circRNA檢測(cè)和描述.pptx

1、,歷史20 年，Scrambled exons 1991322 為什么：不能根據(jù)大小，或從電泳中容易分離（不像miRNA等） 最近，哺乳動(dòng)物細(xì)胞中大量circRNAs，許多是豐富且穩(wěn)定的 產(chǎn)生：exons (exonic circRNA) 或introns (intronic circRNA)，不同產(chǎn)生模式 功能：調(diào)控基因表達(dá)，正在揭示,擴(kuò)增和片段化損壞環(huán)化 circRNA無自由3和5末端，不能被依賴于polyA free RNA末端技術(shù)（RACE，或polyA富集）發(fā)現(xiàn)。 Exon arrangement backsplice 不僅限于circRNAs，早期RNA-Seq將之過濾掉 解決：基

2、于外切酶富集，新的生物信息學(xué)工具，更長reads，更高通量，和rRNA-depleted文庫（而不是polyA富集文庫）。,例子,DCC transcript in human cells 5 exons were shuffled downstream of 3 exons. 除了非經(jīng)典外顯子順序外，外顯子完整，且使用通常的剪接供體和受體位點(diǎn) exon shuffling shuffled transcripts比預(yù)期transcripts豐度少幾個(gè)數(shù)量級(jí)，非polyAed，主要在胞質(zhì)中，在人和大鼠中表達(dá)。 推測(cè)：intramolecular (cis) splicing an exonic

3、 circRNA Backsplice 3與5連接 電鏡也觀測(cè)到了，但是不區(qū)分circRNA和RNA套索,5,3,人、小鼠、大鼠中檢測(cè)到ETS-1、Sry12和cytochrome P450 2C24 (CYPIIC24) - 偶然發(fā)現(xiàn)PCR產(chǎn)物具有backsplice的序列 Sry is usually unspliced, but sites with the canonical splice site GT/AG sequence motifs were involved in the backsplice, suggesting the involvement of the canon

4、ical spliceosome. The splice junctions used in the exonic circRNA forms of ETS-1 and CYPIIC24 used splice donor and acceptor sites also involved in forward splicing 隨后20年也發(fā)現(xiàn)了一些，但是它們通常比它們來源基因的線性產(chǎn)物豐度低很多。直到高通量測(cè)序。,內(nèi)容,鑒定內(nèi)源性circRNAs的方法：分子方法和全基因組方法，集中于各種方法的優(yōu)缺點(diǎn) 來自這些基因組研究的發(fā)現(xiàn)，集中于exonic circRNAs，描述了體內(nèi)circRNAs的

5、生化性質(zhì)，包括驗(yàn)證環(huán)化的方法 討論已知和預(yù)測(cè)的circRNAs功能，推測(cè)其可能應(yīng)用,形成,有backsplice序列是exonic circRNA產(chǎn)物的關(guān)鍵證據(jù) apparent backsplice sequence：序列中外顯子順序相對(duì)于注釋的模板顛倒（reversed） apparent backsplice sequence形成機(jī)制： exonic circRNA， reverse transcriptase template switching tandem duplication and RNA trans-splicing,This may be assessed either

6、by identification of multiple unique reads in deep sequencing data or by divergent qPCR primers. These divergent primers are oriented to amplify away from each other in a genomic context but become convergent and amplify a discrete amplicon when a backsplicing event brings outside sequences together

7、 Alternatively, the presence of the backsplice sequence in the RNA pool may be assessed directly by RNase protection or northern blot probing for the backsplice sequence,Reverse Transcriptase template switching,artifact of cDNA synthesis occurring when an extending cDNA molecule dissociates from its

8、 template RNA resumes extension from another RNA template often in a homology-dependent manner 這產(chǎn)生了包含backsplice產(chǎn)物的虛假證據(jù)，混淆稀有剪接產(chǎn)物分析 template switching is largely random，不期望產(chǎn)生具有一致序列的豐富的cDNA分子 因此cDNA文庫中高豐度的特定backsplice序列提供了序列也出現(xiàn)在RNA模板中的證據(jù),tandem DNA duplications generate duplicated exons within a gene.

9、當(dāng)這些序列轉(zhuǎn)錄時(shí)，mRNA包含一個(gè)明顯的backsplice序列,來自相同基因的兩個(gè)RNA分子共同參與splicing，產(chǎn)生apparent backsplices,環(huán)狀,線性exonic RNAs通常有3 polyA，而環(huán)沒有3末端,鑒定,Exonic circRNAs migrate more slowly in a gel than linear RNA of the same length, and this effect is augmented by increased gel cross-linking However, exonic circRNAs also contain

10、less total nucleotide sequence than full-length, trans-spliced or tandem-duplicated transcripts from the same gene, and therefore will migrate faster in a gel that has low cross-linking,更具有決定意義的實(shí)驗(yàn)是使用弱水解或靶向RNase H降解 circRNA線性化為單個(gè)產(chǎn)物， 線性RNA有多個(gè)產(chǎn)物,2D凝膠電泳 circRNAs：poor migration through highly cross-linke

11、d gels relative to less cross-linked gels,highly cross-linked gels,less cross-linked gels,trap electrophoresis,非線性RNA被捕獲，在外加電場(chǎng)下不動(dòng) 而線性RNA在外加電場(chǎng)下移動(dòng),RNase R exonuclease、tobacco acid Phosphatase、terminator exonuclease treatment有效降解大部分線性RNAs，保留circRNA。,Finally, with sufficiently long sequencing reads or p

12、aired-end reads, it should be possible to identify sequences that are inconsistent with circRNA Each of the above methods have limitations and are best used in combination to validate circRNAs,區(qū)分exonic circRNA和RNA套索,套索RNA在經(jīng)典RNA剪接中形成，大部分是intronic的，在剪接分支點(diǎn)（splicing branch point）有2-5碳連接 套索RNA比想象的更穩(wěn)定 這些穩(wěn)

13、定的套索RNA的3尾巴降解，留下一個(gè)剩余的分子，這種套索產(chǎn)物被稱為circular intronic RNA（intronic circRNA），因出現(xiàn)2-5連接而不同于exonic circRNA（3-5），使用脫支酶可水解2-5連接 套索RNA在上面幾種實(shí)驗(yàn)中難以與exonic circRNA區(qū)分，但是可以通過apparent backsplice序列區(qū)分。circRNA有而套索RNA無,基因組方法,developments in sequencing technology (deeper sequencing with longer read lengths), better algor

14、ithms for mapping RNA to its genomic source ribosomal RNA depletion strategies that enable sequencing of nonpolyadenylated RNA,兩種,從現(xiàn)有轉(zhuǎn)錄模型中候選 剪切比對(duì)算法完成后，通過匹配reads到基因組序列上以鑒定連接,候選，比對(duì)轉(zhuǎn)錄組,using independent mapping of paired-end reads sequenced from opposite ends of a single cDNA fragmentThis approach iden

15、tified an unexpected abundance of fragments in which two read pairs mapped to the same gene but were in the opposite order from that expected from the gene annotation,直接比對(duì)基因組，相當(dāng)于把基因組分成200bp窗口，窗口看做exon,將read分割成片段，然后末端比對(duì),深藍(lán)read比對(duì)到backsplice， 淺藍(lán)read比對(duì)到超過了backsplice的位置（4,5）,CircleSeq,MapSplice，不需要exon順序

16、信息的算法 In mammals, but not archaea, rRNA depletion is required. 1, backsplice-containing reads are identified using a segmented mapping approach 2, reads derived from circular species should be significantly enriched in the RNase Rtreated sample compared to mock treated-control The exons of linear RN

17、As should be depleted by exonuclease digestion, as should splice junctions not present in circRNAs,CircleSeq,缺點(diǎn),requires more input total RNA than sequencing without enrichment and is sensitive to endonuclease contamination It might also be biased against the detection of longer circRNA products, as

18、 a single nicking event would confer exonuclease sensitivity exonuclease protection may extend to some linear products with protective 3 end structures,circRNAs性質(zhì),在細(xì)胞中穩(wěn)定，half-life 48 h （mRNA是10h） 但是在血清中不穩(wěn)定 （ 15s），可能是因?yàn)閏irculating RNA endonucleases Intracellular stability is likely due to circRNA res

19、istance to RNA exonucleases. Possibly due to this stability, some exonic circRNAs have been shown by sequencing read counting methods and qPCR-based methods to be at higher levels than the linear RNA gene product,不包含2-5連接，抗脫支酶（而套索不抗）,序列特征,GT-AG pair of canonical splice sites exonic circRNAs almost a

20、lways use at least one previously annotated splice site the length of a given exon appears to influence circularization. onger-than-average exons, flanked by longer-than-average introns containing inverted tandem repeats that likely promote intron pairing,可能形成機(jī)制,direct backsplicing ，更多,推斷的circRNA功能,

21、miRNA binding protein binding regulation of translation and translation into proteins,miRNA sponges,miRNA sponges或ceRNAs 例如CDR1as有74個(gè)miR-7 seed序列匹配，被Argonaute蛋白緊密結(jié)合（結(jié)合miRNA的蛋白） CDR1as和miR-7在小鼠腦中共表達(dá)，共定位（co-IP） 敲除CDR1as（或者過表達(dá)切CDR1as的miRNA，一樣的），降低了miR-7靶基因的表達(dá) 將CDR1as轉(zhuǎn)到斑馬魚胚胎中（它里邊沒有），顯著降低了中腦大小，模擬了miR-7敲除的表型,但是，CircleSeq表明哺乳動(dòng)物細(xì)胞中非常少的circRNAs包含超過10個(gè)結(jié)合位點(diǎn)/miRNA effective miRNA sponging by exonic circRNA may be relatively unusual, o