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Identification of Novel Genes Coding for Small Expressed RNAs Kang Haiqi From: Science 2001 292:853-858.

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Presentation on theme: "Identification of Novel Genes Coding for Small Expressed RNAs Kang Haiqi From: Science 2001 292:853-858."— Presentation transcript:

1 Identification of Novel Genes Coding for Small Expressed RNAs Kang Haiqi From: Science 2001 292:853-858

2 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)

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

4 miRNASequence (5' to 3')Freq. (%)HeLa cellsMouse kidneyAdult fishFrog ovary S2S2 let-7a * * UGAGGUAGUAGGUUGUAUAGUU10+++ -- let-7b * * UGAGGUAGUAGGUUGUGUGGUU13 let-7c * * UGAGGUAGUAGGUUGUAUGGUU3 let-7d * * AGAGGUAGUAGGUUGCAUAGU2+++ -- let-7e * * UGAGGUAGGAGGUUGUAUAGU2+++ -- let-7f * * UGAGGUAGUAGAUUGUAUAGUU1 miR-15UAGCAGCACAUAAUGGUUUGUG3+++++++/-- miR-16UAGCAGCACGUAAAUAUUGGCG10+++++/- - miR-17ACUGCAGUGAAGGCACUUGU1+++---- miR-18UAAGGUGCAUCUAGUGCAGAUA2+++---- miR-19a * * UGUGCAAAUCUAUGCAAAACUGA1+++-+/--- miR-19b * * UGUGCAAAUCCAUGCAAAACUGA3 miR-20UAAAGUGCUUAUAGUGCAGGUA4+++-+-- miR-21UAGCUUAUCAGACUGAUGUUGA10++++++-- miR-22AAGCUGCCAGUUGAAGAACUGU10+++ ++/-- miR-23AUCACAUUGCCAGGGAUUUCC2+++ +- miR-24UGGCUCAGUUCAGCAGGAACAG4+++++++-- miR-25CAUUGCACUUGUCUCGGUCUGA3++++++-- miR-26a * * UUCAAGUAAUCCAGGAUAGGCU2++++++-- miR-26b * * UUCAAGUAAUUCAGGAUAGGUU1- miR-27UUCACAGUGGCUAAGUUCCGCU2+++ ++-- miR-28AAGGAGCUCACAGUCUAUUGAG2+++ --- miR-29CUAGCACCAUCUGAAAUCGGUU2+++++/--- miR-30CUUUCAGUCGGAUGUUUGCAGC2+++ -- miR-31GGCAAGAUGCUGGCAUAGCUG2+++---- miR-32UAUUGCACAUUACUAAGUUGC1----- miR-33GUGCAUUGUAGUUGCAUUG1----- miR-1UGGAAUGUAAAGAAGUAUGGAG0--+-- miR-7UGGAAGACUAGUGAUUUUGUUGU0+-+/-- miR-9UCUUUGGUUAUCUAGCUGUAUGA0----- miR-10ACCCUGUAGAUCCGAAUUUGU0-+--- * Similar miRNA sequences are difficult to distinguish by Northern blotting because of potential cross-hybridization of probes. Table 2. Human miRNAs

5 3. miRNAs in different developmental stages of D.melanogaster 3.1 miR-3,miR-7 3.2 miR-1,miR-2,miR-8 ～ miR-13 miR-1,miR-3 ～ miR-6,miR-8 ～ miR-11 3.3 miR-1, miR-8, miR-12 3.4 miR-9, miR-11

6 Fig 1 Expression of miRNAs

7 miR-1UGGAAUGUAAAGAAGUAUGGAG32+++++ +++++- miR-2a * * UAUCACAGCCAGCUUUGAUGAGC3 miR-2b * * UAUCACAGCCAGCUUUGAGGAGC3++ +++++++++ miR-3UCACUGGGCAAAGUGUGUCUCA9+++ ----- miR-4AUAAAGCUAGACAACCAUUGA6+++ ----- miR-5AAAGGAACGAUCGUUGUGAUAUG1+++ +/- --- miR-6UAUCACAGUGGCUGUUCUUUUU13+++ +/- --- miR-7UGGAAGACUAGUGAUUUUGUUGU4++++++/- miR-8UAAUACUGUCAGGUAAAGAUGUC3+/- +++ + - miR-9UCUUUGGUUAUCUAGCUGUAUGA7++++++++ +/-- miR-10ACCCUGUAGAUCCGAAUUUGU1++++++++/-+- miR-11CAUCACAGUCUGAGUUCUUGC7+++ +- miR-12UGAGUAUUACAUCAGGUACUGGU7++++ +++++/- miR-13a * * UAUCACAGCCAUUUUGACGAGU1+++ + miR-13b * * UAUCACAGCCAUUUUGAUGAGU0 miR-14UCAGUCUUUUUCUCUCUCCUA1------- let-7UGAGGUAGUAGGUUGUAUAGUU0----+++ - * Similar miRNA sequences are difficult to distinguished by Northern blotting because of potential cross-hybridization of probes. Table 1 D.melanogaster miRNAs

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

9 miR-1UGGAAUGUAAAGAAGUAUGGAG32+++++ +++++- miR-2a * * UAUCACAGCCAGCUUUGAUGAGC3 miR-2b * * UAUCACAGCCAGCUUUGAGGAGC3++ +++++++++ miR-3UCACUGGGCAAAGUGUGUCUCA9+++ ----- miR-4AUAAAGCUAGACAACCAUUGA6+++ ----- miR-5AAAGGAACGAUCGUUGUGAUAUG1+++ +/- --- miR-6UAUCACAGUGGCUGUUCUUUUU13+++ +/- --- miR-7UGGAAGACUAGUGAUUUUGUUGU4++++++/- miR-8UAAUACUGUCAGGUAAAGAUGUC3+/- +++ + - miR-9UCUUUGGUUAUCUAGCUGUAUGA7++++++++ +/-- miR-10ACCCUGUAGAUCCGAAUUUGU1++++++++/-+- miR-11CAUCACAGUCUGAGUUCUUGC7+++ +- miR-12UGAGUAUUACAUCAGGUACUGGU7++++ +++++/- miR-13a * * UAUCACAGCCAUUUUGACGAGU1+++ + miR-13b * * UAUCACAGCCAUUUUGAUGAGU0 miR-14UCAGUCUUUUUCUCUCUCCUA1------- let-7UGAGGUAGUAGGUUGUAUAGUU0----+++ - * Similar miRNA sequences are difficult to distinguished by Northern blotting because of potential cross-hybridization of probes. Table 1 D.melanogaster miRNAs

10 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

11 Fig.2 Genomic organization of miRNA gene clusters

12 Fig 1 Expression of miRNAs

13 miRNASequence (5' to 3')Freq. (%)HeLa cellsMouse kidneyAdult fishFrog ovary S2S2 let-7a * * UGAGGUAGUAGGUUGUAUAGUU10+++ -- let-7b * * UGAGGUAGUAGGUUGUGUGGUU13 let-7c * * UGAGGUAGUAGGUUGUAUGGUU3 let-7d * * AGAGGUAGUAGGUUGCAUAGU2+++ -- let-7e * * UGAGGUAGGAGGUUGUAUAGU2+++ -- let-7f * * UGAGGUAGUAGAUUGUAUAGUU1 miR-15UAGCAGCACAUAAUGGUUUGUG3+++++++/-- miR-16UAGCAGCACGUAAAUAUUGGCG10+++++/- - miR-17ACUGCAGUGAAGGCACUUGU1+++---- miR-18UAAGGUGCAUCUAGUGCAGAUA2+++---- miR-19a * * UGUGCAAAUCUAUGCAAAACUGA1+++-+/--- miR-19b * * UGUGCAAAUCCAUGCAAAACUGA3 miR-20UAAAGUGCUUAUAGUGCAGGUA4+++-+-- miR-21UAGCUUAUCAGACUGAUGUUGA10++++++-- miR-22AAGCUGCCAGUUGAAGAACUGU10+++ ++/-- miR-23AUCACAUUGCCAGGGAUUUCC2+++ +- miR-24UGGCUCAGUUCAGCAGGAACAG4+++++++-- miR-25CAUUGCACUUGUCUCGGUCUGA3++++++-- miR-26a * * UUCAAGUAAUCCAGGAUAGGCU2++++++-- miR-26b * * UUCAAGUAAUUCAGGAUAGGUU1- miR-27UUCACAGUGGCUAAGUUCCGCU2+++ ++-- miR-28AAGGAGCUCACAGUCUAUUGAG2+++ --- miR-29CUAGCACCAUCUGAAAUCGGUU2+++++/--- miR-30CUUUCAGUCGGAUGUUUGCAGC2+++ -- miR-31GGCAAGAUGCUGGCAUAGCUG2+++---- miR-32UAUUGCACAUUACUAAGUUGC1----- miR-33GUGCAUUGUAGUUGCAUUG1----- miR-1UGGAAUGUAAAGAAGUAUGGAG0--+-- miR-7UGGAAGACUAGUGAUUUUGUUGU0+-+/-- miR-9UCUUUGGUUAUCUAGCUGUAUGA0----- miR-10ACCCUGUAGAUCCGAAUUUGU0-+--- * Similar miRNA sequences are difficult to distinguish by Northern blotting because of potential cross-hybridization of probes. Table 2. Human miRNAs

14 6. miRNAs ˊ sequence conservation in D.melanogaster 6.1 miR-1 6.2 mir-1 6.3 mir-1 & let-7 6.4 miR-7(Table 2) 6.5 miR-9, miR-10

15 miRNASequence (5' to 3')Freq. (%)HeLa cellsMouse kidneyAdult fishFrog ovary S2S2 let-7a * * UGAGGUAGUAGGUUGUAUAGUU10+++ -- let-7b * * UGAGGUAGUAGGUUGUGUGGUU13 let-7c * * UGAGGUAGUAGGUUGUAUGGUU3 let-7d * * AGAGGUAGUAGGUUGCAUAGU2+++ -- let-7e * * UGAGGUAGGAGGUUGUAUAGU2+++ -- let-7f * * UGAGGUAGUAGAUUGUAUAGUU1 miR-15UAGCAGCACAUAAUGGUUUGUG3+++++++/-- miR-16UAGCAGCACGUAAAUAUUGGCG10+++++/- - miR-17ACUGCAGUGAAGGCACUUGU1+++---- miR-18UAAGGUGCAUCUAGUGCAGAUA2+++---- miR-19a * * UGUGCAAAUCUAUGCAAAACUGA1+++-+/--- miR-19b * * UGUGCAAAUCCAUGCAAAACUGA3 miR-20UAAAGUGCUUAUAGUGCAGGUA4+++-+-- miR-21UAGCUUAUCAGACUGAUGUUGA10++++++-- miR-22AAGCUGCCAGUUGAAGAACUGU10+++ ++/-- miR-23AUCACAUUGCCAGGGAUUUCC2+++ +- miR-24UGGCUCAGUUCAGCAGGAACAG4+++++++-- miR-25CAUUGCACUUGUCUCGGUCUGA3++++++-- miR-26a * * UUCAAGUAAUCCAGGAUAGGCU2++++++-- miR-26b * * UUCAAGUAAUUCAGGAUAGGUU1- miR-27UUCACAGUGGCUAAGUUCCGCU2+++ ++-- miR-28AAGGAGCUCACAGUCUAUUGAG2+++ --- miR-29CUAGCACCAUCUGAAAUCGGUU2+++++/--- miR-30CUUUCAGUCGGAUGUUUGCAGC2+++ -- miR-31GGCAAGAUGCUGGCAUAGCUG2+++---- miR-32UAUUGCACAUUACUAAGUUGC1----- miR-33GUGCAUUGUAGUUGCAUUG1----- miR-1UGGAAUGUAAAGAAGUAUGGAG0--+-- miR-7UGGAAGACUAGUGAUUUUGUUGU0+-+/-- miR-9UCUUUGGUUAUCUAGCUGUAUGA0----- miR-10ACCCUGUAGAUCCGAAUUUGU0-+--- * Similar miRNA sequences are difficult to distinguish by Northern blotting because of potential cross-hybridization of probes. Table 2. Human miRNAs

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

17 9. Argonaute protein family 9.1 ALG-1, ALG-2 9.2 RISC 9.3 RDE-1,Argonaute 1,QDE-2, Rnase III Dicer 9.4 Argonaute family

18 10. Discussion

19 Thank you !

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