCGTAATG,

ATAAGAATGCGGCCGCCTACTTCTGTAAAGAGACGAGGCTGAA; c14orf28: CCGCTCGAGGTCATCATTGGTTTATATTCGAGCAACAG,

ATAAGAATGCGGCCGCCACAACAACCCTGTGAGGTAGGTAAG;

TTLL11: CCGCTCGAGTCAAGAGGGCTCTCTGGCTTCGTGTAG,

ATAAGAATGCGGCCGCATTCTTACAAGAACCTTTTAAGATAGGTATTC;

3 RHEBL1: CCGCTCGAGGTGTGGGGTAACTGCCTTGCTTCT,

ATAAGAATGCGGCCGCGATTTCTCAATTTATTTGCATATATACATTG;

GSC: CCGCTCGAGCCGCGGGACACTTGCCCGTATTAC,

ATAAGAATGCGGCCGCCATCGCCATCACTTTATTGTACTGTCAC;

SIRT4: CCGCTCGAGGCTGCCTTTGATAGACCCATGCTG,

ATAAGAATGCGGCCGCTATCCACACATTAAGATACCTAGAACC;

CHAC1: CCGCTCGAGCACTCCAGTGCACAAGACAGACTTG,

ATAAGAATGCGGCCGCGTTTTATGGGTAACAGTATGGGACAGAC; c2orf42: CCGCTCGAGCTGACCACTATTACTTTCCCTTAAAGC,

ATAAGAATGCGGCCGCGGAAAAACTAGTGACACTGCTTTATTTG

IRF9: CCGCTCGAGAGAGCAGCAGGCAGCCATTCTGTCC,

ATAAGAATGCGGCCGCTTTAAGTCCTTAGTGAATATCTAAGAGG

GPR137B: CCGCTCGAGTCCTGACAAACCAAGCCTTGGGTAG,

ATAAGAATGCGGCCGCCAGAGGAGGTTTAATAGGATAAGTCAGC

SGK: CCGCTCGAGGACTCTTTCCTCTGAACCCTGTTAGG,

ATAAGAATGCGGCCGCGTGATGGGATGAGGGAAGGATTGTACG

RBJ: CCGCTCGAGGCTCGGACAGCCCTCCTGAAAAAC,

ATAAGAATGCGGCCGCCACTCTGTTTACAAGACATAAACTGG;

ADBR2: CCGCTCGAGGAATTGTAGTACAAATGACTCACTGCTG,

ATAAGAATGCGGCCGCTTACTGTAAACTTTAGACTTTGCTCGG; c9orf150: CCGCTCGAGGAATACTACTGCTTTGGCTAGTGACAG,

DOC2A: CCGCTCGAGTGTCCTCAGCCTGAGTGGACAGCAG,

ATAAGAATGCGGCCGCCCTCGGAGGTGTTTATTGATGCCCAGC;

RNA oligonucleotides: miR-122: UGGAGUGUGACAAUGGUGUUUG,

4 miR-122*: AACGCCAUUAUCACACUAAAUA.

2'-O-methyl oligoribonucleotides (all residues are 2’-O-methyl modified): anti-miR-15a: CACAAACCAUUAUGUGCUGCUA, anti-miR-16: UGUAAACCAUGAUGUGCUGCUA, anti-miR-17: CUACCUGCACUGUAAGCACUUUG, anti-miR-18: CUAUCUGCACUAGAUGCACCUUA, anti-miR-19a: UCAGUUUUGCAUAGAUUUGCACA, anti-miR-19b: UCAGUUUUGCAUGGAUUUGCACA, anti-miR-20a: CUACCUGCACUAUAAGCACUUUA, anti-miR-20b: CUACCUGCACUAUGAGCACUUUG, anti-miR-32: UGCAACUUAGUAAUGUGCAAUA, anti-mir-92: ACAGGCCGGGACAAGUGCAAUA, anti-miR-93: CUACCUGCACGAACAGCACUUUG, anti-mir-196b: CCCAACAACAGGAAACUACCUA, anti-bantam: AUCAGCUUUCAAAAUGAUCUCAdT.

5 Supplemental References

Berninger, P., Gaidatzis, D., van Nimwegen, E., and Zavolan, M. (2008). Computational analysis of small RNA cloning data. Methods 44, 13-21.

Hafner, M., Landgraf, P., Ludwig, J., Rice, A., Ojo, T., Lin, C., Holoch, D., Lim, C., and Tuschl, T. (2008). Identification of microRNAs and other small regulatory RNAs using cDNA library sequencing. Methods 44, 3-12.

Landgraf, P., Rusu, M., Sheridan, R., Sewer, A., Iovino, N., Aravin, A., Pfeffer, S., Rice, A., Kamphorst, A. O., Landthaler, M., et al. (2007). A mammalian microRNA expression atlas based on small RNA library sequencing. Cell 129, 1401-1414.

Meister, G., Landthaler, M., Patkaniowska, A., Dorsett, Y., Teng, G., and Tuschl, T. (2004). Human Argonaute2 mediates RNA cleavage targeted by miRNAs and siRNAs. Mol Cell 15, 185- 197.

Zhang, H., Kolb, F. A., Brondani, V., Billy, E., and Filipowicz, W. (2002). Human Dicer preferentially cleaves dsRNAs at their termini without a requirement for ATP. Embo J 21, 5875- 5885.

6 Supplemental Figure Legend

Figure S1: Double affinity purifications of AGO proteins

FLAG/HA-EIF2C1,-2,-3,-4 were double affinity purified from cell stably expressing the epitope- tagged proteins. The purified proteins were separated by SDS-PAGE and silver-stained.

Purification of AGO proteins was compared to a purification of an extract from the parental Flp-

In T-REx HEK293 cells (C).

Figure S2: Western and Northern analysis of immunoprecipitated proteins and associated miRNA

A. IPs were performed from cells stably expressing the FLAG/HA-protein as indicated. All IPs were performed using anti-FLAG agarose (FA), except for IPs of extracts from FLAG/HA-

EIF2C2 expressing cells, which were carried out with Dynabead Protein G bound anti-FLAG

(FD), anti-HA (HD), and anti-AGO2 (mAD). In addition, an IP was performed with anti-AGO2 bound to Protein G Dynabeads from cells expressing FLAG/HA-QKI.

B. RNA was extracted from IPs, separated by 15% denaturing PAGE, blotted and probed for miR-16. 10 and 4 fmol of synthetic miR-16 were loaded as standard, as well as 20 µg of HEK293 total RNA.

Figure S3: Box plot representation of the distribution of the log2 fold-change (x-axis) of transcripts in the IP versus lysate of individual proteins (indicated on the left). Numbers indicate transcripts that are least 4-fold enriched or underrepresented.

Figure S4: Pairwise correlation plots of the fold change in EIF2C1,-2,-3,-4 IP (x-axis) and

TNRC6A,-B,-C IP or DICER IP (y-axis). Each dot represents an expressed transcript. The

Pearson correlation coefficient is shown at the top of each panel.

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