Supplemental Information. Target-Mediated Protection of Endogenous. MicroRNAs in C. elegans. Inventory of Supplementary Information

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1 Developmental Cell, Volume 20 Supplemental Information Target-Mediated Protection of Endogenous MicroRNAs in C. elegans Saibal Chatterjee, Monika Fasler, Ingo Büssing, and Helge Großhans Inventory of Supplementary Information 1 Supplementary Figure S1: A new RNAi construct permits efficient depletion of xrn-1. Related to Figure 4. Supplementary Materials and Methods

2 Supplementary Figure )*+,-. %&'(0*+,-./ %&'()*+,-./ %&'(0*+,-./ /.+0!"#$ %&'()*+,-./!"#$ 1*23,+4567!"#$%&'(!"#$%&'( Figure S1: A new RNAi construct permits efficient depletion of xrn-1. Related to Figure 4. A) Feeding RNAi using JA:xrn-1(RNAi) bacteria inefficiently depletes xrn-1. xrn-1 mrna levels were determined by RT-qPCR analysis of RNA from N2 worms fed mock RNAi (L4440) or JA:xrn-1(RNAi) bacteria. Result depicted as the average of three independent experiments +/- standard deviation demonstrates that the level of xrn-1 mrna in xrn-1(rnai) sample is decreased to some 50% of the control sample. B) Use of a different xrn-1(rnai) clone covering the entire xrn-1 cdna efficiently depletes XRN-1 protein. Lysates from N2 worms placed either on mock RNAi plates (L4440) or xrn-1(rnai) plates were used for determination of xrn-1 protein level by

3 western blotting using a previously described anti-xrn-1 antibody (Newbury and Woollard, 2004). Little, if any, reduction of XRN-1 protein was seen following xrn- 2(RNAi). Signal quantification was done using Image J. C) Although little mir-241* signal is visible in mock(rnai)-treated animals carrying a control transgene (red arrow; cp. Fig. 4F), image analysis using the histogram function of Image J reveals it to be well above background (arrowhead), permitting quantification.

4 Supplementary Materials and Methods Generation of C. elegans strains. Transgenic strains for this study were generated by microparticle co-bombardment (Praitis et al., 2001) of three plasmids carrying respectively an unc-119 rescue construct, a myo-2::mcherry marker, and tbb-1::gfp fused to artificial mirna target sites (based on a previous design (Pillai et al., 2005) into unc- 119(ed3) to generate HW633: unc-119(ed3); xeex256[tbb-1::gfp::3xmir241*binding sites, myo-2::mcherry, unc-119+], HW639: unc-119(ed3); xeex262[tbb- 1::gfp::3xmir241*mut binding sites, myo-2::mcherry, unc-119+], HW628: unc- 119(ed3); xeex251[tbb-1::gfp::3xlet-7(n2853)binding sites, myo-2::mcherry, unc- 119+]. Relevant transgenic lines were crossed into let-7(n2853) to obtain HW743: let- 7(n2853); xeex251 and HW744: let-7(n2853); xeex262. In Fig. 4F we used HW745: unc-119(ed3); xeex268[tbb-1::gfp::unc-54 unc-119+] as a control, which was generated using a plasmid carrying both an unc-119 rescue sequence and tbb-1::gfp::unc-54. Western blotting. 50 µg of lysate-protein from worms growing on control, xrn-1(rnai) and xrn-2(rnai) plates were subjected to SDS-PAGE and western blotting using an α- XRN-1 antibody as described (Newbury and Woollard, 2004). Plasmids. To obtain the xrn-1(rnai) plasmid, xrn-1 full-length cdna was amplified from total RNA through RT-PCR, and cloned in a TOPO TA vector (Invitrogen), sequence confirmed, subcloned in the L4440 vector, and further transformed into E. coli strain HT115. The MultiSite Three Fragment Vector Construction Kit from Invitrogen was used to create plasmids containing the tbb-1 promoter, a GFP coding sequence and a 3 UTR consisting of three artificial binding sites for the mirna of interest for ectopic mirna target expression in vivo; primer sequences are given below. xrn-2(rnai) and lin-28(rnai) plasmids were described in (Kamath et al., 2003) and (Rual et al., 2004), respectively. lin-28 knockdown (RNAi). To achieve efficient knockdown of lin-28, RNAi was done over two generations. Synchronized N2, lin-14(n355) and lin-14(n536) worms were grown on OP50 plates at 25 C for 10 hrs, then transferred to mock(l4440) or lin- 28(RNAi) plates and grown at 20 C [lin-14 worms have a very small brood size when grown at 25 C] until they turned into gravid adults. The first generation of synchronized L1 worms from the aforementioned sources were again put on the respective RNAi plates (L4440 or lin-28) and grown at 25 C until late L2 stage. Harvested worms were snap frozen into liquid N 2 and subjected to RNA extraction. Oligonucleotides (5-3 ) Northern blotting: let-7(wt): AAC TAT ACA ACC TAC TAC CTC A let-7(n2853): AAC TAT ACA ACC TAC TAT CTC A

5 let-7*: GGT AAG GTA GAA AAT TGC ATA G mir-75: TGA AGC CGG TTG GTA GCT TTA A mir-77: TGG ACA GCT ATG GCC TGA TGA A mir-241: TCA TTT CTC GCA CCT ACC TCA mir-241*: GAT GAA GCA GCT GAG AGA CAA T mir-73*: TGT GGC TCG ATA TGG AAG TCC A mir-58*: GAT GAG ATG CGA AGA GTA GGG CA lin-4: TCA CAC TTG AGG TCT CAG GGA mir-84: TAC AAT ATT ACA TAC TAC CTC A pre-mir-84: GAC ATT ATA GAC AGT CTA CAA TAT TAC ATA CTA CCT CA pre-let-7: CAC CGG TGG TAA TAT TCC AAA CTA TAC AAC pre-lin-4: CA ATA GTA CAC TCA CAC TTG AGG TCT CAG GGA trna Gly : GCT TGG AAG GCA TCC ATG CTG ACC ATT qpcr: xrn-1 Forward CCG CTG GTT GTT AAG GAT GT xrn-1 Reverse CTC CAT TTT CTT GCG GAG AG Pri-let-7 Forward TCC TAG AAC ACA TCT CCC TTT GA Pri-let-7 Reverse CGC AGC TTC GAA GAG TTC TG lin-42 Forward

6 TCT TGT TCA CGT GCA CCT TC lin-42 Reverse GGC TCC GTC TGG CAT AG TAA ama-1 Forward GGA TCG AAG GGA TCG AAG A ama-1 Reverse TGG AAG AAG AAT TCC GAT GG Preparation of templates for in vitro transcription: Pre-mir-241 cassette: G TAA TAC GAC TCA CTA TAG G GAGA CAC CTA CCT CA C TGA TGA GTC CGT GAG GAC GAA ACG GTA CCC GGT ACC GTC TGA GGT AGG TGC (Forward primer: T7 Promoter and HH Ribozyme with sequence complementary to the first 11 nt of mir-241 (bold) and ending with the first 12 nt of mature mir-241 (italics)) and GAT GAA GCA GCT GAG AGA CAA TGA ACG ATT ACT ATA TGG ATG CCG TCA TTT CTC GCA CCT ACC TCA GAC GGT ACC GGG (Reverse primer: pre-mir-241 complementary sequence, 12 nt complementary region to HH Ribozyme) Pre-let-7(n2853) cassette: G TAA TAC GAC TCA CTA TAG G GAGA CTA CTA CCT CAC TGA TGA GTC CGT GAG GAC GAA ACG GTA CCC GGT ACC GTC TGA GAT AGT AGG (Forward primer: T7 Promoter and HH Ribozyme with sequence complementary to the first 11 nt of let-7(n2853) (bold), and ending with the first 12nt of mature let-7(n2853) (italics).) and GGT AAG GTA GAA AAT TGC ATA GTT CAC CGG TGG TAA TAT TCC AAA CTA TAC AAC CTA CTA TCT CA GAC GGT ACC GGG (Reverse primer: pre-let-7(n2853) complementary sequence, 12 nt complementary region to HH Ribozyme) Artificial 3 UTR cassettes: Target sites are underlined, bulged-regions in italics and insertional seed mutations in bold. 3X mir-241 target cassette construction Template sequence: GGGG TCTAGA TCA TTT CTC GCA C TTGAA CT ACC TCA CTCGGAGC TCA TTT CTC GCA C TTGAA CT ACC TCA CTCGGAGC TCA TTT CTC GCA C TTGAA CT ACC TCA GCGGCCGC AAAG

7 Primers for PCR amplification of 3X mir-241 target cassette: GGGG TCTAGA TCA TTT CTC GCA CTTGAAC (Forward primer) and CTTTGCGGCCGC TGAGGTAGTTCAAG (Reverse primer) 3X mir-241* target cassette construction Template sequence: GGGG TCTAGA GAT GAA GCA GCT GA TTGAA G AGA CAA T CTCGGAGC GAT GAA GCA GCT GA TTGAA G AGA CAA T CTCGGAGC GAT GAA GCA GCT GA TTGAA G AGA CAA T GCGGCCGC AAAG Primers for PCR amplification of 3X mir-241* target cassette: GGGG TCTAGA GAT GAA GCA GCT GATTG (Forward primer) and CTT TGC GGC CGC ATT GTC TCT TCA ATC (Reverse primer) 3X mir-241* mutant target cassette construction Template sequence: GGGG TCTAGA GAT GAA GCA GCT GA TTGAA G AGA C CC AAT CTCGGAGC GAT GAA GCA GCT GA TTGAA G AGA C CC AA T CTCGGAGC GAT GAA GCA GCT GA TTGAA G AGA C CC AA T GCGGCCGC AAAG Primers for PCR amplification of 3X mir-241* mutant target cassette: GGGG TCTAGA GAT GAA GCA GCT GATTG (Forward primer) and CTT TGC GGC CGC ATT GGGTC TCT TCA ATC (Reverse primer) 3X let-7* target cassette construction Template Sequence: GGGG TCTAGA GGT AAG GTA GAA AA TTGAA T TGC ATA G CTCGGAGC GGT AAG GTA GAA AA TTGAA T TGC ATA G CTCGGAGC GGT AAG GTA GAA AA TTGAA T TGC ATA G GCGGCCGC AAAG Primers for PCR amplification of 3X let-7* target cassette: GGGG TCTAGA GGT AAG GTA GAA AATTGAAT TG (Forward primer) and CTTTGCGGCCGC C TAT GCA ATT CAA TTT TC (Reverse primer) 3X let-7(n2853) target cassette construction Template sequence: GGGG TCTAGA AAC TAT ACA ACC TA TTGAA C TAT CTC A CTCGGAGC AAC TAT ACA ACC TA TTGAA C TAT CTC A CTCGGAGC AAC TAT ACA ACC TA TTGAA C TAT CTC A GCGGCCGC AAAG

8 Primers for PCR amplification of 3X let-7(n2853) target cassette: GGGG TCTAGA AAC TAT ACA ACC TATTGAAC (Forward primer) and CTTTGCGGCCGC TGAGATAGTTCAATAG (Reverse primer) Gateway cloning of 3 UTRs for in vivo expression The same templates as described above for the in vitro targets were used with the following primers attb2r mir-241* fw GW: GGG GAC AGC TTT CTT GTA CAA AGT GGA CGA TGA AGC AGC TGA TTG AAG attb3 mir-241* rev GW: GGG GAC AAC TTT GTA TAA TAA AGT TGC ATT GTC TCT TCA ATC AGC attb3 mir-241*mut rev GW: GGG GAC AAC TTT GTA TAA TAA AGT TGC ATT GGG TCT CTT CAA TCA G attb2r n2853 fw GW: GGG GAC AGC TTT CTT GTA CAA AGT GGG AAA CTA TAC AAC CTA TTG AAC attb3 n2853 rev GW: GGG GAC AAC TTT GTA TAA TAA AGT TGC TGA GAT AGT TCA ATA G References Kamath, R.S., Fraser, A.G., Dong, Y., Poulin, G., Durbin, R., Gotta, M., Kanapin, A., Le Bot, N., Moreno, S., et al. (2003). Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Nature 421, Newbury, S., and Woollard, A. (2004). The 5'-3' exoribonuclease xrn-1 is essential for ventral epithelial enclosure during C. elegans embryogenesis. RNA 10, Praitis, V., Casey, E., Collar, D., and Austin, J. (2001). Creation of low-copy integrated transgenic lines in Caenorhabditis elegans. Genetics 157, Rual, J.F., Ceron, J., Koreth, J., Hao, T., Nicot, A.S., Hirozane-Kishikawa, T., Vandenhaute, J., Orkin, S.H., Hill, D.E., et al. (2004). Toward improving Caenorhabditis elegans phenome mapping with an ORFeome-based RNAi library. Genome Res 14,