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

题目：非典型开放阅读框的翻译控制黏膜免疫

作者以及单位：

Ruaidhrí Jackson, Lina Kroehling, Alexandra Khitun, [...], 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：

Nature volume 564, pages434–438 (2018)

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 potential^1,2. 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’. Although the idea that such non-canonical ORFs can encode functional proteins is controversial^3,4, 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 mis annotation 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.

哺乳动物的蛋白质编码基因组注释是不完整的。开放阅读框（ORF）的任意大小限制和对作为翻译唯一起始子的甲硫氨酸密码子的绝对需求，使得对潜在的重要非典型的蛋白质编码转录物的识别受到限制。在这里，我们对于对细菌感染产生免疫应答的巨噬细胞使用了无偏倚的转录学方法，说明了与大量RNA有关的核糖体之前被注释为“非蛋白编码”。尽管非典型的ORF能够编码功能蛋白这种观点是有争议的，但是我们鉴定了一系列短的、非ATG起始的ORF，它们能够产生稳定的、在空间结构上不同的蛋白质。值得注意的是，对于在细菌侵染期间和结肠炎期间的黏膜免疫系统，我们展示了“隐藏”在长非编码RNA AW112010 中的新ORF的翻译对免疫系统的环境和谐是很重要的。这项工作扩大了我们对蛋白质编码基因组的理解，证明了非典型ORF的蛋白质产物对于活体内的免疫应答非常关键。因此，我们提出将非经典的含ORF基因错误注释成非编码RNA可能会掩盖许多以前未发现的蛋白质编码基因在免疫和疾病中的重要作用。

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) 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.**P < 0.01, ***P < 0.001, ****P < 0.0001, unpaired two-tailed t-test.

图1：细菌感染驱动了广泛的与核糖体相关的非编码RNA
a-f, RiboTagLysM小鼠的对照组（NT）和用LPS处理的小鼠中的巨噬细胞。RNA-seq数据显示两种独立的生物学重复的组合。
a, 在LPS不同时间处理后的差异表达核糖体相关RNA，红色表示下调，蓝色表示上调，圆图（左）的数字表示所处的染色体编号。
b, 从RiboTag RNA-seq中注释的蛋白编码基因百分比。
c, b图中的蛋白非编码基因进一步细分。
d, 基于核糖体相关性区分可检测到的巨噬细胞lncRNA，红色表示较相关，蓝色是较不相关。
e,f, 火山图和热图，分析在LPS处理的巨噬细胞中与核糖体相关的lncRNA
g, qPCR分析4种样品（对照、6h/24h LPS处理、伤寒沙门菌感染）的核糖体相关RNA
h, qPCR分析24小时后的伤寒沙门菌感染结肠组织中的核糖体相关RNA。