The translation of non-canonical open reading frames controls mucosal immunity
题目：非经典开放阅读框的翻译调控黏膜免疫
作者及单位：
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
发表期刊及时间：
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’==（注意此处是与核糖体结合的RNA注释为非编码，而不是核糖体注释为非编码）. 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， 哺乳动物编码蛋白质的基因组注释并不完善。 武断的限制 开放阅读框(ORF)的大小并把蛋氨酸密码子作为翻译唯一启动子的绝 对要求， 这二者都限制了重要的非标准蛋白编码转录本的鉴定。 本文 选取了对细菌侵染应答后的巨噬细胞， 使用了无偏转录组学的方法， 研究发现， ==以往被认为‘不编码蛋白质’ 的RNA其实大量的与核糖体相结合==。尽管关于非标准 ORFs 能编码功能性蛋白的观点还饱受争议， 但是我们鉴定到了很多短的、 非 ATG 起始的 ORFs， 它们能产生稳固且空间上差异的蛋白质。 尤其是， 我们发现存在一类新的 ORF， 它' 隐藏'于长非编码 RNA Aw112010 内， 这类 ORF 的翻译对于细菌感染 、和结肠炎期间协调粘膜免疫来说是至关重要的。 此项工作扩展了我们对编码蛋白的基因组的认知， 还论证了非标准 ORFs 产生的蛋白质产 物确实对机体免疫反应至关重要。 本文因此提出： 将含有非标准 ORF 的基因错误的注释为非编码 RNAs 后， 许多以前未发现能编码蛋白质 的基因在免疫和疾病中的重要作用会被掩盖。
图表选析：
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)== （感染复数）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 标准误）P < 0.01, ****P < 0.001, *****P < 0.0001, unpaired two-tailed t-test.
图 1：细菌感染驱动大量核糖体与非编码 RNA结合。 a-f， 用 LPS 刺激或未处理的来自 RiboTagLysM小鼠的骨髓巨噬细胞（BMDMS）。 RNA 通过 RNA-seq获得。数据用两个独立生物重复结合的形式进行展示。 a.环形图显示了 6 个小时 和 24 个小时 LPS 刺激后差异表达的核糖体结合转录本。红色代表上调，蓝色代表下调。 从外到内每个轨道分别代表：染色体位置；12810 个已知的编码蛋白质转录组；1176 个 LncRNAs；1107 个假基因；413 个其他非编码 RNA。 B. 按照来自 RiboTag RNA-seq 注释 的编码蛋白质基因（FPKM≥1）， 百分比饼图被分成两部分。 C.图 b 中非编码蛋白质基因被 进一步分类。 D. BMDM 中识别出的与核糖体结合的 lncRNA分层。在 RiboTag RNA-seq 中 FPKM 大于等于 1 的核糖体结合的lncRNA在红色区域中标出。蓝色部分代表未在 RiboTag RNA-seq 中发现，但在传统 RNA-seq中 FPKM 大于等于 0.01 的 lncRNA。 e,f, 在 BMDM 中使用 LPS 刺激后火山图（e）和热力图（f）分析。 G. 未处理 BMDM，用 LPS （10 ng ml−1）刺激的 BMDM 和用 1 感染复数的 S. Typhimurium 感染六个小时后 的 BMDMs 进行 q-PCR 分析。 h, 用 2 × 108CFU 的 S. Typhimurium饲养 RiboTagLysM 小鼠。 24 小时后，提取结肠样本并裂解。分离出巨噬细胞线粒体结合 RNA，并使用 qPCR 进行分 析。数据以七个生物学重复的形式展示。 数据的平均数、标准误和非配对双尾t 检验结果 **P < 0.01, ***P < 0.001, ****P < 0.0001）在图中被展示出来。
==multiplicity of infection (MOI)== ：感染复数，其含义是感染时噬菌体与细菌的数量比值，也就是平均每个细菌感染噬菌体的数量。
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==（核糖体谱） 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 核糖体释放分值) analysis was conducted on transcripts identified by RiboProfiling. Purple broken lines represent the 95th percentile of the 3ʹ 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，野生型BMDM未经处理或用LPS（10ng ml-1）刺激6小时，并进行核糖体谱分析。数据代表两个生物学重复。 a，蛋白质编码基因和lncRNA的PME值。 PME截止值≥0.6％表示被认为是蛋白质编码的转录物。 b，对RiboProfiling鉴定的转录本进行翻译效率（TE）和RRS分析。紫色虚线代表已知蛋白质编码基因的3'UTR的第95百分位数，并区分编码和非编码转录物。 从RibORF和/或RiboCode鉴定的lncRNA中利用RRS和翻译效率值进行c，起始密码子使用和d，ORF大小的分类。 e，排在前面的差异调节的LPS刺激的lncRNA ORF的热图。 f，用空载体（EV）或Aw112010Flag ORF转染的HEK293细胞。用DAPI，鬼笔环肽和抗Flag对细胞染色。比例尺，9微米;原始放大倍率，×60和×100。 g，野生型（WT）和Aw112010HA BMDM未经处理或用LPS（10ng ml-1）刺激，产生蛋白质裂解物并对血细胞凝集素和β-微管蛋白进行蛋白质印迹分析。数据代表三个生物学重复。 h，产生Aw112010HA BMDM，用LPS刺激6小时并进行血细胞凝集素免疫沉淀。将纯化的裂解物进行质谱分析。 MS1提取的离子色谱图中的前体离子峰对应于掺入合成同位素标记的肽标准品（上图），并且在同一样品中共同洗脱与内源Aw112010肽（下图）一致的峰。在肽序列的上方和下方指示鉴定的片段离子（b和y离子，红色）。数据代表两个生物学重复。
==ribosome profiling==（核糖体谱）：所有与核糖体结合的RNA序列测定
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).
图3. AW112010 编码的非经典 ORF翻译对于黏膜免疫至关重要 a-e 10 个野生型小鼠和 11 个 Aw112010 终止的小鼠在伤寒链球菌 （1 千菌落形成单位） 感染前 24 小时均口服链霉素(20 毫克) a 感染后测量的体重减少量 b.感染后 24 小时在野生型和 Aw112010 终止小鼠粪便中存在的鼠 伤寒沙门氏菌 CFU 计数。 c.感染后 96h 野生型和 Aw112010终止小鼠盲肠中鼠伤寒沙门氏菌 CFU 计数。 d、 e、 野生型和 Aw112010 终止小鼠感染 96h 后， 小鼠肝脏(d)和 脾脏(e)中鼠伤寒沙门氏菌 CFU 计数。 f.被 100CFUs 的伤寒沙门氏菌感染 72 小时后，野生型和 Aw112010 终止小鼠的脾脏中巨噬细胞（F4/80， 绿色） 、 B 细胞（B220， 紫色） 和沙门氏菌（抗沙门氏菌， 红色） 的共聚焦免疫染色。 图像代表三个 独立的生物复制品。 比例尺， 9μm； 原始放大倍数， ×60。 g. 在口灌胃感染 100CFU 后的野生型(n=10)和 Aw112010 终止 (n=10)小鼠的存活曲线分析 h、i、在饮用水中给予 2.5%葡聚糖硫酸酯钠 5 天的野生型 Wild-typ 和 Aw112010 终止小鼠。 h、 在 12 天内测定野生型(n=11)和 Aw112010Stop(n=12)小鼠的体 重损失。 i、 测定野生型(n=10)和 Aw112010 终止(n=12)小鼠的结肠长度。 b –e 中的水平条表示平均细菌数。 h 和 i 中的错误栏表示复制的 s.e.m.P<0.05 * 显著、 P<0.01 很显著、 P<0.001*非常显著、 P<0.0001 ****极其显著、 非参数 Mann–Whitney 检验(b–e)、 log-rank 对数秩检验(g)和未配对的双尾 t 检验(a,h,i)。
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.
图4. W112010 编码的非经典 ORF翻译是白介素12产生的必备条件 A： BMDMs 用细胞松弛素 cytochalasin D (CD) (10 μM)预处理 1h， LPS (10 ng ml-1) 预处 理 6 h，或不处理。pHrodo BioParticles 被执行 1h，使用流式细胞术对细胞 CD11b and pHrodo 表达进行评估。 散点图代表了三个独立实验。 B： 用伤寒链球菌感染 BMDMs 6 h，细胞裂解， CFUs 测定。 C: BMDMs 用 LPS (100 ng ml-1)预处理 5h， S. Typhimurium 感染 1h， 测定乳酸脱氢酶(LDH) 的释放量 D： BMDMs 被 LPS (10 ng ml-1)刺激。 Il12b 表达被 qPCR 决定。 E: 小鼠腹腔注射 6h PBS (n = 5) 或 LPS (n = 6, WT 和 Aw112010Stop) (10 mg kg-1). ELISA 检测血清 IL-12p40 水平.
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.
扩展数据图 3| 用不同分析方法对预测的具有蛋白编码 能力的lncRNA 进行统计分析。 A. 对核糖体谱数据的 riboCode 分析发现 lncRNA 中有 85 个 ORF 具有编码蛋白的潜力。 B. RibORF、 RRS 和翻译效率鉴定的非标准 ORF 和使用核糖体谱表达 lncRNA的 BMDM的 RiboCode 分析策略比较。C. 野生型 BMDM 不经 LPS（10ng ml-1） 处理或刺激 6h， 进行核糖体谱分析。数据代表了两个生物复制体。 用 RibORF、 RiboCode、 RRS 和翻译效率分析鉴定的 LPS 诱导不同调控的基因的火山图。
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.
扩展数据图 4| 过表达非标准 ORF 显示了不同的亚细胞定位。 A. 使用编码非标 准 ORF（GM7160 和 GM9895）的 500ng 标记质粒转染HEK293 细胞。细胞被固定，并被 DAP（I 蓝色， 核）、 鬼笔环肽（红色， 细胞骨架 F-actin） 和 anti-Flag（绿色， 目的 ORF） 着色。原始放大， 60 和100。数据代表了三个或多个独立的实验。
翻译小组：
叶名琛、王俊豪、黄敬潼、陈凯星、邓峻玮、黄子亮、倪豪辰、郑凌伶
