circRNA检测和描述.pptx

1、,历史20 年，Scrambled exons 1991322 为什么：不能根据大小，或从电泳中容易分离（不像miRNA等） 最近，哺乳动物细胞中大量circRNAs，许多是丰富且稳定的 产生：exons (exonic circRNA) 或introns (intronic circRNA)，不同产生模式 功能：调控基因表达，正在揭示,扩增和片段化损坏环化 circRNA无自由3和5末端，不能被依赖于polyA free RNA末端技术（RACE，或polyA富集）发现。 Exon arrangement backsplice 不仅限于circRNAs，早期RNA-Seq将之过滤掉 解决：基

2、于外切酶富集，新的生物信息学工具，更长reads，更高通量，和rRNA-depleted文库（而不是polyA富集文库）。,例子,DCC transcript in human cells 5 exons were shuffled downstream of 3 exons. 除了非经典外显子顺序外，外显子完整，且使用通常的剪接供体和受体位点 exon shuffling shuffled transcripts比预期transcripts丰度少几个数量级，非polyAed，主要在胞质中，在人和大鼠中表达。 推测：intramolecular (cis) splicing an exonic

3、 circRNA Backsplice 3与5连接 电镜也观测到了，但是不区分circRNA和RNA套索,5,3,人、小鼠、大鼠中检测到ETS-1、Sry12和cytochrome P450 2C24 (CYPIIC24) - 偶然发现PCR产物具有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 随后20年也发现了一些，但是它们通常比它们来源基因的线性产物丰度低很多。直到高通量测序。,内容,鉴定内源性circRNAs的方法：分子方法和全基因组方法，集中于各种方法的优缺点 来自这些基因组研究的发现，集中于exonic circRNAs，描述了体内circRNAs的

5、生化性质，包括验证环化的方法 讨论已知和预测的circRNAs功能，推测其可能应用,形成,有backsplice序列是exonic circRNA产物的关键证据 apparent backsplice sequence：序列中外显子顺序相对于注释的模板颠倒（reversed） apparent backsplice sequence形成机制： 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 这产生了包含backsplice产物的虚假证据，混淆稀有剪接产物分析 template switching is largely random，不期望产生具有一致序列的丰富的cDNA分子 因此cDNA文库中高丰度的特定backsplice序列提供了序列也出现在RNA模板中的证据,tandem DNA duplications generate duplicated exons within a gene.

9、当这些序列转录时，mRNA包含一个明显的backsplice序列,来自相同基因的两个RNA分子共同参与splicing，产生apparent backsplices,环状,线性exonic RNAs通常有3 polyA，而环没有3末端,鉴定,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,更具有决定意义的实验是使用弱水解或靶向RNase H降解 circRNA线性化为单个产物， 线性RNA有多个产物,2D凝胶电泳 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,非线性RNA被捕获，在外加电场下不动 而线性RNA在外加电场下移动,RNase R exonuclease、tobacco acid Phosphatase、terminator exonuclease treatment有效降解大部分线性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,区分exonic circRNA和RNA套索,套索RNA在经典RNA剪接中形成，大部分是intronic的，在剪接分支点（splicing branch point）有2-5碳连接 套索RNA比想象的更稳定 这些稳

13、定的套索RNA的3尾巴降解，留下一个剩余的分子，这种套索产物被称为circular intronic RNA（intronic circRNA），因出现2-5连接而不同于exonic circRNA（3-5），使用脱支酶可水解2-5连接 套索RNA在上面几种实验中难以与exonic circRNA区分，但是可以通过apparent backsplice序列区分。circRNA有而套索RNA无,基因组方法,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,两种,从现有转录模型中候选 剪切比对算法完成后，通过匹配reads到基因组序列上以鉴定连接,候选，比对转录组,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,直接比对基因组，相当于把基因组分成200bp窗口，窗口看做exon,将read分割成片段，然后末端比对,深蓝read比对到backsplice， 浅蓝read比对到超过了backsplice的位置（4,5）,CircleSeq,MapSplice，不需要exon顺序

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,缺点,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性质,在细胞中稳定，half-life 48 h （mRNA是10h） 但是在血清中不稳定 （ 15s），可能是因为circulating 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连接，抗脱支酶（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,可能形成机制,direct backsplicing ，更多,推断的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个miR-7 seed序列匹配，被Argonaute蛋白紧密结合（结合miRNA的蛋白） CDR1as和miR-7在小鼠脑中共表达，共定位（co-IP） 敲除CDR1as（或者过表达切CDR1as的miRNA，一样的），降低了miR-7靶基因的表达 将CDR1as转到斑马鱼胚胎中（它里边没有），显著降低了中脑大小，模拟了miR-7敲除的表型,但是，CircleSeq表明哺乳动物细胞中非常少的circRNAs包含超过10个结合位点/miRNA effective miRNA sponging by exonic circRNA may be relatively unusual, o