2018-12-17
Next-generation sequencing reveals two populations of damage-induced small RNAs at endogenous DNA double-strand breaks
題目：
新一代測(cè)序技術(shù)揭示了在內(nèi)源DNA雙鏈斷裂中兩種損傷誘導(dǎo)的RNA
發(fā)表刊物及時(shí)間：
Nucleic Acids Research, Volume 46, Issue 22, 14 December 2018, Pages 11869–11882, https://doi.org/10.1093/nar/gky1107
作者：
[Franziska Bonath](javascript:;) [Judit Domingo-Prim](javascript:;) [Marcel Tarbier](javascript:;)[Marc R Friedl?nder](javascript:;) [Neus Visa](javascript:;)
通訊作者及單位：
Marc R Friedl?nder
Science for Life Laboratory, Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, SE-106 91 Stockholm, Sweden
Neus Visa
Department of Molecular Biosciences, The Wenner-Gren Institute, Stockholm University, SE-106 91 Stockholm, Sweden
摘要：
Recent studies suggest that transcription takes place at DNA double-strand breaks (DSBs), that transcripts at DSBs are processed by Drosha and Dicer into damage-induced small RNAs (diRNAs), and that diRNAs are required for DNA repair. However, diRNAs have been mostly detected in reporter constructs or repetitive sequences, and their existence at endogenous loci has been questioned by recent reports. Using the ==homing endonuclease（自制的核酸內(nèi)切酶）== I-PpoI, we have investigated diRNA production in genetically unperturbed human and mouse cells. I-PpoI is an ideal tool to clarify the requirements for diRNA production because it induces DSBs in different types of loci: the repetitive 28S locus, unique genes and intergenic loci. We show by extensive sequencing that the rDNA locus produces substantial levels of diRNAs, whereas unique genic and intergenic loci do not. Further characterization of diRNAs emerging from the 28S locus reveals the existence of two diRNA subtypes. Surprisingly, Drosha and its partner DGCR8 are dispensable for diRNA production and only one diRNAs subtype depends on Dicer processing. Furthermore, we provide evidence that diRNAs are incorporated into Argonaute. Our findings provide direct evidence for diRNA production at endogenous loci in mammalian cells and give insights into RNA processing at DSBs.
最近的研究表明福也，DNA雙鏈斷裂（DSBs）中會(huì)發(fā)生轉(zhuǎn)錄，DSB的轉(zhuǎn)錄本由Drosha和Dicer加工成損傷誘導(dǎo)的小RNA（diRNA），用于修復(fù)DNA析苫。但是贺纲，最近diRNA大多多數(shù)在報(bào)告構(gòu)建體或重復(fù)序列當(dāng)中被檢測(cè)到案训，并且最近的報(bào)道針對(duì)它們?cè)趦?nèi)源基因座的存在性也提出了質(zhì)疑企锌。我們?cè)谶z傳未受干擾的人和小鼠的細(xì)胞中利用自制的內(nèi)切酶I-Ppol研究了diRNA的產(chǎn)生角寸。I-Ppol 是一個(gè)闡明diRNA產(chǎn)生的理想工具乖酬，因?yàn)樗诓煌?lèi)型的基因座中誘導(dǎo)DSB：重復(fù)的28S基因座死相、獨(dú)立的基因和基因間區(qū)域。我們用大量的測(cè)序展示了rDNA基因座會(huì)產(chǎn)生高水平的diRNA咬像，然而獨(dú)立的基因和基因間區(qū)則不會(huì)算撮。通過(guò)進(jìn)一步描繪28S基因座產(chǎn)生的diRNA揭示了兩種diRNA亞型的存在。令 人驚奇的是县昂，Drosha和它的伴侶DGCR8在diRNA的產(chǎn)生中是非必需的肮柜，只有一種diRNA亞型是依賴(lài) 于Dicer加工。而且倒彰，我們有證據(jù)表明diRNA被整合到Argonaute當(dāng)中审洞。我們的研究結(jié)果為哺乳動(dòng)物細(xì)胞可以產(chǎn)生內(nèi)源基因座的雙鏈RNA提供了直接證據(jù)，并為雙鏈斷裂過(guò)程中的RNA 加工提供了見(jiàn)解待讳。
==homing endonuclease（自制的核酸內(nèi)切酶）==：自己表達(dá)純化出來(lái)的核酸內(nèi)切酶芒澜，而不是從商業(yè)化公司購(gòu)買(mǎi)獲得
圖表選析
Figure 1. DiRNAs originate from endogenous DSBs in human cells. (A) Schematic representation of the 28S rDNA (upper part) and definition of areas around DSBs (lower part). The arrow indicates the location of the I-PpoI target site. The lower panel defines the position and polarity of the transcripts analysed. Upstream and downstream are relative to the I-PpoI cleavage site. Sense and antisense are according to the canonical transcription start of the rDNA locus (arrow). Sense transcripts have the same polarity as the 28S rRNA. (B) Small RNA reads from HeLa cells transfected with a plasmid that expressed I-PpoI analysed at different times after transfection, as indicated. The reads were mapped to the 28S rDNA locus and normalized to total collapsed 28S rRNA read counts. The grey vertical line in each plot indicates the position of the I-PpoI cleavage site in the 28S locus. The top (blue) and bottom (red) parts of the plot show the coverage in the sense and antisense strands, respectively. The reads in the sense strand (blue) are assumed to be predominantly degradation products of rRNA. (C) Quantification of diRNA reads in the 28S rDNA locus expressed as reads per million (RPM) of total collapsed reads. The plot includes only upstream, antisense reads. (D) Length distributions of antisense small RNAs derived from the 28S rDNA locus, upstream region, at different times after transfection with the I-PpoI expression plasmid. The black bars highlight reads of 21 and 22 nt. The percentage of the 21–22 nt population is displayed. Enrichment of the 21–22 nt fraction is significant at 16 h (P = 1.85 × 10?5) and 24 h (P = 1.77 × 10?11) in an exact binomial statistical test.
圖1. DiRNA起源于人細(xì)胞中的內(nèi)源性DSB。 （A）28S rDNA（上部）的示意圖和并定義了DSB周?chē)膮^(qū)域（下部）创淡。箭頭表示I-PpoI靶向的位置痴晦。 下圖定義了分析的轉(zhuǎn)錄本的位置和極性。上游和下游相對(duì)于I-PpoI切割位點(diǎn)琳彩。正向和方向是根據(jù)rDNA基因座的經(jīng)典轉(zhuǎn)錄起始（箭頭）誊酌。正向轉(zhuǎn)錄本具有與28S rRNA相同的極性。 （B）如所示露乏，在轉(zhuǎn)染后不同時(shí)間分析 表達(dá)I-PpoI的質(zhì)粒轉(zhuǎn)染的HeLa細(xì)胞的小RNA讀數(shù)碧浊。將讀數(shù)定位于28S rDNA基因座并標(biāo)準(zhǔn)化為完全折疊的28S rRNA讀數(shù)計(jì)數(shù)。每個(gè)圖中的灰色垂直線表示I-PpoI切割位點(diǎn)在28S基因座中的位置瘟仿。圖中的頂部（藍(lán)色） 和底部（紅色）部分分別顯示有義鏈和反義鏈的覆蓋范圍箱锐。有義鏈（藍(lán) 色）中的讀數(shù)被認(rèn)為主要是rRNA的降解產(chǎn)物。 （C）28S rDNA基因座中diRNA 讀數(shù)的定量表示為每百萬(wàn)讀數(shù)（RPM）的總折疊讀數(shù)猾骡。該圖僅包括上游反義讀數(shù)瑞躺。 （D）在用I-PpoI表達(dá)質(zhì)粒轉(zhuǎn)染后不同時(shí)間源自28S rDNA基因 座上游區(qū)域的反義小RNA的長(zhǎng)度分布敷搪。黑條突出顯示21和22 nt的讀數(shù)。 顯示21-22 nt種類(lèi)的百分比幢哨。在精確的二項(xiàng)式統(tǒng)計(jì)檢驗(yàn)中赡勘，在21小時(shí)（P = 1.85×10-5）和24小時(shí)（P = 1.77×10-11），21-22nt組分的富集是顯著的捞镰。
Figure 2. DiRNAs originate from endogenous DSBs in murine cells. (A) Small RNA reads from mESC cells transfected with a plasmid that expressed I-PpoI analysed 36 h after transfection. The reads were mapped to the 28S rDNA locus and normalized to spike-ins. The grey vertical lines indicate the position of the I-PpoI cleavage site in the 28S locus. The top (blue) and bottom (red) part of the plot show the coverage in the sense and antisense strands, respectively. (B) Length distributions of antisense small RNAs derived from the 28S rDNA locus, upstream region, at 36 h after transfection with the I-PpoI expression plasmid. The black bars highlight reads of 21 and 22 nt. The percentage of the 21–22 nt population is displayed. (C) Quantification of diRNA reads in the 28S rDNA locus expressed as collapsed reads per million ==spike-ins==. The plot includes only upstream, antisense reads.
圖2. 在鼠類(lèi)細(xì)胞中闸与， diRNAs 源于內(nèi)源性 DSBs. (A)表達(dá) I-PpoI 的質(zhì)粒轉(zhuǎn)染小鼠胚胎干細(xì)胞細(xì)胞 36h 后小 RNAs 讀數(shù)的分析。讀數(shù)被映射到 28S rDNA 位點(diǎn)岸售， 并標(biāo)準(zhǔn)化為尖峰践樱。 灰色垂直線表示 I-PpoI 裂解位點(diǎn) 在 28S 位點(diǎn)的位置。 上半部分（藍(lán)色） 和底部（紅色） 部分分別顯示了正義和反義鏈的覆蓋范圍 (B)在 I-PpoI 表達(dá)質(zhì)粒轉(zhuǎn)染后 36h凸丸， 來(lái)自上游區(qū)域 28S rDNA 位點(diǎn)的反義小 RNA 的長(zhǎng)度分布拷邢。黑色柱突出顯示 21 和 22nt 的讀數(shù)。顯示了 21-22nt 分布的百分比屎慢。 (C)量化 28S rDNA 位點(diǎn)的 diRNA 讀數(shù)瞭稼， 表達(dá)為每百萬(wàn)個(gè)spike-in折疊讀數(shù)。 只包括上游的反義讀數(shù)腻惠。
==spike-ins==：沒(méi)有特別翻譯环肘，往往是一段已知序列，在實(shí)驗(yàn)后根據(jù)這段序列的數(shù)量對(duì)其它序列進(jìn)行定量校準(zhǔn)集灌，相當(dāng)于外標(biāo)的control
Figure 3. DiRNAs arise from repetitive, but not from non-repetitive genomic loci in mammalian cells. (A) Schematic definition of repetitive ribosomal (rDNA), genic and intergenic regions used in (B and C). I-PpoI sites in genic regions can be located in exons or introns. I-PpoI sites defined as intergenic do not overlap with any annotated gene. (B and C) Total read counts of collapsed reads mapped to the 28S rDNA locus or to genic and intergenic loci in HeLa cells (B) or mESCs (C). The genome coordinates of the loci included in the analysis are listed in Supplementary Tables S2 and S3. For an overview of underlying samples see Supplementary Table S1. Read counts per locus are listed in Supplementary Table S4. (D) The efficiency of I-PpoI cleavage at selected loci was quantified by qPCR using primers spanning the I-PpoI motif on genomic DNA and expressed as percentage of DSBs. The analysis was carried out 24 h (for Ryr2, Dab1 and SLCO5A1) or 36 h (for AAMDC, chr7 and 28S rDNA) after transfection. The ARPP locus is not cleaved by I-PpoI and was used for normalization.
圖3. DiRNA來(lái)自哺乳動(dòng)物細(xì)胞中的重復(fù)區(qū)域悔雹，而不是非重復(fù)的基因組位點(diǎn)。 （A）（B和C）中使用的重復(fù)核糖體（rDNA）欣喧，基因和基因間區(qū)域的示意圖腌零。基因區(qū)域中的I-PpoI位點(diǎn)可位于外顯子或內(nèi)含子中续誉。定義為基因間的I-PpoI位點(diǎn)不與任何注釋基因重疊莱没。 （B和C）在HeLa細(xì)胞（B）或mESC（C）中映射到28S rDNA基因座或基因和基因間基因座的折疊讀數(shù)的總讀數(shù)初肉。包括在分析中的基因座的基因組坐標(biāo)列于補(bǔ)充表S2和S3中酷鸦。有關(guān)基礎(chǔ)樣本的概述，請(qǐng)參見(jiàn)補(bǔ)充表S1牙咏。每個(gè)基因座的讀數(shù)計(jì)數(shù)列于補(bǔ)充表S4中臼隔。 （D）通過(guò)qPCR使用跨越基因組DNA上的I-PpoI基序的引物定量I-PpoI切割在所選基因座處的效率，并表示為DSB的百分比妄壶。在轉(zhuǎn)染后24小時(shí)（對(duì)于Ryr2摔握，Dab1和SLCO5A1）或36小時(shí)（對(duì)于AAMDC，chr7和28S rDNA）進(jìn)行分析丁寄。 ARPP基因座未被I-PpoI切割并用于標(biāo)準(zhǔn)化氨淌。
Figure 6. Endogenous diRNAs are incorporated into Argonaute. (A) Coverage of collapsed small RNA reads mapping to the 28S rDNA normalized to ==reads per million== (RPM,每百萬(wàn)讀數(shù)) in Input and Argonaute RIP samples from either mock transfected (uncut) or I-PpoI expressing cells (cut). Coverage from three (antibody A) or two (antibody B) biological replicates were averaged. The grey vertical lines indicate the I-PpoI cleavage site. (B) Argonaute incorporation rates for miRNAs (black), snoRNAs (blue), tRNAs (green) or reads mapping to the antisense (diRNAs, red) or sense strand of the 28S rDNA (yellow). The Argonaute incorporation rate is estimated by fold-change (RIP/Input) normalized to the median miRNA fold-change. The plot shows averages from three (antibody A) or two (antibody B) biological replicates. Grey bars indicate the standard deviations. (C) Length distributions of diRNAs produced at the 28S rDNA (upstream, antisense) in Input or Argonaute RIP samples, as indicated in the figure. The percentage of the 21–22 nt population in each sample is displayed.
圖 6.內(nèi)源性 diRNA 結(jié)合 Argonaute泊愧。（A）小 RNA 讀數(shù)匹配到 28S rDNA 的的覆蓋率， 其標(biāo)準(zhǔn)化為每百萬(wàn)讀數(shù)作為 Input盛正。 和來(lái)自 模擬轉(zhuǎn)染（未切割）或 I-PpoI 表達(dá)細(xì)胞（切割） 的 Argonaute RIP 樣品删咱。將來(lái)自三個(gè)（抗體 A）或兩個(gè)（抗體 B）生物學(xué)重復(fù)的覆 蓋率取平均值『荔荩灰色垂直線表示 I-PpoI 切割位點(diǎn)痰滋。（B） miRNA （黑色）， snoRNA（藍(lán)色）续崖， tRNA（綠色） 或映射到 28S rDNA （黃色）的反義（diRNA敲街，紅色）或有義鏈的讀數(shù)與 Argonaute 結(jié) 合率。通過(guò)標(biāo)準(zhǔn)化為中值 miRNA 倍數(shù)變化的倍數(shù)變化（RIP /輸 入）估計(jì) Argonaute 結(jié)合率严望。該圖顯示來(lái)自三個(gè)（抗體 A）或兩 個(gè)（抗體 B）生物學(xué)重復(fù)的平均值多艇。灰色條表示標(biāo)準(zhǔn)偏差像吻。 （C） 如圖所示墩蔓，在 Input 或 Argonaute RIP 樣品中 28S rDNA（上游， 反義）產(chǎn)生的 diRNA 的長(zhǎng)度分布萧豆。 顯示每個(gè)樣本中 21-22nt 種群 的百分比奸披。
==注意：此圖的B和C標(biāo)反了==
Figure 7. Model for the biogenesis of Dicer-dependent and Dicer-independent diRNAs at endogenous DSBs in the mammalian genome. See main text for details.
圖7. 在哺乳動(dòng)物基因組中內(nèi)源 DSB的Dicer依賴(lài)和Dicer非依賴(lài)的diRNA的生物發(fā)生模型。詳細(xì)見(jiàn)原文
We have revealed the existence of two different diRNA populations that result from the processing of dilncRNAs at repetitive, transcribed loci and we propose a model for their biogenesis. According to this model, the free DNA ends at the DSB recruit RNAPII, which results in the synthesis of dilncRNAs (Figure 7, box 1). We have been able to detect dilncRNAs extending from DSBs in both the 28S rDNA locus and the Ryr2 gene. The newly made dilncRNAs are able to anneal with already existing transcripts made at the same locus, and double-stranded RNAs are produced. These double-stranded RNAs can be processed by Dicer into diRNAs with the characteristic length of 21–22 nt (Figure 7, box 2).
我們已經(jīng)揭示了兩個(gè)不同的diRNA群體的存在涮雷，這些diRNA群體是由重復(fù)的轉(zhuǎn)錄基因座上的dilncRNA 的加工產(chǎn)生的阵面，我們提出了它們的生物發(fā)生的模型。根據(jù)該模型洪鸭，游離DNA終止于DSB招募RNAPII样刷， 其導(dǎo)致dilncRNA的合成（圖7，第一格）览爵。我們已經(jīng)能夠檢測(cè)到28S rDNA基因座和Ryr2基因中從 DSB延伸的dilncRNA置鼻。新生成的dilncRNA能夠與已存在的同一個(gè)基因座產(chǎn)生的轉(zhuǎn)錄物結(jié)合，并產(chǎn)生 雙鏈RNA蜓竹。這些雙鏈RNA可由Dicer加工成具有21-22nt特征長(zhǎng)度的diRNA（圖7箕母，第二格）。 如其他人所示（15,51）俱济，DNA-RNA雜合體在DSBs形成嘶是，這意味著dilncRNAs也與模板DNA結(jié)合。此 外蛛碌，我們的模型提出聂喇，一部分dilncRNAs可能被不同的RNA內(nèi)切酶和RNA外切酶降解，這導(dǎo)致在Dicer 缺失細(xì)胞中觀察到的具有大范圍不同長(zhǎng)度的diRNA-Is群體（圖7蔚携，第三格）希太。因此克饶，野生型細(xì)胞的 diRNA長(zhǎng)度譜是diRNA-Ds和diRNA-Is的譜的組合。
翻譯小組：
葉名琛誊辉、渠夢(mèng)葳彤路、王俊豪、鄧俊瑋芥映、黃敬潼洲尊、黃子亮、常彥琪奈偏、李碧琪坞嘀、陳凱星、 鄭凌伶
