Supplementary Figure 1 Asymmetrical function of 5p and 3p arms of mir-181 and mir-30 families and mir-142 and mir-154. (a) Control experiments using mirna sensor vector and empty pri-mirna overexpression vector in HEK
293T cells. (b) Luciferase reporter assays showing activities of 5p and 3p arms of mouse mir-181, -30 families and mir- 142, -154. Samples are from HEK 293T cells transfected with mirna sensor vector and pri-mirna expression vector of indicated mirnas (48 hr after transfection). An inhibitory effect of each arm is summarized in Fig. 1b. (c) Luciferase reporter assays in HepG2 and HCT116 cells showing activities of 5p and 3p arms of mouse mir- 181, -30 families and mir-142, -154. (d) Luciferase reporter assays in HEK 293T cells showing activities of 5p and 3p arms of human mir-30 family. (e) Effects of mutation in a catalytic residue of Ago2 on 5p/3p arm ratios. mirna reads of 5p and 3p arms in wild-type and Ago2 D597A mutant fetal livers (Cheloufi, S. et al., Nature. 465, 584-9, 2010) are plotted. This analysis suggests that 5p arm bias is not associated with the slicer activity of Ago2. (f) Luciferase reporter assays showing examples of 3p arm-dominant mirnas. Samples are from HEK 293T cells transfected with mirna sensor vector and pri-mirna expression vector of indicated mirnas (48 hr after transfection). Error bars, s.d. (n = 3 technical replicates).
Supplementary Figure 2 Validation of asymmetry codes by a lattice of pri-mirnas. (a) Control experiments using mirna sensor vector and empty pri-mirna overexpression vector in HEK 293T cells. (b) Luciferase reporter assays showing activities of 5p and 3p arms of a lattice of pri-mirnas. Samples are from HEK 293T cells transfected with mirna sensor vector and pri-mirna expression vector of indicated mirnas (48 hr after transfection). A relative inhibitory effect of each arm is summarized in Fig. 1f. Error bars, s.d. (n = 3 technical replicates).
Supplementary Figure 3 The PAZ domain is dispensable for asymmetric selection. (a) Overall structure of Ago2 with single mature mirna strand (4F3T) (Elkayam, E. et al., Cell. 150, 100-10, 2012). (b) A stochastic mode of initial recognition of RNA duplexes by either MID or PAZ domain.
(c) Sequences and thermodynamic stability profiles of symmetrical RNA duplexes used in Fig. 3 and Fig. 4. The terminal sequences of these duplexes were designed to show similar thermodynamic stability profiles. 5 -nt identity and thermodynamic stability profiles of these symmetrical 2nt overhang RNA duplexes covered four nucleotides and 0-2 nt mismatches (MM 0-2). Three end nucleotides and mutated nucleotides are highlighted in red and blue, respectively. (d) Preparation of GST-PAZ and GST-MID protein fragments. (e) Competitor experiments. Radiolabelled RNA duplexes, GST-PAZ and unlabeled competitors were incubated, UV photo-crosslinked and analyzed by PAGE and autoradiography. Quantification result is shown in the bottom. (f) Immunoblot analysis showing expression of Ago2 mutants (F294A, L339A, and R277A-R280A-K335A) in HEK 293T cells.
Supplementary Figure 4 Asymmetric selection is not affected by mutation in the RTTPQT motif. (a) Asymmetry sensing by MID domain. UV photo-crosslinking experiments showing binding properties of MID domain for symmetrical RNA duplexes. Radiolabelled RNA duplexes, GST-MID and unlabeled competitors were incubated, UV photo-crosslinked and analyzed by PAGE and autoradiography. Quantification
result is shown in right. (b) Prediction of RNA-binding motif in Ago2 MID domain by BindN program (Wang, L. et al., Nucleic Acids Res. 34, W243-8, 2006). The predicted RNA-binding residues are labeled with +. (c) Structural comparison of human Ago2 with mir-20a (4F3T) and T. thermophilus Ago with guide DNA and target RNA duplex (3F73) (Elkayam, E. et al., Cell. 150, 100-10, 2012; Wang, Y. et al., Nature. 456, 921-6, 2008). Nucleotide selection loop and 554-RTTPQT-559 motif are highlighted in blue and orange, respectively. (d) UV photo-crosslinking experiments showing binding properties of RTTPQT motif mutant for symmetrical RNA duplexes. (e) Immunoblot analysis showing expression of Ago2 RTTPQT mutant in HEK 293T cells. (f) RIP-qRT-PCR analysis showing effects of mutation in RTTPQT motif of Ago2 on asymmetry sensing for type 1 mir-154 (both arm type). Samples are from HEK 293T cells transfected with Ago2 mutant expression vector and pri-mirna expression vector. Error bars, s.d. (n = 3 technical replicates).
Supplementary Figure 5 Recognition of TS and 5 -nt rules by conserved amino acid residues in the MID domain. (a) Proposed mechanisms for detection of thermodynamic stability of RNA duplex ends by phosphate-binding pocket (tract).
(b) Sequences of MID and PIWI domains including G-drive (phosphate-binding pocket (tract), pink) and N- drive (nucleotide selection loop, blue) in Ago1-4. N, P, and S indicate nucleobase, phosphate, and ribose, respectively. (c) Immunoblot analysis showing expression of G-drive and N-drive mutants of Ago2 in HEK 293T cells. (d) RIP-qRT-PCR analysis showing effects of mutations in PAZ domain and MID domain on asymmetry sensing for type 1 mir-216a (5p arm type). Samples are from HEK 293T cells transfected with Ago2 mutant expression vector and pri-mirna expression vector. 5p arm of type 1 mir-216a is predominantly selected by both TS rule and 5 -nt rule. Two G-drive mutants (K566A and R792A C793A) and two N-drive mutants (524- GKT-526 to GKGT and 523-PGKTP-527 to AAAAA) reduced 5p/3p ratios. Error bars, s.d. (n = 3 technical replicates). (e) Preparation of GST-MID mutants.
Supplementary Figure 6 Role of 5 -nt identity in RISC stabilization. (a) Design of sirna duplexes used in Fig. 5a. Sequences are shown in Supplementary Table 4. (b) A lattice of pri-mirnas used for RIP-qRT-PCR analysis of Ago2 mutants in Fig. 5b. (c) Predicted pattern of altered mirna asymmetry caused by mutations in G-drive and N-drive. (d) Luciferase reporter assays showing role of 5 -nt identity in initial RNA loading and RISC dissociation. Samples are from HEK 293T cells were transfected with mirna sensor vector and pri-mirna expression vector (mouse pri-mir-216a (5p arm type) with 5p arms with different 5 -nts). To evaluate the role of 5 -nt identity in RISC stabilization and/or dissociation, 24 hr after transfection, cells were treated with Actinomycin D (1 mg/ml) and subjected to luciferase reporter assay. (e) RIP-qRT-PCR analysis showing role of 5 -nt identity in RISC stabilization. Samples were from HEK 293T cells transfected with pcdna3-flag-ago2 and pri-mirna expression vector (pri-mir-216a (5p arm type) with 5p arms with different 5 -nts) and treated with/without Actinomycin D (1 mg/ml, 24 hr). (f) Prediction of inhibitory activities of both arms of diverse mirnas (46 pri-mirnas, 92 strands) shown in Fig. 6b by equation (iii). FR indicates fold repression. Exponential curves were fitted. Error bars, s.d. (n = 3 technical replicates).
Supplementary Figure 7 Effects of MID-domain mutation on asymmetry pattern. (a) Immunoblot analysis showing showing expression of Flag-tagged WT or mutant Ago2 in mouse Agoknockout ES cells. (b) Comparison of qrt-pcr analysis and small RNA sequencing for small RNAs interacting with Flag-tagged WT Ago2 or Ago2 mutants in mouse Ago-knockout ES cells shown in (a). The results for mir-140 (both arm type), in which TS and 5 -nt rules favor 5p and 3p arms, respectively, are shown. (c) Small RNA sequencing analysis showing asymmetry patterns of small RNAs interacting with Flag-tagged WT Ago2 or Ago2 mutants (G-drive mutants (Q548A and R792A C793A) and N-drive mutants (524-GKT-526 to GKGT and 523-PGKTP-527 to AAAAA)) in mouse Ago-knockout ES cells. MiRNAs were classified into 8 groups according to 5 -nt identity, as in Fig. 6a. y axis indicates log2 strand ratio. (d) Univariate linear regression analysis using G for mirna asymmetry patterns. Correlation coefficients between strand ratio and G were summarized. Mutation of K566 in the phosphate-binding pocket strongly attenuated global dependence on TS rule, in consistent with the most remarkable effect of this mutation on TS rule (Fig. 4 and Fig. 5). As for other phosphate-binding pocket mutants, R792A C793A mutant showed a weak but similar trend to reduce dependence on TS rule, and Q548A mutant showed a complex pattern, but reduced dependence on TS rule in several groups.
Supplementary Figure 8 Effects of cancer-associated variations on asymmetric activities of mir-142 and mir-146a. (a) Details of mir-142 asymmetry. According to genome-wide high-throughput mirna profiling (Chiang, H.R. et al., Genes Dev. 24, 992-1009, 2010; Griffiths-Jones, S. et al., Nucleic Acids Res. 36, D154-8, 2008; Park, J.E. et al., Nature. 475, 201-5, 2011), processing of pre-mir-142 shows considerable heterogeneity. Application of our model to each mirna duplex can explain potent activities of both arms of mir-142 precursors. In this figure, we adopted conventional 22nt length processing to infer identities of each mirna duplex from the results of deep sequencing. (b) Proposed roles of variations in mir-142 and mir-146a duplexes on the asymmetric biogenesis. (c) Effects of mir-142 mutation on expression levels of mir-142 and mir-142 targets in TCGA AML data. In AML database, two 3p variants and two 5p variants dominate mir-142 pool, suggesting that the processing of mir-142 shows a complex pattern of mirna duplexes in this cell type. *P < 0.05 by two-tailed Mann-Whitney test. (d and e) Impacts of genotypes (GG, CG, and CC) of mir-146a SNP (rs2910164) on patient survival in liver hepatocellular carcinoma (d) and skin cutaneous melanoma (e) in the TCGA database. The P values were calculated with two-tailed log-rank test. (f and g) Effects of mir-146a genotypes (GG, CG, and CC) on mir-146a 5p/3p ratios in liver hepatocellular carcinoma (f) and skin cutaneous melanoma (g) in the TCGA database. Boxes represent the median and interquartile range (IQR). Error bars, 1.5X IQR. **P < 0.01; ***P < 0.001 by two-tailed Mann-Whitney test. (h) GSEA showing upregulation of mir-146a-5p targets in the HCC patients with mir-146a CC genotype. In melanoma, the significant alteration of mir-146a-5p targets by mir-146a genotype was not observed.
Supplementary Table 1. Sequences of mir-181 and mir-30 families and mir-142 and mir-154. For mir-142, 5p and 3p arms in mirbase are shown. See details of mir-142 asymmetry in Supplementary Fig. 8a. mirna mir-181a-1 mir-181a-2 mir-181c mir-181b-1 mir-181d mir-30a mir-30d mir-30e mir-30c-1 mir-30c-2 mir-30b mir-384 mir-142 mir-154 Sequence 5p arm Loop 3p arm UUGCUUCAGUGAACAUUCAACGCUGUCGGUGAGUUUGG--AAUUCAAAUA----AAAACCAUCGACCGUUGAUUGUACCCUAUAGC -----CCAUGGAACAUUCAACGCUGUCGGUGAGUUUGG--GAUUCAAAAACAAAAAAACCACCGACCGUUGACUGUACCUUGG--- GGGUUUGGGGGAACAUUCAAC-CUGUCGGUGAGUUUGGGCAGCUCAGACA------AACCAUCGACCGUUGAGUGGACCCCGAGGC AGGUCACAAUCAACAUUCAUUGCUGUCGGUGGGUUG-AACUGUGUAGAAA-----AGCUCACUGAACAAUGAAUGCAA-CUGUGGC -----ACAAUUAACAUUCAUUGUUGUCGGUGGGUUGUGAGGAGGCAGCCA-----GACCCACCGGGGGAUGAAUGUCA-CUGU--- 5p arm Loop 3p arm --GCG--AC-UGUAAACA--UCCUCGACUGGAAGCUGUGAAGCCAC--AAAUGGGC--UUUCAGUCGGAUGUUUGCAGCUGC---- CUGUG--UC-UGUAAACA--UCCCCGACUGGAAGCUGU-AAGCCAC--AGCCAAGC--UUUCAGUCAGAUGUUUGCUGCUACUGGC CUUUGCUAC-UGUAAACA--UCCUUGACUGGAAGCUGU-AAGGUGUUGAGAGGAGC--UUUCAGUCGGAUGUUUACAGCGGCAGGC UGUAG-UGUGUGUAAACA--UCCUACACUCUCAGCUGU-GAGCU-C--AAGGUGGC--UGGGAGAGGGUUGUUUACUCCUUCUGCC GACAGAUAU-UGUAAACA--UCCUACACUCUCAGCUGU-GAAAAGU--AAGAAAGC--UGGGAGAAGGCUGUUUACUCUCUCUGCC UUCAG-UUCAUGUAAACA--UCCUACACU--CAGCUGU-CAUACAU--GCGUUGGC--UGGGAUGUGGAUGUUUACGUCAGCUGUC CAGGA--AU-UGUAAACAAUUCCUAGGC---AAUGUGU-AUAAUGUU--GGUAAGUCAUUCCUAGAAAUUGUUCACAAUGCCUGUA 5p arm Loop 3p arm ACCCAUAAAGUAGAAAGCACUACUAACAGCACUGGAGGGUGUAGUGUUUCCUACUUUAUGGAUG GAAGAUAGGUUAUCCGUGUUGCCUUCGCUUUAUUCGUGACGAAUCAUACACGGUUGACCUAUUUUU
Supplementary Table 2. Sequences of mouse wild-type and mutant mirna precursors used in Fig. 1e and Fig. 1f. 5p/3p arms and mutated nucleotides are highlighted in yellow and blue, respectively. mirna mir-154 (Type1) mir-154 (Type2, WT) mir-154 (Type3) mir-154 (Type4) mir-193b (Type1) mir-193b (Type2) mir-193b (Type3) mir-193b (Type4, WT) mir-292 (Type1) mir-292 (Type2, WT) mir-292 (Type3) mir-292 (Type4) mir-384 (Type1) mir-384 (Type2, WT) mir-384 (Type3) mir-384 (Type4) mir-181c (Type1) mir-181c (Type2, WT) mir-181c (Type3) mir-181c (Type4) mir-383 (Type1, WT) mir-383 (Type2) mir-383 (Type3) mir-383 (Type4) mir-216a (Type1, WT) mir-216a (Type2) mir-216a (Type3) mir-216a (Type4) mir-191 (Type1) mir-191 (Type2) mir-191 (Type3, WT) mir-191 (Type4) Sequence 5p arm Loop 3p arm GAAGAUAGGUUAUCCGUGUUGCCUgCGCUUUAUUCGUGACGcAUCAUACACGGUUGACCUAUUUUU GAAGAUAGGUUAUCCGUGUUGCCUUCGCUUUAUUCGUGACGAAUCAUACACGGUUGACCUAUUUUU GAAGAcAGGUUAUCCGUGUUGCCUgCGCUUUAUUCGUGACGcAUCAUACACGGUUGACCUgUUUUU GAAGAcAGGUUAUCCGUGUUGCCUUCGCUUUAUUCGUGACGAAUCAUACACGGUUGACCUgUUUUU AGAAUuGGGGUUUUGAGGGCGAGAgGAGUUUGUGUUUUAUCCcACUGGCCCACAAAGUCCCaCUUUUGGGGUCA AGAAUuGGGGUUUUGAGGGCGAGATGAGUUUGUGUUUUAUCCAACUGGCCCACAAAGUCCCaCUUUUGGGGUCA AGAAUCGGGGUUUUGAGGGCGAGAgGAGUUUGUGUUUUAUCCcACUGGCCCACAAAGUCCCGCUUUUGGGGUCA AGAAUCGGGGUUUUGAGGGCGAGAUGAGUUUGUGUUUUAUCCAACUGGCCCACAAAGUCCCGCUUUUGGGGUCA GUGAUACUCAAACUGGGGGCUCUUgUGGAUUUUCAUCGGAAGAcAAGUGCCGCCAGGUUUUGAGUGUCACCGGUUG GUGAUACUCAAACUGGGGGCUCUUUUGGAUUUUCAUCGGAAGAAAAGUGCCGCCAGGUUUUGAGUGUCACCGGUUG GUGAUgCUCAAACUGGGGGCUCUUgUGGAUUUUCAUCGGAAGAcAAGUGCCGCCAGGUUUUGAGcGUCACCGGUUG GUGAUgCUCAAACUGGGGGCUCUUUUGGAUUUUCAUCGGAAGAAAAGUGCCGCCAGGUUUUGAGcGUCACCGGUUG GAAUUGUAAACAAUUCCUAGGCAAgGUGUAUAAUGUUGGUAAGUCcUUCCUAGAAAUUGUUCACAAUGCCUGUAACA GAAUUGUAAACAAUUCCUAGGCAAUGUGUAUAAUGUUGGUAAGUCAUUCCUAGAAAUUGUUCACAAUGCCUGUAACA GAAUcGUAAACAAUUCCUAGGCAAgGUGUAUAAUGUUGGUAAGUCcUUCCUAGAAAUUGUUCACgAUGCCUGUAACA GAAUcGUAAACAAUUCCUAGGCAAUGUGUAUAAUGUUGGUAAGUCAUUCCUAGAAAUUGUUCACgAUGCCUGUAACA GGGGAACAUUCAACCUGUCGGUGAGgUUGGGCAGCUCAGACAAcCCAUCGACCGUUGAGUGGACCCCGAGGCC GGGGAACAUUCAACCUGUCGGUGAGUUUGGGCAGCUCAGACAAACCAUCGACCGUUGAGUGGACCCCGAGGCC GGGGgACAUUCAACCUGUCGGUGAGgUUGGGCAGCUCAGACAAcCCAUCGACCGUUGAGUGGACCCCGAGGCC GGGGgACAUUCAACCUGUCGGUGAGUUUGGGCAGCUCAGACAAACCAUCGACCGUUGAGUGGACCCCGAGGCC CUCAGAUCAGAAGGUGACUGUGGCUUUGGGUGGAUAUUAAUCAGCCACAGCACUGCCUGGUCAGAAAGAG CUCAGAUCAGAAGGUGACUGUGuCUUUGGGUGGAUAUUAAUCAGaCACAGCACUGCCUGGUCAGAAAGAG CUCgGAUCAGAAGGUGACUGUGGCUUUGGGUGGAUAUUAAUCAGCCACAGCACUGCCUGGUCAGAAAGAG CUCgGAUCAGAAGGUGACUGUGuCUUUGGGUGGAUAUUAAUCAGaCACAGCACUGCCUGGUCAGAAAGAG UUGGUUUAAUCUCAGCUGGCAACUGUGAGAUGUCCCUAUCAUUCCUCACAGUGGUCUCUGGGAUUAUGCUAA UUGGUUUAAUCUCAGCUGGCAACUGUaAGAUGUCCCUAUCAUUCCUuACAGUGGUCUCUGGGAUUAUGCUAA UUGGUUcAAUCUCAGCUGGCAACUGUGAGAUGUCCCUAUCAUUCCUCACAGUGGUCUCUGGGAUUgUGCUAA UUGGUUcAAUCUCAGCUGGCAACUGUaAGAUGUCCCUAUCAUUCCUuACAGUGGUCUCUGGGAUUgUGCUAA AGCGGGaAACGGAAUCCCAAAAGCAGCUGUUGUCUCCAGAGCAUUCCAGCUGCACUUGGAUUUCGUUCCCUGCU AGCGGGaAACGGAAUCCCAAAAGCAGaUGUUGUCUCCAGAGCAUUCCAuCUGCACUUGGAUUUCGUUCCCUGCU AGCGGGCAACGGAAUCCCAAAAGCAGCUGUUGUCUCCAGAGCAUUCCAGCUGCACUUGGAUUUCGUUCCCUGCU AGCGGGCAACGGAAUCCCAAAAGCAGaUGUUGUCUCCAGAGCAUUCCAuCUGCACUUGGAUUUCGUUCCCUGCU
Supplementary Table 3. Sequences of wild-type and mutant mirna precursors used in Fig. 1g. 5p/3p arms, 9-12nt region, and unpaired nucleotides are highlighted in yellow, red, and blue, respectively. Mutated nucleotides are indicated by lowercase. mirna mir-181c (WT) mir-181c (CM) mir-181c (9-10nt MM) mir-181c (11-12nt MM) mir-384 (WT) mir-384 (CM) mir-384 (9-10nt MM) mir-384 (11-12nt MM) mir-146a (WT, CM) mir-146a (9-10nt MM) mir-146a (11-12nt MM) Sequence 5p arm Loop 3p arm GGGAACAUUCAACCUGUCGGUGAGUUUGGGCAGCUCAGACAAACCAUCGACCGUUGAGUGGACCCCGAG GGGAACAUUCAAC_gGUCGGUG_GUUUGGGCAGCUCAGACAAACCAUCGACCGUUGAGUGGACCCCGAG GGGAACAUUCAug_gGUCGGUG_GUUUGGGCAGCUCAGACAAACCAUCGACCGUUGAGUGGACCCCGAG GGGAACAUUCAAC_ccUCGGUG_GUUUGGGCAGCUCAGACAAACCAUCGACCGUUGAGUGGACCCCGAG 5p arm Loop 3p arm AAUUGUAAACAAUUCCUAGGCAAUGUGUAUAAUGUUGGUAAGUCAUUCCUAGAAAUUGUUCACAAUGCCU AAUUGUgAACAAUUuCUAGG_AAUGUGUAUAAUGUUGGUAAGUCAUUCCUAGAAAUUGUUCACAAUGCCU AAUUGUgAACAccUuCUAGG_AAUGUGUAUAAUGUUGGUAAGUCAUUCCUAGAAAUUGUUCACAAUGCCU AAUUGUgAACAAUaaCUAGG_AAUGUGUAUAAUGUUGGUAAGUCAUUCCUAGAAAUUGUUCACAAUGCCU 5p arm Loop 3p arm AGCUCUGAGAACUGAAUUCCAUGGGUUAUAUCAAUGUCAGACCUGUGAAAUUCAGUUCUUCAGCU AGCUCUGAGAACUcuAUUCCAUGGGUUAUAUCAAUGUCAGACCUGUGAAAUUCAGUUCUUCAGCU AGCUCUGAGAACUGAccUCCAUGGGUUAUAUCAAUGUCAGACCUGUGAAAUUCAGUUCUUCAGCU
Supplementary Table 4. Sequences of sirna duplexes used in Fig. 5a. Sequences and thermodynamic stability profiles of asymmetrical 21-nt RNA duplexes used in Fig. 5. Control sirna duplex was described in a previous report (Noland, C.L. et al., RNA. 5, 639-48, 2013). Mutated nucleotides are highlighted in red. ΔΔG(A-B) Set Control A-i A-ii A-iii A-iv B-i B-ii B-iii B-iv asymmetrical 21-nt RNA duplex (top: strand A, bottom: strand B) ΔΔG(1nt) ΔΔG(2nt) ΔΔG(3nt) ΔΔG(4nt) UCGAAGUAUUCCGCGUACGUU UUAGCUUCAUAAGGCGCAUGC 0.5 0.3-0.8 0.5 UCGAAGUAUUCCGCGUAGCUU UUAGCUUCAUAAGGCGCAUGC -2.5-4.9-5.5-4.2 UCGAAGUAUUCCGCGUACCUU UUAGCUUCAUAAGGCGCAUGC -2.5-1.6-2.7-1.4 UCGAAGUAUUCCGCGUACGUU UUCGCUUCAUAAGGCGCAUGC 3 2.3 1.2 2.5 UCGAAGUAUUCCGCGUACGUU UUCCCUUCAUAAGGCGCAUGC 3 5.2 3 4.3 UCGAAGUAUUCCGCUUUUGUU UUAGCUUCAUAAGGCGAAAAC 0.2-1.3-2.8-2.8 UCGAAGUAUUCCGCGUAUGUU UUAGCUUCAUAAGGCGCAUAC 0.2-1.1-2.2-0.9 UUGAAGUAUUCCGCGUACGUU UUAACUUCAUAAGGCGCAUGC 2 2.1 1 2.3 UUUAAGUAUUCCGCGUACGUU UUAAAUUCAUAAGGCGCAUGC 2 3.3 3.3 4.6
Supplementary Table 5. Primers used in this study Primers for the construction of pri-mirna expression vectors. Primer sequence (The lowercase nucleotides indicate BamHI and XhoI sites) mirna Forward (5 to 3 ) Reverse (5 to 3 ) mouse pri-mir-181a-1 CGggatccCTTTGACACAGCACAAAGTGGA CCGctcgagGGCCACAGTTGCATTCATTGTT mouse pri-mir-181a-2 CGggatccCAGATATCCACTTTTAGTCAGCA CCGctcgagGCATTTGGTACTATGATGGCACT mouse pri-mir-181b-1 CGggatccACATCTCTGCCTCACAGGTTGCT CCGctcgagCCATTTGTAACCCCCAGGAGTAT mouse pri-mir-181c CGggatccGAGACAGGCACATTATCATCTCT CCGctcgagAAGGGTTCTATCCTCTTTCCCA mouse pri-mir-181d CGggatccTCCCAACTCCAGTTATCCAAGAA CCGctcgagCTGAGCAAACATCATCCCCCCA mouse pri-mir-30a CGggatccTTATGGCCAACAGTAATGGGTGA CCGctcgagGAAGCACTGTCTTATTTGTTT mouse pri-mir-30b CGggatccAGAGAGCATGAAAGAGAGAACGA CCGctcgagAACCTGAAGCTAGGCTTAAGGCT mouse pri-mir-30c-1 CGggatccGGTCTCAGTACCTTGAGAAG CCGctcgagTCACAACACACTGAGAGCTC mouse pri-mir-30c-2 CGggatccTCCTGGCATGTGCAAAACAG CCGctcgagTCTCAGTCTCCTGAGTAGTG mouse pri-mir-30d CGggatccGTGTCCATGTGGAGTAACATAGA CCGctcgagGATAAACTTGTACTGACACGGCA mouse pri-mir-30e CGggatccGTAGTATACTGTTTAGAGTCA CCGctcgagCATCACAGATCTTACCACCTT mouse pri-mir-384 CGggatccTGAAAGGAATTTTCCTTGGCA CCGctcgagCCTGCATCACCAAAGTACTAT mouse pri-mir-142 CGggatccCTCATCTGGCGCCATGTTGAGT CCGctcgagATTCGAGAGAGCGGCTGTGGTGT mouse pri-mir-154 CGggatccTTAGACACTGTATCCTTGGCAGT CCGctcgagGTGGATGACTACAGAATACTCCA human pri-mir-30a CGggatccTAGAAACTAGAAGCTCGGTG CCGctcgagCACATTTCCTCATTTCTCTTACG human pri-mir-30b CGggatccGAAAAATTAGCCGGGCATAG CCGctcgagACTACTCCTACTGCAACCATG human pri-mir-30c-1 CGggatccGTCATGGAAGTGCACTTTAG CCGctcgagCTATCAGGGAGAGAGGAAACAG human pri-mir-30c-2 CGggatccATGCCTGGGTATTAGGGCCAAAC CCGctcgagGAATCTTACATGGTAACCCAACC human pri-mir-30d CGggatccGGAGAAATTGCACTTGGTGAAC CCGctcgagACGGCATATCCTGTTATTAGG human pri-mir-30e CGggatccTATCCTTAGAAGCAGTCATTC CCGctcgagGAGTTAGCACAATGAGAGTGAGC mouse pri-mir-222 CGggatccATCATATGCCCCAGTACAGG CCGctcgagTCAAACTCTGGATGACTTCC mouse pri-mir-223 CGggatccATCCCCGTTTTTGTTTGGAG CCGctcgagGCAGTCCATGGCATTTTCAC mouse pri-mir-124 CGggatccATGCGGTGGTCCTTAGCTCA CCGctcgagCTTGCATAGATCCGTGTTTC mouse pri-mir-27a CGggatccTAGGTGCTACACTCCGCTCCCA CCGctcgagTTTCCAGGTCTTGCTCACGGCA mouse pri-mir-143 CGggatccAAACAGGGGAGCCACAGGTT CCGctcgagGTTGAGAAGGGTTCCGAGGGT mouse pri-mir-193b CGggatccTTAGACACAGTATCTCCCTCCA CCGctcgagGTTTGTTCCGAGAATTTGGCA mouse pri-mir-292a CGggatccTCTCTTGAGCCTGAATGAGA CCGctcgagCGGCTCCTTTATGAACGCGGA mouse pri-mir-383 CGggatccAAGCTTTTATGCACTGCCCA CCGctcgagGTTATTTTCCCTTTACAGCCTA mouse pri-mir-216a CGggatccGTGTCTATTCACACTTGATAGTGT CCGctcgagTCACAGAGAAAGGACTTTGTGT mouse pri-mir-191 CGggatccCTTGGGACTCACAGGGCTAA CCGctcgagAGCTACTCTCCCTCCATGAA human pri-mir-146a CGggatccGCTCAAGAGATCCACCCACA CCGctcgagACTTGGAACCCTGCTTAGCA Primers for the construction of mirna sensor vectors. Primer sequence (Sensor-oligo-forward: XhoI-SacI-(miRNA-complementary sequence)-noti) mirna Forward (5 to 3 ) Reverse (5 to 3 )
mir-181a-1-5p TCGAGAGTAGAGCTCTAGTACTCACCGACAGCGTTGAATGTTGC GGCCGCAACATTCAACGCTGTCGGTGAGTACTAGAGCTCTACTC mir-181a-1-3p TCGAGAGTAGAGCTCTAGTGGTACAATCAACGGTCGATGGTGC GGCCGCACCATCGACCGTTGATTGTACCACTAGAGCTCTACTC mir-181a-2-5p identical to mir-181a-1-5p identical to mir-181a-1-5p mir-181a-2-3p TCGAGAGTAGAGCTCTAGTCAAGGTACAGTCAACGGTCGGTGC GGCCGCACCGACCGTTGACTGTACCTTGACTAGAGCTCTACTC mir-181b-1-5p TCGAGAGTAGAGCTCTAGTACCCACCGACAGCAATGAATGTTGC GGCCGCAACATTCATTGCTGTCGGTGGGTACTAGAGCTCTACTC mir-181b-1-3p TCGAGAGTAGAGCTCTAGTGCATTCATTGTTCAGTGAGGC GGCCGCCTCACTGAACAATGAATGCACTAGAGCTCTACTC mir-181c-5p TCGAGAGTAGAGCTCTAGTACTCACCGACAGGTTGAATGTTGC GGCCGCAACATTCAACCTGTCGGTGAGTACTAGAGCTCTACTC mir-181c-3p TCGAGAGTAGAGCTCTAGTGGTCCACTCAACGGTCGATGGTGC GGCCGCACCATCGACCGTTGAGTGGACCACTAGAGCTCTACTC mir-181d-5p TCGAGAGTAGAGCTCTAGTACCCACCGACAACAATGAATGTTGC GGCCGCAACATTCATTGTTGTCGGTGGGTACTAGAGCTCTACTC mir-181d-3p TCGAGAGTAGAGCTCTAGTTGACATTCATCCCCCGGTGGGGC GGCCGCCCCACCGGGGGATGAATGTCAACTAGAGCTCTACTC mir-30a-5p TCGAGAGTAGAGCTCTAGTCTTCCAGTCGAGGATGTTTACAGC GGCCGCTGTAAACATCCTCGACTGGAAGACTAGAGCTCTACTC mir-30a-3p TCGAGAGTAGAGCTCTAGTGCTGCAAACATCCGACTGAAAGGC GGCCGCCTTTCAGTCGGATGTTTGCAGCACTAGAGCTCTACTC mir-30b-5p TCGAGAGTAGAGCTCTAGTAGCTGAGTGTAGGATGTTTACAGC GGCCGCTGTAAACATCCTACACTCAGCTACTAGAGCTCTACTC mir-30b-3p (mouse) TCGAGAGTAGAGCTCTAGTGACGTAAACATCCACATCCCAGGC GGCCGCCTGGGATGTGGATGTTTACGTCACTAGAGCTCTACTC mir-30b-3p (human) TCGAGAGTAGAGCTCTAGTGAAGTAAACATCCACCTCCCAGGC GGCCGCCTGGGAGGTGGATGTTTACTTCACTAGAGCTCTACTC mir-30c-1-5p TCGAGAGTAGAGCTCTAGTGCTGAGAGTGTAGGATGTTTACAGC GGCCGCTGTAAACATCCTACACTCTCAGCACTAGAGCTCTACTC mir-30c-1-3p TCGAGAGTAGAGCTCTAGTGGAGTAAACAACCCTCTCCCAGGC GGCCGCCTGGGAGAGGGTTGTTTACTCCACTAGAGCTCTACTC mir-30c-2-5p identical to mir-30c-1-5p identical to mir-30c-1-5p mir-30c-2-3p TCGAGAGTAGAGCTCTAGTAGAGTAAACAACCCTCTCCCAGGC GGCCGCCTGGGAGAGGGTTGTTTACTCTACTAGAGCTCTACTC mir-30d-5p TCGAGAGTAGAGCTCTAGTCTTCCAGTCGGGGATGTTTACAGC GGCCGCTGTAAACATCCCCGACTGGAAGACTAGAGCTCTACTC mir-30d-3p TCGAGAGTAGAGCTCTAGTGCAGCAAACATCTGACTGAAAGGC GGCCGCCTTTCAGTCAGATGTTTGCTGCACTAGAGCTCTACTC mir-30e-5p TCGAGAGTAGAGCTCTAGTCTTCCAGTCAAGGATGTTTACAGC GGCCGCTGTAAACATCCTTGACTGGAAGACTAGAGCTCTACTC mir-30e-3p TCGAGAGTAGAGCTCTAGTGCTGTAAACATCCGACTGAAAGGC GGCCGCCTTTCAGTCGGATGTTTACAGCACTAGAGCTCTACTC mir-384-5p TCGAGAGTAGAGCTCTAGTACATTGCCTAGGAATTGTTTACAGC GGCCGCTGTAAACAATTCCTAGGCAATGTACTAGAGCTCTACTC mir-384-3p TCGAGAGTAGAGCTCTAGTATTGTGAACAATTTCTAGGAATGC GGCCGCATTCCTAGAAATTGTTCACAATACTAGAGCTCTACTC mir-142-5p TCGAGAGTAGAGCTCTAGTAGTAGTGCTTTCTACTTTATGGC GGCCGCCATAAAGTAGAAAGCACTACTACTAGAGCTCTACTC mir-142-3p TCGAGAGTAGAGCTCTAGTTCCATAAAGTAGGAAACACTACAGC GGCCGCTGTAGTGTTTCCTACTTTATGGAACTAGAGCTCTACTC mir-154-5p TCGAGAGTAGAGCTCTAGTCGAAGGCAACACGGATAACCTAGC GGCCGCTAGGTTATCCGTGTTGCCTTCGACTAGAGCTCTACTC mir-154-3p TCGAGAGTAGAGCTCTAGTAATAGGTCAACCGTGTATGATTGC GGCCGCAATCATACACGGTTGACCTATTACTAGAGCTCTACTC mir-222-5p TCGAGAGTAGAGCTCTAGTAGGATCTACACTGGCTACTGAGC GGCCGCTCAGTAGCCAGTGTAGATCCTACTAGAGCTCTACTC mir-222-3p TCGAGAGTAGAGCTCTAGTACCCAGTAGCCAGATGTAGCTGC GGCCGCAGCTACATCTGGCTACTGGGTACTAGAGCTCTACTC mir-223-5p TCGAGAGTAGAGCTCTAGTCAACTCAGCTTGTCAAATACACGGC GGCCGCCGTGTATTTGACAAGCTGAGTTGACTAGAGCTCTACTC mir-223-3p TCGAGAGTAGAGCTCTAGTTGGGGTATTTGACAAACTGACAGC GGCCGCTGTCAGTTTGTCAAATACCCCAACTAGAGCTCTACTC mir-124-5p TCGAGAGTAGAGCTCTAGTATCAAGGTCCGCTGTGAACACGGC GGCCGCCGTGTTCACAGCGGACCTTGATACTAGAGCTCTACTC mir-124-3p TCGAGAGTAGAGCTCTAGTGGCATTCACCGCGTGCCTTAGC GGCCGCTAAGGCACGCGGTGAATGCCACTAGAGCTCTACTC mir-27a-5p TCGAGAGTAGAGCTCTAGTTGCTCACAAGCAGCTAAGCCCTGC GGCCGCAGGGCTTAGCTGCTTGTGAGCAACTAGAGCTCTACTC mir-27a-3p TCGAGAGTAGAGCTCTAGTGCGGAACTTAGCCACTGTGAAGC GGCCGCTTCACAGTGGCTAAGTTCCGCACTAGAGCTCTACTC mir-143-5p TCGAGAGTAGAGCTCTAGTCCAGAGATGCAGCACTGCACCGC GGCCGCGGTGCAGTGCTGCATCTCTGGACTAGAGCTCTACTC mir-143-3p TCGAGAGTAGAGCTCTAGTGAGCTACAGTGCTTCATCTCAGC GGCCGCTGAGATGAAGCACTGTAGCTCACTAGAGCTCTACTC Type1 mir-154-5p TCGAGAGTAGAGCTCTAGTCGCAGGCAACACGGATAACCTAGC GGCCGCTAGGTTATCCGTGTTGCCTGCGACTAGAGCTCTACTC Type1 mir-154-3p TCGAGAGTAGAGCTCTAGTAATAGGTCAACCGTGTATGATGGC GGCCGCCATCATACACGGTTGACCTATTACTAGAGCTCTACTC
Type3 mir-154-5p TCGAGAGTAGAGCTCTAGTCGCAGGCAACACGGATAACCTGGC GGCCGCCAGGTTATCCGTGTTGCCTGCGACTAGAGCTCTACTC Type3 mir-154-3p TCGAGAGTAGAGCTCTAGTAACAGGTCAACCGTGTATGATGGC GGCCGCCATCATACACGGTTGACCTGTTACTAGAGCTCTACTC Type4 mir-154-5p TCGAGAGTAGAGCTCTAGTCGAAGGCAACACGGATAACCTGGC GGCCGCCAGGTTATCCGTGTTGCCTTCGACTAGAGCTCTACTC Type4 mir-154-3p TCGAGAGTAGAGCTCTAGTAACAGGTCAACCGTGTATGATTGC GGCCGCAATCATACACGGTTGACCTGTTACTAGAGCTCTACTC Type1 mir-193b-5p TCGAGAGTAGAGCTCTAGTTCCTCTCGCCCTCAAAACCCCAGC GGCCGCTGGGGTTTTGAGGGCGAGAGGAACTAGAGCTCTACTC Type1 mir-193b-3p TCGAGAGTAGAGCTCTAGTAGTGGGACTTTGTGGGCCAGTGGC GGCCGCCACTGGCCCACAAAGTCCCACTACTAGAGCTCTACTC Type2 mir-193b-5p TCGAGAGTAGAGCTCTAGTTCATCTCGCCCTCAAAACCCCAGC GGCCGCTGGGGTTTTGAGGGCGAGATGAACTAGAGCTCTACTC Type2 mir-193b-3p TCGAGAGTAGAGCTCTAGTAGTGGGACTTTGTGGGCCAGTTGC GGCCGCAACTGGCCCACAAAGTCCCACTACTAGAGCTCTACTC Type3 mir-193b-5p TCGAGAGTAGAGCTCTAGTTCCTCTCGCCCTCAAAACCCCGGC GGCCGCCGGGGTTTTGAGGGCGAGAGGAACTAGAGCTCTACTC Type3 mir-193b-3p TCGAGAGTAGAGCTCTAGTAGCGGGACTTTGTGGGCCAGTGGC GGCCGCCACTGGCCCACAAAGTCCCGCTACTAGAGCTCTACTC Type4 mir-193b-5p (WT) TCGAGAGTAGAGCTCTAGTTCATCTCGCCCTCAAAACCCCGGC GGCCGCCGGGGTTTTGAGGGCGAGATGAACTAGAGCTCTACTC Type4 mir-193b-3p (WT) TCGAGAGTAGAGCTCTAGTAGCGGGACTTTGTGGGCCAGTTGC GGCCGCAACTGGCCCACAAAGTCCCGCTACTAGAGCTCTACTC Type1 mir-292a-5p TCGAGAGTAGAGCTCTAGTCACAAGAGCCCCCAGTTTGAGTGC GGCCGCACTCAAACTGGGGGCTCTTTTGACTAGAGCTCTACTC Type1 mir-292a-3p TCGAGAGTAGAGCTCTAGTACACTCAAAACCTGGCGGCACTTGGC GGCCGCAAAGTGCCGCCAGGTTTTGAGTGTACTAGAGCTCTACTC Type2 mir-292a-5p (WT) TCGAGAGTAGAGCTCTAGTCAAAAGAGCCCCCAGTTTGAGTGC GGCCGCACTCAAACTGGGGGCTCTTGTGACTAGAGCTCTACTC Type2 mir-292a-3p (WT) TCGAGAGTAGAGCTCTAGTACACTCAAAACCTGGCGGCACTTTGC GGCCGCCAAGTGCCGCCAGGTTTTGAGTGTACTAGAGCTCTACTC Type3 mir-292a-5p TCGAGAGTAGAGCTCTAGTCACAAGAGCCCCCAGTTTGAGCGC GGCCGCGCTCAAACTGGGGGCTCTTGTGACTAGAGCTCTACTC Type3 mir-292a-3p TCGAGAGTAGAGCTCTAGTACGCTCAAAACCTGGCGGCACTTGGC GGCCGCCAAGTGCCGCCAGGTTTTGAGCGTACTAGAGCTCTACTC Type4 mir-292a-5p TCGAGAGTAGAGCTCTAGTCAAAAGAGCCCCCAGTTTGAGCGC GGCCGCGCTCAAACTGGGGGCTCTTTTGACTAGAGCTCTACTC Type4 mir-292a-3p TCGAGAGTAGAGCTCTAGTACGCTCAAAACCTGGCGGCACTTTGC GGCCGCAAAGTGCCGCCAGGTTTTGAGCGTACTAGAGCTCTACTC Type1 mir-384-5p TCGAGAGTAGAGCTCTAGTACCTTGCCTAGGAATTGTTTACAGC GGCCGCTGTAAACAATTCCTAGGCAAGGTACTAGAGCTCTACTC Type1 mir-384-3p TCGAGAGTAGAGCTCTAGTATTGTGAACAATTTCTAGGAAGGC GGCCGCCTTCCTAGAAATTGTTCACAATACTAGAGCTCTACTC Type3 mir-384-5p TCGAGAGTAGAGCTCTAGTACCTTGCCTAGGAATTGTTTACGGC GGCCGCCGTAAACAATTCCTAGGCAAGGTACTAGAGCTCTACTC Type3 mir-384-3p TCGAGAGTAGAGCTCTAGTATCGTGAACAATTTCTAGGAAGGC GGCCGCCTTCCTAGAAATTGTTCACGATACTAGAGCTCTACTC Type4 mir-384-5p TCGAGAGTAGAGCTCTAGTACATTGCCTAGGAATTGTTTACGGC GGCCGCCGTAAACAATTCCTAGGCAATGTACTAGAGCTCTACTC Type4 mir-384-3p TCGAGAGTAGAGCTCTAGTATCGTGAACAATTTCTAGGAATGC GGCCGCATTCCTAGAAATTGTTCACGATACTAGAGCTCTACTC Type1 mir-181c-5p TCGAGAGTAGAGCTCTAGTAACCTCACCGACAGGTTGAATGTTGC GGCCGCAACATTCAACCTGTCGGTGAGGTTACTAGAGCTCTACTC Type1 mir-181c-3p TCGAGAGTAGAGCTCTAGTGGTCCACTCAACGGTCGATGGGGC GGCCGCCCCATCGACCGTTGAGTGGACCACTAGAGCTCTACTC Type3 mir-181c-5p TCGAGAGTAGAGCTCTAGTAACCTCACCGACAGGTTGAATGTCGC GGCCGCAACATTCAACCTGTCGGTGAGGTTACTAGAGCTCTACTC Type3 mir-181c-3p identical to Type1 mir-181c-3p identical to Type1 mir-181c-3p Type4 mir-181c-5p TCGAGAGTAGAGCTCTAGTAAACTCACCGACAGGTTGAATGTCGC GGCCGCGACATTCAACCTGTCGGTGAGTTTACTAGAGCTCTACTC Type4 mir-181c-3p identical to Type2 mir-181c-3p identical to Type2 mir-181c-3p Type1 mir-383-5p (WT) TCGAGAGTAGAGCTCTAGTAGCCACAGTCACCTTCTGATCTGC GGCCGCAGATCAGAAGGTGACTGTGGCTACTAGAGCTCTACTC Type1 mir-383-3p (WT) TCGAGAGTAGAGCTCTAGTTCTGACCAGGCAGTGCTGTGGGC GGCCGCCCACAGCACTGCCTGGTCAGAACTAGAGCTCTACTC Type2 mir-383-5p TCGAGAGTAGAGCTCTAGTAGACACAGTCACCTTCTGATCTGC GGCCGCAGATCAGAAGGTGACTGTGTCTACTAGAGCTCTACTC Type2 mir-383-3p TCGAGAGTAGAGCTCTAGTTCTGACCAGGCAGTGCTGTGTGC GGCCGCACACAGCACTGCCTGGTCAGAACTAGAGCTCTACTC Type3 mir-383-5p TCGAGAGTAGAGCTCTAGTAGCCACAGTCACCTTCTGATCCGC GGCCGCGGATCAGAAGGTGACTGTGGCTACTAGAGCTCTACTC Type3 mir-383-3p identical to Type1 mir-383-3p identical to Type1 mir-383-3p Type4 mir-383-5p TCGAGAGTAGAGCTCTAGTAGACACAGTCACCTTCTGATCCGC GGCCGCGGATCAGAAGGTGACTGTGTCTACTAGAGCTCTACTC Type4 mir-383-3p identical to Type2 mir-383-3p identical to Type2 mir-383-3p Type1 mir-216a-5p (WT) TCGAGAGTAGAGCTCTAGTCTCACAGTTGCCAGCTGAGATTAGC GGCCGCTAATCTCAGCTGGCAACTGTGAGACTAGAGCTCTACTC
Type1 mir-216a-3p (WT) TCGAGAGTAGAGCTCTAGTCATAATCCCAGAGACCACTGTGGC GGCCGCCACAGTGGTCTCTGGGATTATGACTAGAGCTCTACTC Type2 mir-216a-5p TCGAGAGTAGAGCTCTAGTCTTACAGTTGCCAGCTGAGATTAGC GGCCGCTAATCTCAGCTGGCAACTGTAAGACTAGAGCTCTACTC Type2 mir-216a-3p TCGAGAGTAGAGCTCTAGTCATAATCCCAGAGACCACTGTAGC GGCCGCTACAGTGGTCTCTGGGATTATGACTAGAGCTCTACTC Type3 mir-216a-5p TCGAGAGTAGAGCTCTAGTCTCACAGTTGCCAGCTGAGATTGGC GGCCGCCAATCTCAGCTGGCAACTGTGAGACTAGAGCTCTACTC Type3 mir-216a-3p TCGAGAGTAGAGCTCTAGTCACAATCCCAGAGACCACTGTGGC GGCCGCCACAGTGGTCTCTGGGATTGTGACTAGAGCTCTACTC Type4 mir-216a-5p TCGAGAGTAGAGCTCTAGTCTTACAGTTGCCAGCTGAGATTGGC GGCCGCCAATCTCAGCTGGCAACTGTAAGACTAGAGCTCTACTC Type4 mir-216a-3p TCGAGAGTAGAGCTCTAGTCACAATCCCAGAGACCACTGTAGC GGCCGCTACAGTGGTCTCTGGGATTGTGACTAGAGCTCTACTC Type1 mir-191-5p TCGAGAGTAGAGCTCTAGTCAGCTGCTTTTGGGATTCCGTTTGC GGCCGCAAACGGAATCCCAAAAGCAGCTGACTAGAGCTCTACTC Type1 mir-191-3p identical to Type3 mir-191-3p identical to Type3 mir-191-3p Type2 mir-191-5p TCGAGAGTAGAGCTCTAGTCATCTGCTTTTGGGATTCCGTTTGC GGCCGCAAACGGAATCCCAAAAGCAGATGACTAGAGCTCTACTC Type2 mir-191-3p TCGAGAGTAGAGCTCTAGTGGGAACGAAATCCAAGTGCAGAGC GGCCGCTCTGCACTTGGATTTCGTTCCCACTAGAGCTCTACTC Type3 mir-191-5p (WT) TCGAGAGTAGAGCTCTAGTCAGCTGCTTTTGGGATTCCGTTGGC GGCCGCCAACGGAATCCCAAAAGCAGCTGACTAGAGCTCTACTC Type3 mir-191-3p (WT) TCGAGAGTAGAGCTCTAGTGGGAACGAAATCCAAGTGCAGCGC GGCCGCGCTGCACTTGGATTTCGTTCCCACTAGAGCTCTACTC Type4 mir-191-5p TCGAGAGTAGAGCTCTAGTCATCTGCTTTTGGGATTCCGTTGGC GGCCGCCAACGGAATCCCAAAAGCAGATGACTAGAGCTCTACTC Type4 mir-191-3p identical to Type2 mir-191-3p identical to Type2 mir-191-3p mir-181c-cm-5p TCGAGAGTAGAGCTCTAGTAAACCACCGACCGTTGAATGTTGC GGCCGCAACATTCAACGGTCGGTGGTTTACTAGAGCTCTACTC mir-181c-9-10nt-mm-5p TCGAGAGTAGAGCTCTAGTAAACCACCGACCCATGAATGTTGC GGCCGCAACATTCATGGGTCGGTGGTTTACTAGAGCTCTACTC mir-181c-11-12nt-mm-5p TCGAGAGTAGAGCTCTAGTAAACCACCGAGGGTTGAATGTTGC GGCCGCAACATTCAACCCTCGGTGGTTTACTAGAGCTCTACTC mir-384-cm-5p TCGAGAGTAGAGCTCTAGTACATTCCTAGAAATTGTTCACAGC GGCCGCTGTGAACAATTTCTAGGAATGTACTAGAGCTCTACTC mir-384-9-10nt-mm-5p TCGAGAGTAGAGCTCTAGTACATTCCTAGAAGGTGTTCACAGC GGCCGCTGTGAACACCTTCTAGGAATGTACTAGAGCTCTACTC mir-384-11-12nt-mm-5p TCGAGAGTAGAGCTCTAGTACATTCCTAGTTATTGTTCACAGC GGCCGCTGTGAACAATAACTAGGAATGTACTAGAGCTCTACTC mir-146a-5p TCGAGAGTAGAGCTCTAGTAACCCATGGAATTCAGTTCTCAGC GGCCGCTGAGAACTGAATTCCATGGGTTACTAGAGCTCTACTC mir-146a-9-10nt-mm-5p TCGAGAGTAGAGCTCTAGTAACCCATGGAATAGAGTTCTCAGC GGCCGCTGAGAACTCTATTCCATGGGTTACTAGAGCTCTACTC mir-146a-11-12nt-mm-5p TCGAGAGTAGAGCTCTAGTAACCCATGGAGGTCAGTTCTCAGC GGCCGCTGAGAACTGACCTCCATGGGTTACTAGAGCTCTACTC mir-216a-5p (5 -nt = U) identical to Type1 mir-216a-5p identical to Type1 mir-216a-5p mir-216a-5p (5 -nt = A) TCGAGAGTAGAGCTCTAGTCTCACAGTTGCCAGCTGAGATTTGC GGCCGCAAATCTCAGCTGGCAACTGTGAGACTAGAGCTCTACTC mir-216a-5p (5 -nt = G) TCGAGAGTAGAGCTCTAGTCTCACAGTTGCCAGCTGAGATTCGC GGCCGCGAATCTCAGCTGGCAACTGTGAGACTAGAGCTCTACTC mir-216a-5p (5 -nt = C) identical to Type3 mir-216a-5p identical to Type3 mir-216a-5p mir-142-3p-mut1 TCGAGAGTAGAGCTCTAGTTCCATAAAGTAGGAAACACCACAGC GGCCGCTGTGGTGTTTCCTACTTTATGGAACTAGAGCTCTACTC mir-142-3p-mut2 TCGAGAGTAGAGCTCTAGTTCCATAAAGTAGGAAACGCTACAGC GGCCGCTGTAGCGTTTCCTACTTTATGGAACTAGAGCTCTACTC mir-142-3p-mut3 TCGAGAGTAGAGCTCTAGTTCCATAAAGTAGGAAAGACTACAGC GGCCGCTGTAGTCTTTCCTACTTTATGGAACTAGAGCTCTACTC mir-146a-3p-g TCGAGAGTAGAGCTCTAGTCTGAAGAACTGAATTTCACAGGGC GGCCGCCCTGTGAAATTCAGTTCTTCAGACTAGAGCTCTACTC mir-146a-3p-c TCGAGAGTAGAGCTCTAGTCTGAAGAACTGAATTTCAGAGGGC GGCCGCCCTCTGAAATTCAGTTCTTCAGACTAGAGCTCTACTC Primers for the construction of vectors for recombinant proteins. Forward (5 to 3 ) Reverse (5 to 3 ) GST-PAZ GgaattcTGGAAAATGATGCTGAATATT ATAAGAATGCGGCCgcTTATGTGTTGAAACTTGCAC GST-MID GgaattcGTCCAGGGCGTCTGGGACAT ATAAGAATGCGGCCgcTTAGTGAGTGACGTCTGCTCC RT-PCR primers. mirna 5 to 3
Type1 mir-154-5p Type1 mir-154-3p Type2 mir-154-5p (WT) Type2 mir-154-3p (WT) Type3 mir-154-5p Type3 mir-154-3p Type4 mir-154-5p Type4 mir-154-3p Type1 mir-292a-5p Type1 mir-292a-3p Type2 mir-292a-5p (WT) Type2 mir-292a-3p (WT) Type3 mir-292a-5p Type3 mir-292a-3p Type4 mir-292a-5p Type4 mir-292a-3p Type1 mir-383-5p (WT) Type1 mir-383-3p (WT) Type2 mir-383-5p Type2 mir-383-3p Type3 mir-383-5p Type3 mir-383-3p Type4 mir-383-5p Type4 mir-383-3p Type1 mir-191-5p Type1 mir-191-3p Type2 mir-191-5p Type2 mir-191-3p Type3 mir-191-5p (WT) Type3 mir-191-3p (WT) Type4 mir-191-5p Type4 mir-191-3p Control strand-a (Figure 5) Control strand-b (Figure 5) A-i strand-a (Figure 5) A-ii strand-a (Figure 5) A-iii strand-b (Figure 5) A-iv strand-b (Figure 5) B-i strand-a (Figure 5) B-i strand-b (Figure 5) B-ii strand-a (Figure 5) TAGGTTATCCGTGTTGCCTGCG CATCATACACGGTTGACCTATT TAGGTTATCCGTGTTGCCTTCG AATCATACACGGTTGACCTATT CAGGTTATCCGTGTTGCCTGCG CATCATACACGGTTGACCTGTT CAGGTTATCCGTGTTGCCTTCG AATCATACACGGTTGACCTGTT ACTCAAACTGGGGGCTCTTGTG CAAGTGCCGCCAGGTTTTGAGTGT ACTCAAACTGGGGGCTCTTTTG AAAGTGCCGCCAGGTTTTGAGTGT GCTCAAACTGGGGGCTCTTGTG CAAGTGCCGCCAGGTTTTGAGCGT GCTCAAACTGGGGGCTCTTTTG AAAGTGCCGCCAGGTTTTGAGCGT AGATCAGAAGGTGACTGTGGCT CCACAGCACTGCCTGGTCAGA AGATCAGAAGGTGACTGTGTCT ACACAGCACTGCCTGGTCAGA GGATCAGAAGGTGACTGTGGCT CCACAGCACTGCCTGGTCAGA GGATCAGAAGGTGACTGTGTCT ACACAGCACTGCCTGGTCAGA AAACGGAATCCCAAAAGCAGCTG GCTGCACTTGGATTTCGTTCCC AAACGGAATCCCAAAAGCAGATG TCTGCACTTGGATTTCGTTCCC CAACGGAATCCCAAAAGCAGCTG GCTGCACTTGGATTTCGTTCCC CAACGGAATCCCAAAAGCAGATG TCTGCACTTGGATTTCGTTCCC TCGAAGTATTCCGCGTACGTT CGTACGCGGAATACTTCGATT TCGAAGTATTCCGCGTAGCTT TCGAAGTATTCCGCGTACCTT CGTACGCGGAATACTTCGCTT CGTACGCGGAATACTTCCCTT TCGAAGTATTCCGCTTTTGTT CAAAAGCGGAATACTTCGATT TCGAAGTATTCCGCGTATGTT
B-ii strand-b (Figure 5) B-iii strand-a (Figure 5) B-iii strand-b (Figure 5) B-iv strand-a (Figure 5) B-iv strand-b (Figure 5) Type1 mir-216a-5p (WT) Type1 mir-216a-3p (WT) mir-216a-5p (5 -nt = U) mir-216a-5p (5 -nt = A) mir-216a-5p (5 -nt = G) mir-216a-5p (5 -nt = C) mir-142-5p mir-142-3p mir-142-3p-mut1 mir-142-3p-mut2 mir-142-3p-mut3 mir-146a-5p mir-146a-3p-g mir-146a-3p-c CATACGCGGAATACTTCGATT TTGAAGTATTCCGCGTACGTT CGTACGCGGAATACTTCAATT TTTAAGTATTCCGCGTACGTT CGTACGCGGAATACTTAAATT TAATCTCAGCTGGCAACTGTGAG CACAGTGGTCTCTGGGATTATG TAATCTCAGCTGGCAACTGTGAG AAATCTCAGCTGGCAACTGTGAG GAATCTCAGCTGGCAACTGTGAG CAATCTCAGCTGGCAACTGTGAG CATAAAGTAGAAAGCACTACT TGTAGTGTTTCCTACTTTATGGA TGTGGTGTTTCCTACTTTATGGA TGTAGCGTTTCCTACTTTATGGA TGTAGTCTTTCCTACTTTATGGA TGAGAACTGAATTCCATGGGTT CCTCTGAAATTCAGTTCTTCAG CCTGTGAAATTCAGTTCTTCAG