WilsonKao 10 發表於 January 12, 2007 檢舉 Share 發表於 January 12, 2007 從我自己的BBS個版上轉來的，沒關係吧: P其實是因為我那邊太冷清了，希望放在比較熱門一點的地方，在這個高手比比皆是的地方或許可以找到一些解答。http://www.nature.com/focus/rnai/animations/index.html在用生圖找論文的時候逛到的。細節和精緻程度自然不能跟之前的白血球動畫比，個人認為製作這個動畫的公司不是沒能 力做到，但是還是沒有去做，實在是可惜。有幾個地方我有疑問:1.在差不多0:52左右的時候，是不是要補上例如說exportin之類的東西?2.之後的transcription似乎沒有出現5'-UTR，還有他們之間的互動，tRNA更沒有出現3.在2:12的時候，是先跟protein結合在一起，之後才unwind，形成RISC，我之前看到的說法好像是先unwind...此外，protein要怎麼辨識那一股才是mRNA-complementary?這點我目前還沒找到解釋。4.2:35時，只有mRNA被cleavage，我想再找找一些更詳細的說法。5.2:55時，那個RNase是exonuclease?連nucelotide都變成ribose和base?6.在3:23左右講到RDRP的時候，有說是根據"aberrant"當作template來做一個antisese strand的，那何謂aberrant? 7.4:04講到的，真菌和哺乳類的RDRP需要siRNA作為primer，template是沒被處理過的mRNA，植物的(3:23提到的)不需要他作為primer。那是說，abberant指的是現成的siRNA?如果是這樣代表說RDRP會辨識那種長度的RNA囉?那如果它指的abberant是degraded mRNA，那怎麼確定它的對象是degraded的片段還是一般的mRNA?那這樣功能分歧的RDRP在演化上的意義是什麼?8.動畫中說RISC是在中間做mRNA cleavage，那究竟就單純是中間還是有辨識位?9.Dicer切割出來的產物兩端一定要多兩個bp的原因是什麼?10.A. thaliana和其進親沒有Drosha(這篇動畫裡沒提到)，但是據說有幾種Dicer有NLS，那當初生物演化出如此分歧的原因/因素是?那有何辦法確定他們在何處分歧? 鏈接文章 分享到其他網站
訪客 發表於 January 13, 2007 檢舉 Share 發表於 January 13, 2007 7.4:04講到的，真菌和哺乳類的RDRP需要siRNA作為primer，template是沒被處理過的mRNA，植物的(3:23提到的)不需要他作為primer。那是說，abberant指的是現成的siRNA?如果是這樣代表說RDRP會辨識那種長度的RNA囉?那如果它指的abberant是degraded mRNA，那怎麼確定它的對象是degraded的片段還是一般的mRNA?那這樣功能分歧的RDRP在演化上的意義是什麼?我對ri只有一點粗淺的認知不過我記得目前是相信動物和植物獨立演化出ri，差異可能是這樣來的 鏈接文章 分享到其他網站
WilsonKao 10 發表於 January 24, 2007 作者 檢舉 Share 發表於 January 24, 2007 針對第1，第8和第9個問題:引用一段review(MicroRNAs: SMALL RNAs WITH A BIG ROLE IN GENE REGULATION, Lin He and Gregory J.Hannon, Nature Vol5, July 2004):(關於為何引用此篇review的理由將在引用文中以粗體標明)In addition, efficient Dicer cleavage also requires the presence of the overhang and a minimal stem length (D. Siolas and G.J.H., unpublished observations), indicating a model in which the Dicer PAZ domain might recognize the end of the Drosha cleavage product, and therefore position the site of the second RNase-III cleavage on the stem of the miRNA precursors.Dicer cleavage generates mature miRNAs that range from 21 to 25 nucleotides, and such differences in size possibly result from the presence of bulges and mismatches on the pre-miRNA stem.(根據此篇review的建議，以上敘述可參考60. Carmell, M. A. & Hannon, G. J. RNase III enzymes and the initiation of gene silencing. Nature Struct. Mol. Biol. 11, 214–218 (2004).)...(中略)Although plant miRNAs seem to be produced from long, primary transcripts, the maturation of miRNAs in plants must, by necessity, occur differently from miRNA maturation in animals, as no plant Drosha homologue has yet been found. However, the Dicer family inArabidopsis thaliana has a level of complexity that has not been observed in other organisms studied so far. There are four Dicer homologues in A. thaliana — DCL1, DCL2, DCL3 and DCL4 — two of which (DCL1 and DCL4) contain nuclear localization signals. Therefore, it seems possible that Drosha function might be carried out by one or more specialized Dicer enzymes in plants. The Dicer homologue DCL1 contains two nuclear localization signals, and predominates the nuclei when expressed as a GFP-fusion proteinin transient-transfection studies. In plants that are deficient for DCL1, the production of mature miRNAs is reduced for all miRNAs examined, yet no accumulation is detected for the corresponding pre-miRNAs.These findings imply that DCL1 might catalyse both the Drosha- and Dicer-like cleavages for the maturation of some, if not most, miRNAs inside the nucleus.Consistent with this model, mature miRNAs seem to be produced within the nucleus in plants, because nuclear expression of P19, a viral protein that represses the ccumulation of siRNAs and miRNAs, results in a significant reduction in mature miRNAs,whereas the cytoplasmic expression of P19 has no such effects. It is not clear whether other Dicer homologues in plants are also involved in miRNA maturation.However, it has been speculated that the presence of multiple plant Dicer enzymes might explain the complexity of siRNA species in plants.Unlike siRNAs in animals, which are usually 21–22 nucleotides long, plants generate two classes of siRNA: 21–22- and ~25-bp-long siRNAs. Given the different classes of small dsRNAs found in plants, it is possible that each Dicer homologue regulates separate cleavage events to generate specific species of dsRNA duplexes.The functional specificity of different Dicer enzymes in organisms with multiple Dicer homologues has recently been indicated by a series of genetic and biochemical studies in Drosophila melanogaster . Two Dicer homologues have been identified in flies: Dicer1 and Dicer2 .Deficiency in Dicer1 disrupts the processing of pre-miRNAs, whereas loss of Dicer2 affects the production of siRNAs, but not miRNA maturation. These findings are consistent with the fact that the PAZ domain is only present in Dicer1, but absent in Dicer2, given a model in which the PAZ domain recognizes the staggered ends of pre-miRNAs.(根據此篇review的建議，以上敘述可參考62. Papp, I. et al. Evidence for nuclear processing of plant microRNA and short interfering RNA precursors. Plant Physiol.132, 1382–1390 (2003).63. Park, W., Li, J., Song, R., Messing, J. & Chen, X. CARPEL FACTORY, a Dicer homolog, and HEN1, a novel protein, act in microRNA metabolism in Arabidopsis thaliana. Curr. Biol.12, 1484–1495 (2002).64. Timmons, L. The long and short of siRNAs. Mol. Cell 10, 435–437 (2002).66. Pham, J. W., Pellino, J. L., Lee, Y. S., Carthew, R. W. & Sontheimer, E. J. A Dicer-2-dependent 80S complex cleaves targeted mRNAs during RNAi in Drosophila. Cell 117, 83–94 (2004).67. Lee, Y. S. et al. Distinct roles for Drosophila Dicer-1 and Dicer-2 in the siRNA/miRNA silencing pathways. Cell 117, 69–81 (2004))...(中略)The effector complex for miRNAs shares core components with that of siRNAs, so much so that both are collectively referred to as the RNA-induced silencing complex (RISC).(這就是我為什麼認為引用這篇其實是在講miRNA的review是合理的原因。)The target specificity, and probably also the functional efficiency, of a miRNA requires that the mature miRNA strand from the miRNA:miRNA* duplex be selectively incorporated into the RISC for target recognition. The miRNA* strand, on the other hand, is probably degraded rapidly on its exclusion from the RISC, as the recovery rate of miRNA*s from endogenous tissues is ~100-fold lower than that of miRNAs.As Dicer processes the pre-miRNA into the miRNA:miRNA* duplex, the stability of the 5′ ends of the two arms of the miRNA:miRNA* duplex is usually different. Although mature miRNAs can reside on either strand of the hairpin stem, it is almost always derived from the strand with the less stable 5′ end compared with the miRNA* strand. These findings indicate that the relative instability at the 5′ end of the mature miRNA might facilitate its preferential incorporation into the RISC. The selective assembly of the mature miRNA into the RISC probably reflects the relative ease of unwinding from one end of the miRNA: miRNA* duplex.Therefore, the thermodynamic properties of the miRNA precursor determine the asymmetrical RISC assembly, and therefore, the target specificity for posttranscriptional inhibition. However, in rare cases in which miRNA and miRNA* have similar 5′-end stability, each arm of the miRNA precursor is predicted to be assembled into the RISC at similar frequencies. This prediction has been confirmed by similar recovery rates for such miRNAs and miRNA*s from endogenous tissues. This thermodynamic model also applies to the asymmetrical assembly of the siRNA duplex, in which the strand of siRNA with the less stable 5′ end is preferentially assembled into the RISC complex to target mRNA cleavage. Altogether, there seems to be a common thermodynamic mechanism that regulates the asymmetric assembly of siRNA or miRNA from the dsRNA duplexes, which safeguards specificity towards corresponding targets.(根據此篇review的建議，以上敘述可參考71. Schwarz, D. S. et al. Asymmetry in the assembly of the RNAi enzyme complex. Cell 115, 199–208 (2003).72. Khvorova, A., Reynolds, A. & Jayasena, S. D. Functional siRNAs and miRNAs exhibit strand bias. Cell 115, 209–216 (2003).This paper, together with reference 71, characterized the regulatory mechanism of the asymmetric assembly of siRNA/miRNA into the RISC complex.)所以在A. thaliana的情況下，處理內生性的miRNA的情況下的確是需要類似exportin的東西來運出來，這是第一個問題。根據引用文的開頭一段，說明說Dicer有辨識相關切割位置的特性，且Dicer之PAZ domain會與pre-miRNA產生弱作用力。但後面的段落告訴我們這個特性使得不同的Dicer對於不同來源的dsRNA會有不同的敏感度，如D. melanogaster 的Dicer1對於pre-miRNA可能有比較多的交互作用，Dicer2則跟siRNA的生成有比較多的關係。這算是回答第八個問題的一部分。根據引用文最後幾段的說明，多出幾個bp(overhangs)的原因跟辨識有關，但是詳細機制有待釐清? 這是找到針對第九個問題最有關聯的說明，詳細情況仍需要找資料佐證。最後，我必須說，我對於強者這兩個字是無緣的(羞)，只是找資料來回答自己的無聊人士XD其他的答案仍需要時間和運氣的等待，或是有其他真強者願意提供資料和想法來解答這些問題: P然後，我相信由此可知用英文的確是方便和不得已，然後同樣的內容誰發都一樣，所以應該不會引起什麼爭議了吧(笑)希望大家是專注在內容不是在形式上挑剔囉: P 鏈接文章 分享到其他網站
--◎笨笨糖◎-- 10 發表於 January 24, 2007 檢舉 Share 發表於 January 24, 2007 呃，你沒去畢旅耶lll謝謝喔XD(英文...看來我要慢慢看了) 鏈接文章 分享到其他網站
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