Identification of Novel Genes Coding for Small Expressed RNAs Kang Haiqi From: Science :

1.siRNA and stRNA:two distinct pathways of posttranscriptional regulation DNA mRNAprotein dsRNA precursorsiRNA Developmental timing Degradation stRNA(-) single-strand Dicer double-strand (sequence- specific)

2. Isolate miRNA dsRNA 5 ˊ,3 ˊ adapter PCR concatamerization cloningsequencing

miRNASequence (5' to 3')Freq. (%)HeLa cellsMouse kidneyAdult fishFrog ovary S2S2 let-7a * * UGAGGUAGUAGGUUGUAUAGUU let-7b * * UGAGGUAGUAGGUUGUGUGGUU13 let-7c * * UGAGGUAGUAGGUUGUAUGGUU3 let-7d * * AGAGGUAGUAGGUUGCAUAGU let-7e * * UGAGGUAGGAGGUUGUAUAGU let-7f * * UGAGGUAGUAGAUUGUAUAGUU1 miR-15UAGCAGCACAUAAUGGUUUGUG /-- miR-16UAGCAGCACGUAAAUAUUGGCG /- - miR-17ACUGCAGUGAAGGCACUUGU miR-18UAAGGUGCAUCUAGUGCAGAUA miR-19a * * UGUGCAAAUCUAUGCAAAACUGA1+++-+/--- miR-19b * * UGUGCAAAUCCAUGCAAAACUGA3 miR-20UAAAGUGCUUAUAGUGCAGGUA miR-21UAGCUUAUCAGACUGAUGUUGA miR-22AAGCUGCCAGUUGAAGAACUGU /-- miR-23AUCACAUUGCCAGGGAUUUCC miR-24UGGCUCAGUUCAGCAGGAACAG miR-25CAUUGCACUUGUCUCGGUCUGA miR-26a * * UUCAAGUAAUCCAGGAUAGGCU miR-26b * * UUCAAGUAAUUCAGGAUAGGUU1- miR-27UUCACAGUGGCUAAGUUCCGCU miR-28AAGGAGCUCACAGUCUAUUGAG miR-29CUAGCACCAUCUGAAAUCGGUU2+++++/--- miR-30CUUUCAGUCGGAUGUUUGCAGC miR-31GGCAAGAUGCUGGCAUAGCUG miR-32UAUUGCACAUUACUAAGUUGC miR-33GUGCAUUGUAGUUGCAUUG miR-1UGGAAUGUAAAGAAGUAUGGAG miR-7UGGAAGACUAGUGAUUUUGUUGU0+-+/-- miR-9UCUUUGGUUAUCUAGCUGUAUGA miR-10ACCCUGUAGAUCCGAAUUUGU * Similar miRNA sequences are difficult to distinguish by Northern blotting because of potential cross-hybridization of probes. Table 2. Human miRNAs

3. miRNAs in different developmental stages of D.melanogaster 3.1 miR-3,miR miR-1,miR-2,miR-8 ～ miR-13 miR-1,miR-3 ～ miR-6,miR-8 ～ miR miR-1, miR-8, miR miR-9, miR-11

Fig 1 Expression of miRNAs

miR-1UGGAAUGUAAAGAAGUAUGGAG miR-2a * * UAUCACAGCCAGCUUUGAUGAGC3 miR-2b * * UAUCACAGCCAGCUUUGAGGAGC miR-3UCACUGGGCAAAGUGUGUCUCA miR-4AUAAAGCUAGACAACCAUUGA miR-5AAAGGAACGAUCGUUGUGAUAUG1+++ +/- --- miR-6UAUCACAGUGGCUGUUCUUUUU /- --- miR-7UGGAAGACUAGUGAUUUUGUUGU /- miR-8UAAUACUGUCAGGUAAAGAUGUC3+/ miR-9UCUUUGGUUAUCUAGCUGUAUGA /-- miR-10ACCCUGUAGAUCCGAAUUUGU /-+- miR-11CAUCACAGUCUGAGUUCUUGC miR-12UGAGUAUUACAUCAGGUACUGGU /- miR-13a * * UAUCACAGCCAUUUUGACGAGU miR-13b * * UAUCACAGCCAUUUUGAUGAGU0 miR-14UCAGUCUUUUUCUCUCUCCUA let-7UGAGGUAGUAGGUUGUAUAGUU * Similar miRNA sequences are difficult to distinguished by Northern blotting because of potential cross-hybridization of probes. Table 1 D.melanogaster miRNAs

4.mir-3 ～ mir-6 Fig.2 Genomic organization of miRNA gene clusters

miR-1UGGAAUGUAAAGAAGUAUGGAG miR-2a * * UAUCACAGCCAGCUUUGAUGAGC3 miR-2b * * UAUCACAGCCAGCUUUGAGGAGC miR-3UCACUGGGCAAAGUGUGUCUCA miR-4AUAAAGCUAGACAACCAUUGA miR-5AAAGGAACGAUCGUUGUGAUAUG1+++ +/- --- miR-6UAUCACAGUGGCUGUUCUUUUU /- --- miR-7UGGAAGACUAGUGAUUUUGUUGU /- miR-8UAAUACUGUCAGGUAAAGAUGUC3+/ miR-9UCUUUGGUUAUCUAGCUGUAUGA /-- miR-10ACCCUGUAGAUCCGAAUUUGU /-+- miR-11CAUCACAGUCUGAGUUCUUGC miR-12UGAGUAUUACAUCAGGUACUGGU /- miR-13a * * UAUCACAGCCAUUUUGACGAGU miR-13b * * UAUCACAGCCAUUUUGAUGAGU0 miR-14UCAGUCUUUUUCUCUCUCCUA let-7UGAGGUAGUAGGUUGUAUAGUU * Similar miRNA sequences are difficult to distinguished by Northern blotting because of potential cross-hybridization of probes. Table 1 D.melanogaster miRNAs

5. miRNAs in different tissues 5.1 miR-15, miR-16(Fig 2B) 5.2 mir-17 ～ mir-20 (Fig 2B) 5.3 Fig1B & Table 2

Fig.2 Genomic organization of miRNA gene clusters

Fig 1 Expression of miRNAs

miRNASequence (5' to 3')Freq. (%)HeLa cellsMouse kidneyAdult fishFrog ovary S2S2 let-7a * * UGAGGUAGUAGGUUGUAUAGUU let-7b * * UGAGGUAGUAGGUUGUGUGGUU13 let-7c * * UGAGGUAGUAGGUUGUAUGGUU3 let-7d * * AGAGGUAGUAGGUUGCAUAGU let-7e * * UGAGGUAGGAGGUUGUAUAGU let-7f * * UGAGGUAGUAGAUUGUAUAGUU1 miR-15UAGCAGCACAUAAUGGUUUGUG /-- miR-16UAGCAGCACGUAAAUAUUGGCG /- - miR-17ACUGCAGUGAAGGCACUUGU miR-18UAAGGUGCAUCUAGUGCAGAUA miR-19a * * UGUGCAAAUCUAUGCAAAACUGA1+++-+/--- miR-19b * * UGUGCAAAUCCAUGCAAAACUGA3 miR-20UAAAGUGCUUAUAGUGCAGGUA miR-21UAGCUUAUCAGACUGAUGUUGA miR-22AAGCUGCCAGUUGAAGAACUGU /-- miR-23AUCACAUUGCCAGGGAUUUCC miR-24UGGCUCAGUUCAGCAGGAACAG miR-25CAUUGCACUUGUCUCGGUCUGA miR-26a * * UUCAAGUAAUCCAGGAUAGGCU miR-26b * * UUCAAGUAAUUCAGGAUAGGUU1- miR-27UUCACAGUGGCUAAGUUCCGCU miR-28AAGGAGCUCACAGUCUAUUGAG miR-29CUAGCACCAUCUGAAAUCGGUU2+++++/--- miR-30CUUUCAGUCGGAUGUUUGCAGC miR-31GGCAAGAUGCUGGCAUAGCUG miR-32UAUUGCACAUUACUAAGUUGC miR-33GUGCAUUGUAGUUGCAUUG miR-1UGGAAUGUAAAGAAGUAUGGAG miR-7UGGAAGACUAGUGAUUUUGUUGU0+-+/-- miR-9UCUUUGGUUAUCUAGCUGUAUGA miR-10ACCCUGUAGAUCCGAAUUUGU * Similar miRNA sequences are difficult to distinguish by Northern blotting because of potential cross-hybridization of probes. Table 2. Human miRNAs

6. miRNAs ˊ sequence conservation in D.melanogaster 6.1 miR mir mir-1 & let miR-7(Table 2) 6.5 miR-9, miR-10

miRNASequence (5' to 3')Freq. (%)HeLa cellsMouse kidneyAdult fishFrog ovary S2S2 let-7a * * UGAGGUAGUAGGUUGUAUAGUU let-7b * * UGAGGUAGUAGGUUGUGUGGUU13 let-7c * * UGAGGUAGUAGGUUGUAUGGUU3 let-7d * * AGAGGUAGUAGGUUGCAUAGU let-7e * * UGAGGUAGGAGGUUGUAUAGU let-7f * * UGAGGUAGUAGAUUGUAUAGUU1 miR-15UAGCAGCACAUAAUGGUUUGUG /-- miR-16UAGCAGCACGUAAAUAUUGGCG /- - miR-17ACUGCAGUGAAGGCACUUGU miR-18UAAGGUGCAUCUAGUGCAGAUA miR-19a * * UGUGCAAAUCUAUGCAAAACUGA1+++-+/--- miR-19b * * UGUGCAAAUCCAUGCAAAACUGA3 miR-20UAAAGUGCUUAUAGUGCAGGUA miR-21UAGCUUAUCAGACUGAUGUUGA miR-22AAGCUGCCAGUUGAAGAACUGU /-- miR-23AUCACAUUGCCAGGGAUUUCC miR-24UGGCUCAGUUCAGCAGGAACAG miR-25CAUUGCACUUGUCUCGGUCUGA miR-26a * * UUCAAGUAAUCCAGGAUAGGCU miR-26b * * UUCAAGUAAUUCAGGAUAGGUU1- miR-27UUCACAGUGGCUAAGUUCCGCU miR-28AAGGAGCUCACAGUCUAUUGAG miR-29CUAGCACCAUCUGAAAUCGGUU2+++++/--- miR-30CUUUCAGUCGGAUGUUUGCAGC miR-31GGCAAGAUGCUGGCAUAGCUG miR-32UAUUGCACAUUACUAAGUUGC miR-33GUGCAUUGUAGUUGCAUUG miR-1UGGAAUGUAAAGAAGUAUGGAG miR-7UGGAAGACUAGUGAUUUUGUUGU0+-+/-- miR-9UCUUUGGUUAUCUAGCUGUAUGA miR-10ACCCUGUAGAUCCGAAUUUGU * Similar miRNA sequences are difficult to distinguish by Northern blotting because of potential cross-hybridization of probes. Table 2. Human miRNAs

7. Evolutionary relation of miRNA 8. stem-loop structure of miRNA

9. Argonaute protein family 9.1 ALG-1, ALG RISC 9.3 RDE-1,Argonaute 1,QDE-2, Rnase III Dicer 9.4 Argonaute family

10. Discussion

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