The translation of non-canonical open reading frames controls mucosal immunity

題目：非經(jīng)典開放閱讀框的翻譯調(diào)控黏膜免疫

作者及單位：

Ruaidhrí Jackson, [...], Richard A. Flavell

Richard A. Flavell

• Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA
• Howard Hughes Medical Institute, Yale University, New Haven, CT, USA

發(fā)表期刊及時間：

Naturevolume 564, pages434–438 (2018) Published: 12 December 2018

摘要：

The annotation of the mammalian protein-coding genome is incomplete. Arbitrary size restriction of open reading frames (ORFs) and the absolute requirement for a methionine codon as the sole initiator of translation have constrained the identification of potentially important transcripts with non-canonical protein-coding potential1,2. Here, using unbiased transcriptomic approaches in macrophages that respond to bacterial infection, ==we show that ribosomes associate with a large number of RNAs that were previously annotated as ‘non-protein coding’==（注意此處是與核糖體結(jié)合的RNA注釋為非編碼嘁扼，而不是核糖體注釋為非編碼）. Although the idea that such non-canonical ORFs can encode functional proteins is controversial3,4, we identify a range of short and non-ATG-initiated ORFs that can generate stable and spatially distinct proteins. Notably, we show that the translation of a new ORF ‘hidden’ within the long non-coding RNA Aw112010 is essential for the orchestration of mucosal immunity during both bacterial infection and colitis. This work expands our interpretation of the protein-coding genome and demonstrates that proteinaceous products generated from non-canonical ORFs are crucial for the immune response in vivo. We therefore propose that the misannotation of non-canonical ORF-containing genes as non-coding RNAs may obscure the essential role of a multitude of previously undiscovered protein-coding genes in immunity and disease.

目前涛酗， 哺乳動物編碼蛋白質(zhì)的基因組注釋并不完善。 武斷的限制 開放閱讀框(ORF)的大小并把蛋氨酸密碼子作為翻譯唯一啟動子的絕 對要求碾褂， 這二者都限制了重要的非標準蛋白編碼轉(zhuǎn)錄本的鑒定。 本文 選取了對細菌侵染應(yīng)答后的巨噬細胞叁温， 使用了無偏轉(zhuǎn)錄組學的方法瘟裸， 研究發(fā)現(xiàn)陶衅， ==以往被認為‘不編碼蛋白質(zhì)’ 的RNA其實大量的與核糖體相結(jié)合==。盡管關(guān)于非標準 ORFs 能編碼功能性蛋白的觀點還飽受爭議褂痰， 但是我們鑒定到了很多短的亩进、 非 ATG 起始的 ORFs， 它們能產(chǎn)生穩(wěn)固且空間上差異的蛋白質(zhì)缩歪。 尤其是归薛， 我們發(fā)現(xiàn)存在一類新的 ORF， 它' 隱藏'于長非編碼 RNA Aw112010 內(nèi)匪蝙， 這類 ORF 的翻譯對于細菌感染 苟翻、和結(jié)腸炎期間協(xié)調(diào)粘膜免疫來說是至關(guān)重要的。 此項工作擴展了我們對編碼蛋白的基因組的認知骗污， 還論證了非標準 ORFs 產(chǎn)生的蛋白質(zhì)產(chǎn) 物確實對機體免疫反應(yīng)至關(guān)重要崇猫。 本文因此提出： 將含有非標準 ORF 的基因錯誤的注釋為非編碼 RNAs 后， 許多以前未發(fā)現(xiàn)能編碼蛋白質(zhì) 的基因在免疫和疾病中的重要作用會被掩蓋需忿。

圖表選析：

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Fig. 1: Bacterial infection drives widespread ribosomal association with non-coding RNAs.

a–f, BMDMs from RiboTagLysM mice were non-treated (NT) or stimulated with LPS (1 ng ml?1). RNA was subjected to RNA-seq. Data are presented as a combination of two independent biological replicates. a, Circos plot shows differentially expressed ribosome-associated transcripts after 6 and 24 h LPS stimulation. Red denotes upregulation; blue denotes downregulation. Each track from the periphery to the core represents: chromosomes location; 12,820 known protein-coding transcripts; 1,176 lncRNAs; 1,107 pseudogenes; and 413 other non-coding RNAs. b, Pie chart of the percentage breakdown of protein-coding genes annotated from RiboTag RNA-seq (fragments per kilobase of transcript per million mapped reads (FPKM) ≥ 1). c, The non-protein coding genes in b are further classified. d, Stratification of detectable BMDM lncRNAs based on ribosome association. Ribosome-associated lncRNAs with an FPKM of ≥ 1 in RiboTag RNA-seq are represented in the red exploded section. Blue section depicts lncRNAs not found in RiboTag RNA-seq, but with an FPKM of ≥ 0.01 in conventional RNA-seq. e, f, Volcano plot (e) and heat map analysis (f) of lncRNAs associated with ribosomes after LPS stimulation in BMDMs. g, qPCR analysis of ribosome-associated transcripts of non-treated BMDMs or those stimulated with LPS (10 ng ml?1) or infected with S. Typhimurium at a ==multiplicity of infection (MOI)== （感染復數(shù)）of 1 for 6 h. Data are presented as six biological replicates, and fold expression was calculated from each individual non-treated sample. h, RiboTagLysM mice were gavaged with 2 × 10⁸ CFUs of S. Typhimurium. After 24 h, colonic tissue was extracted and lysed. Macrophage ribosome-associated RNA was isolated and qPCR analysis was conducted. Data are presented as seven biological replicates. Data are mean and ==s.e.m.==（Standard Error of Mean 標準誤）P < 0.01, ****P < 0.001, *****P < 0.0001, unpaired two-tailed t-test.

圖 1：細菌感染驅(qū)動大量核糖體與非編碼 RNA結(jié)合诅炉。 a-f， 用 LPS 刺激或未處理的來自 RiboTagLysM小鼠的骨髓巨噬細胞（BMDMS）屋厘。 RNA 通過 RNA-seq獲得涕烧。數(shù)據(jù)用兩個獨立生物重復結(jié)合的形式進行展示。 a.環(huán)形圖顯示了 6 個小時 和 24 個小時 LPS 刺激后差異表達的核糖體結(jié)合轉(zhuǎn)錄本汗洒。紅色代表上調(diào)议纯，藍色代表下調(diào)。 從外到內(nèi)每個軌道分別代表：染色體位置溢谤；12810 個已知的編碼蛋白質(zhì)轉(zhuǎn)錄組瞻凤；1176 個 LncRNAs憨攒；1107 個假基因；413 個其他非編碼 RNA阀参。 B. 按照來自 RiboTag RNA-seq 注釋 的編碼蛋白質(zhì)基因（FPKM≥1）肝集， 百分比餅圖被分成兩部分。 C.圖 b 中非編碼蛋白質(zhì)基因被 進一步分類蛛壳。 D. BMDM 中識別出的與核糖體結(jié)合的 lncRNA分層杏瞻。在 RiboTag RNA-seq 中 FPKM 大于等于 1 的核糖體結(jié)合的lncRNA在紅色區(qū)域中標出。藍色部分代表未在 RiboTag RNA-seq 中發(fā)現(xiàn)衙荐，但在傳統(tǒng) RNA-seq中 FPKM 大于等于 0.01 的 lncRNA捞挥。 e,f, 在 BMDM 中使用 LPS 刺激后火山圖（e）和熱力圖（f）分析。 G. 未處理 BMDM忧吟，用 LPS （10 ng ml?1）刺激的 BMDM 和用 1 感染復數(shù)的 S. Typhimurium 感染六個小時后 的 BMDMs 進行 q-PCR 分析砌函。 h, 用 2 × 10⁸CFU 的 S. Typhimurium飼養(yǎng) RiboTagLysM 小鼠。 24 小時后瀑罗，提取結(jié)腸樣本并裂解胸嘴。分離出巨噬細胞線粒體結(jié)合 RNA，并使用 qPCR 進行分 析斩祭。數(shù)據(jù)以七個生物學重復的形式展示劲蜻。 數(shù)據(jù)的平均數(shù)嘴纺、標準誤和非配對雙尾t 檢驗結(jié)果 **P < 0.01, ***P < 0.001, ****P < 0.0001）在圖中被展示出來。

==multiplicity of infection (MOI)== ：感染復數(shù)，其含義是感染時噬菌體與細菌的數(shù)量比值悦冀，也就是平均每個細菌感染噬菌體的數(shù)量履因。

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Fig. 2: LPS triggers genome wide differential translation of non-canonical ORFs in lncRNAs.

a–e, Wild-type BMDMs were non-treated or stimulated with LPS (10 ng ml?1) for 6 h and ==ribosome profiling==（核糖體譜） was conducted. Data are representative of two biological replicates. a, ==PME==（percentage of maximum entropy 最大熵百分比） values for protein-coding genes and lncRNAs. A PME cut-off value of ≥0.6% represents transcripts considered to be protein coding. b, Translation efficiency (TE) and ==RRS==(ribosome release score 核糖體釋放分值) analysis was conducted on transcripts identified by RiboProfiling. Purple broken lines represent the 95th percentile of the 3? UTRs of known protein-coding genes and discriminates coding and non-coding transcripts. c, d, Categorization of start codon usage (c) and ORF size (d) in RibORF-and/or RiboCode-identified lncRNAs with coding RRS and translation efficiency values. e, Heat map of the top differentially regulated LPS-stimulated lncRNA ORFs. f, HEK293 cells transfected with empty vector (EV) or Aw112010Flag ORF. Cells were stained with DAPI, phalloidin and anti-Flag. Scale bars, 9 μm; original magnifications, ×60 and ×100. g, Wild-type (WT) and Aw112010HA BMDMs were non-treated or stimulated with LPS (10 ng ml?1), protein lysates were generated and western blot analysis was conducted for haemagglutinin and β-tubulin. Data are representative of three biological replicates. h, Aw112010HA BMDMs were generated, stimulated with LPS for 6 h and subjected to haemagglutinin immunoprecipitation. Purified lysates were subject to mass spectrometry analysis. Precursor ion peaks in the MS1 extracted ion chromatogram corresponding to a spiked in synthetic isotopically labelled peptide standard (top) and co-elution of a peak consistent with the endogenous Aw112010 peptide (bottom) in the same sample. Identified fragment ions (b and y ions, red) are indicated above and below the peptide sequence. Data are representative of two biological replicates.

a-e跪腹，野生型BMDM未經(jīng)處理或用LPS（10ng ml-1）刺激6小時畏梆，并進行核糖體譜分析。數(shù)據(jù)代表兩個生物學重復坏挠。 a芍躏，蛋白質(zhì)編碼基因和lncRNA的PME值。 PME截止值≥0.6％表示被認為是蛋白質(zhì)編碼的轉(zhuǎn)錄物降狠。 b对竣，對RiboProfiling鑒定的轉(zhuǎn)錄本進行翻譯效率（TE）和RRS分析。紫色虛線代表已知蛋白質(zhì)編碼基因的3'UTR的第95百分位數(shù)榜配，并區(qū)分編碼和非編碼轉(zhuǎn)錄物否纬。 從RibORF和/或RiboCode鑒定的lncRNA中利用RRS和翻譯效率值進行c，起始密碼子使用和d蛋褥，ORF大小的分類临燃。 e，排在前面的差異調(diào)節(jié)的LPS刺激的lncRNA ORF的熱圖。 f膜廊，用空載體（EV）或Aw112010Flag ORF轉(zhuǎn)染的HEK293細胞乏沸。用DAPI，鬼筆環(huán)肽和抗Flag對細胞染色溃论。比例尺屎蜓，9微米;原始放大倍率痘昌，×60和×100钥勋。 g，野生型（WT）和Aw112010HA BMDM未經(jīng)處理或用LPS（10ng ml-1）刺激辆苔，產(chǎn)生蛋白質(zhì)裂解物并對血細胞凝集素和β-微管蛋白進行蛋白質(zhì)印跡分析算灸。數(shù)據(jù)代表三個生物學重復。 h驻啤，產(chǎn)生Aw112010HA BMDM菲驴，用LPS刺激6小時并進行血細胞凝集素免疫沉淀。將純化的裂解物進行質(zhì)譜分析骑冗。 MS1提取的離子色譜圖中的前體離子峰對應(yīng)于摻入合成同位素標記的肽標準品（上圖）赊瞬，并且在同一樣品中共同洗脫與內(nèi)源Aw112010肽（下圖）一致的峰。在肽序列的上方和下方指示鑒定的片段離子（b和y離子贼涩，紅色）巧涧。數(shù)據(jù)代表兩個生物學重復。

==ribosome profiling==（核糖體譜）：所有與核糖體結(jié)合的RNA序列測定

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Fig. 3: Translation of the non-canonical Aw112010 encoded ORF is essential for mucosal immunity.

a–e, Wild-type (n = 10) and Aw112010Stop (n = 11) mice were administered streptomycin (20 mg) by oral gavage 24 h before S. Typhimurium infection (1 × 103 CFUs). a, Weight loss was measured after infection. b, Enumeration of S. Typhimurium CFUs present in the faeces of wild-type and Aw112010Stop mice 24 h after infection. c, Enumeration of S. Typhimurium CFUs in the caecum of wild-type and Aw112010Stop mice 96 h after infection. d, e, Enumeration of S. Typhimurium CFUs in the liver (d) and spleen (e) of wild-type and Aw112010Stop mice 96 h after infection. f, Confocal immunostaining of macrophages (F4/80, green), B cells (B220, purple) and Salmonella (anti-Salmonella, red) in the spleens of wild-type and Aw112010Stop mice infected with 1 × 102 CFUs of S. Typhimurium 72 h after gavage. Images are representative of three independent biological replicates. Scale bars, 9 μm; original magnification, ×60. g, Survival curve analysis of wild-type (n = 10) and Aw112010Stop (n = 10) mice infected with 1 × 102 CFUs via oral gavage. h, i, Wild-type and Aw112010Stop cohoused littermate mice were administered 2.5% DSS in their drinking water for 5 days. h, Weight loss from wild-type (n = 11) and Aw112010Stop (n = 12) mice was measured over 12 days. i, Colon length was measured from wild-type (n = 10) and Aw112010Stop (n = 12) mice. Horizontial bars in b–e represent the mean bacterial count. Error bars in h and i denote s.e.m. of replicates. P < 0.05, P < 0.01, ****P < 0.001, *****P < 0.0001, nonparametric Mann–Whitney test (b–e), log-rank test (g), and unpaired two tailed t-test (a, h*, i).

圖3. AW112010 編碼的非經(jīng)典 ORF翻譯對于黏膜免疫至關(guān)重要 a-e 10 個野生型小鼠和 11 個 Aw112010 終止的小鼠在傷寒鏈球菌 （1 千菌落形成單位） 感染前 24 小時均口服鏈霉素(20 毫克) a 感染后測量的體重減少量 b.感染后 24 小時在野生型和 Aw112010 終止小鼠糞便中存在的鼠 傷寒沙門氏菌 CFU 計數(shù)遥倦。 c.感染后 96h 野生型和 Aw112010終止小鼠盲腸中鼠傷寒沙門氏菌 CFU 計數(shù)谤绳。 d、 e袒哥、 野生型和 Aw112010 終止小鼠感染 96h 后缩筛， 小鼠肝臟(d)和 脾臟(e)中鼠傷寒沙門氏菌 CFU 計數(shù)。 f.被 100CFUs 的傷寒沙門氏菌感染 72 小時后堡称，野生型和 Aw112010 終止小鼠的脾臟中巨噬細胞（F4/80瞎抛， 綠色） 、 B 細胞（B220却紧， 紫色） 和沙門氏菌（抗沙門氏菌桐臊， 紅色） 的共聚焦免疫染色。 圖像代表三個 獨立的生物復制品啄寡。 比例尺豪硅， 9μm； 原始放大倍數(shù)挺物， ×60懒浮。 g. 在口灌胃感染 100CFU 后的野生型(n=10)和 Aw112010 終止 (n=10)小鼠的存活曲線分析 h、i、在飲用水中給予 2.5%葡聚糖硫酸酯鈉 5 天的野生型 Wild-typ 和 Aw112010 終止小鼠砚著。 h次伶、 在 12 天內(nèi)測定野生型(n=11)和 Aw112010Stop(n=12)小鼠的體 重損失。 i稽穆、 測定野生型(n=10)和 Aw112010 終止(n=12)小鼠的結(jié)腸長度冠王。 b –e 中的水平條表示平均細菌數(shù)。 h 和 i 中的錯誤欄表示復制的 s.e.m.P<0.05 * 顯著舌镶、 P<0.01 很顯著柱彻、 P<0.001*非常顯著、 P<0.0001 ****極其顯著餐胀、 非參數(shù) Mann–Whitney 檢驗(b–e)哟楷、 log-rank 對數(shù)秩檢驗(g)和未配對的雙尾 t 檢驗(a,h,i)。

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Fig. 4: Translation of the Aw112010 non-canonical ORF encoded protein is required for IL-12 production.

a, BMDMs were pretreated with cytochalasin D (CD) (10 μM) for 1 h, LPS (10 ng ml?1) for 6 h, or non-treated. pHrodo BioParticles were administered for 1 h and cells were assessed for CD11b and pHrodo expression by flow cytometry. Plots are representative of three independent experiments. b, BMDMs were infected with S. Typhimurium for 6 h. Cells were lysed and CFUs were determined. c, BMDMs were pretreated with LPS (100 ng ml?1) for 5 h and infected with S. Typhimurium for 1 h, and the release of lactate dehydrogenase (LDH) was measured. RU, relative units. d, BMDMs were stimulated with LPS (10 ng ml?1), and Il12bexpression was determined by qPCR. e, Mice were administered PBS (n = 5) or LPS (n = 6, WT and Aw112010Stop) (10 mg kg?1) for 6 h via intraperitoneal injection. Serum levels of IL-12p40 were determined by ELISA. f, BMDMs were stimulated with LPS (10 ng ml?1), and Aw112010 expression was determined by qPCR. g, BMDMs were treated with cycloheximide (CHX; 50 μg ml?1) or nonsense-mediated decay inhibitor (iNMD; 50 μM) for 6 h, and Aw112010 expression was determined by qPCR. Fold change was determined relative to vehicle samples. h, Predicted minimal free energy of RNA folding of wild-type and mutant (Mut) Aw112010 mRNA. i, j, BMDMs were subjected to electroporation with indicated plasmids. BMDMs were stimulated with LPS (10 ng ml?1) for 6 h. i, Western blot conducted for Aw112010–Flag and β-tubulin. j, Il12b mRNA was determined by qPCR. Error bars denote s.e.m. Data in b and c are from three independent experiments conducted with three biological and three technical replicates. Data in d are from three biological replicates, and fold change in expression was calculated relative to the non-treated wild-type sample. Data in f and g are from four independent experiments. Data in i and j are from three biological replicates. Fold expression in j is calculated from a single wild-type EV NT replicate for wild-type cells, and a single Aw112010Stop EV NT replicate for Aw112010Stop cells. *P < 0.05, P < 0.01, ****P < 0.001, *****P < 0.0001, unpaired two-tailed t-test.

圖4. W112010 編碼的非經(jīng)典 ORF翻譯是白介素12產(chǎn)生的必備條件 A： BMDMs 用細胞松弛素 cytochalasin D (CD) (10 μM)預處理 1h否灾， LPS (10 ng ml-1) 預處 理 6 h卖擅，或不處理。pHrodo BioParticles 被執(zhí)行 1h墨技，使用流式細胞術(shù)對細胞 CD11b and pHrodo 表達進行評估惩阶。 散點圖代表了三個獨立實驗。 B： 用傷寒鏈球菌感染 BMDMs 6 h扣汪，細胞裂解断楷， CFUs 測定。 C: BMDMs 用 LPS (100 ng ml-1)預處理 5h私痹， S. Typhimurium 感染 1h脐嫂， 測定乳酸脫氫酶(LDH) 的釋放量 D： BMDMs 被 LPS (10 ng ml-1)刺激。 Il12b 表達被 qPCR 決定紊遵。 E: 小鼠腹腔注射 6h PBS (n = 5) 或 LPS (n = 6, WT 和 Aw112010Stop) (10 mg kg-1). ELISA 檢測血清 IL-12p40 水平.

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Extended Data Fig. 3: Breakdown of different analytical approaches to predict protein coding lncRNAs.

a, RiboCode analysis of ribosome-profiling data identifies 85 ORFs within lncRNAs with protein-coding potential. b, Comparison of non-canonical ORFs identified by RibORF, RRS and translation efficiency, and RiboCode analytical strategies from BMDMs expressing lncRNA using ribosome profiling. c, Wild-type BMDMs were non-treated or stimulated with LPS (10 ng ml?1) for 6 h and ribosome profiling was conducted. Data are representative of two biological replicates. Volcano plot of LPS-induced differentially regulated genes identified by RibORF, RiboCode, RRS and translation efficiency analysis.

擴展數(shù)據(jù)圖 3| 用不同分析方法對預測的具有蛋白編碼 能力的lncRNA 進行統(tǒng)計分析账千。 A. 對核糖體譜數(shù)據(jù)的 riboCode 分析發(fā)現(xiàn) lncRNA 中有 85 個 ORF 具有編碼蛋白的潛力。 B. RibORF暗膜、 RRS 和翻譯效率鑒定的非標準 ORF 和使用核糖體譜表達 lncRNA的 BMDM的 RiboCode 分析策略比較匀奏。C. 野生型 BMDM 不經(jīng) LPS（10ng ml-1） 處理或刺激 6h， 進行核糖體譜分析学搜。數(shù)據(jù)代表了兩個生物復制體娃善。 用 RibORF、 RiboCode瑞佩、 RRS 和翻譯效率分析鑒定的 LPS 誘導不同調(diào)控的基因的火山圖聚磺。

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Extended Data Fig. 4: Overexpression of non-canonical ORFs reveals distinct subcellular localization.

a, HEK293 cells were transfected with 500 ng of Flag-tagged plasmids encoding the non-canonical ORFs GM7160 and GM9895. Cells were fixed and stained with DAPI (blue, nucleus), phalloidin (red, cytoskeletal F-actin) and anti-Flag (green, ORF of interest). Original magnifications, ×60 and ×100. Data are representative of three or more independent experiments.

擴展數(shù)據(jù)圖 4| 過表達非標準 ORF 顯示了不同的亞細胞定位。 A. 使用編碼非標 準 ORF（GM7160 和 GM9895）的 500ng 標記質(zhì)粒轉(zhuǎn)染HEK293 細胞炬丸。細胞被固定瘫寝，并被 DAP（I 藍色蜒蕾， 核）、 鬼筆環(huán)肽（紅色焕阿， 細胞骨架 F-actin） 和 anti-Flag（綠色咪啡， 目的 ORF） 著色。原始放大暮屡， 60 和100撤摸。數(shù)據(jù)代表了三個或多個獨立的實驗。

翻譯小組：

葉名琛褒纲、王俊豪准夷、黃敬潼、陳凱星外厂、鄧峻瑋冕象、黃子亮代承、倪豪辰汁蝶、鄭凌伶