Most "dark matter" transcripts are associated with known genes

A series of reports over the last few years have indicated that a much larger portion of the mammalian genome is transcribed than can be accounted for by currently annotated genes, but the quantity and nature of these additional transcripts remains unclear. Here, we have used data from single- and paired-end RNA-Seq and tiling arrays to assess the quantity and composition of transcripts in PolyA+ RNA from human and mouse tissues. Relative to tiling arrays, RNA-Seq identifies many fewer transcribed regions ("seqfrags") outside known exons and ncRNAs. Most nonexonic seqfrags are in introns, raising the possibility that they are fragments of pre-mRNAs. The chromosomal locations of the majority of intergenic seqfrags in RNA-Seq data are near known genes, consistent with alternative cleavage and polyadenylation site usage, promoter- and terminator-associated transcripts, or new alternative exons; indeed, reads that bridge splice sites identified 4,544 new exons, affecting 3,554 genes. Most of the remaining seqfrags correspond to either single reads that display characteristics of random sampling from a low-level background or several thousand small transcripts (median length = 111 bp) present at higher levels, which also tend to display sequence conservation and originate from regions with open chromatin. We conclude that, while there are bona fide new intergenic transcripts, their number and abundance is generally low in comparison to known exons, and the genome is not as pervasively transcribed as previously reported. Genome-wide expression profiling of human and mouse total and polyA+ RNA from four different tissue sources (brain, heart, liver, testes) was performed using Affymetrix tiling microarrays (Human & Mouse tiling 2.0R array set; 7 arrays per genome). BAR files: HBP - Human Brain PolyA+ RNA; HBT - Human Brain Total RNA; HLP - Human Liver PolyA+ RNA; HLT - Human Liver Total RNA; HHP - Human Heart PolyA+ RNA; HHT - Human Heart Total RNA; HTP - Human Testes PolyA+ RNA; HTT - Human Testes Total RNA; MBP - Mouse Brain PolyA+ RNA; MBT - Mouse Brain Total RNA; MLP - Mouse Liver PolyA+ RNA; MLT - Mouse Liver Total RNA; MHP - Mouse Heart PolyA+ RNA; MHT - Mouse Heart Total RNA; MTP - Mouse Testes PolyA+ RNA; MTT - Mouse Testes Total RNA过去数年的一系列研究报告显示，哺乳动物基因组中被转录的区域占比远高于现有注释基因所能覆盖的范围，但这些额外转录本的数量与本质仍不明确。本研究利用单端与双端RNA测序（RNA-Seq）以及镶嵌微阵列（tiling array），对人类与小鼠组织中聚腺苷酸化RNA（PolyA+ RNA）的转录本数量与组成进行了评估。相较于镶嵌微阵列，RNA-Seq在已知外显子和非编码RNA（ncRNA）之外鉴定到的转录区域（下称"seqfrags"）要少得多。绝大多数非外显子seqfrags位于内含子区域，这提示它们可能是前体信使RNA（pre-mRNA）的片段。RNA-Seq数据中大部分基因间区seqfrags的染色体位置紧邻已知基因，这与可变切割及多聚腺苷酸化位点使用、启动子与终止子相关转录本，或是新型可变外显子的情况相符；事实上，跨越剪接位点的测序读段（reads）共鉴定出4544个新型外显子，涉及3554个基因。剩余的大部分seqfrags要么对应少量呈现低水平背景随机采样特征的单条读段，要么对应数千条表达水平较高的小型转录本（中位长度为111 bp），这类转录本往往表现出序列保守性，且起源于染色质开放区域。我们的研究结论为：尽管确实存在真实可信的新型基因间区转录本，但相较于已知外显子，其数量与丰度普遍较低，且基因组并未如此前报道那般广泛转录。本研究采用Affymetrix镶嵌微阵列（Human & Mouse tiling 2.0R array set；每个基因组对应7张芯片），对来自四种不同组织来源（脑、心脏、肝脏、睾丸）的人类与小鼠总RNA及PolyA+ RNA进行了全基因组表达谱分析。BAR格式文件说明：HBP - 人类大脑聚腺苷酸化RNA；HBT - 人类大脑总RNA；HLP - 人类肝脏聚腺苷酸化RNA；HLT - 人类肝脏总RNA；HHP - 人类心脏聚腺苷酸化RNA；HHT - 人类心脏总RNA；HTP - 人类睾丸聚腺苷酸化RNA；HTT - 人类睾丸总RNA；MBP - 小鼠大脑聚腺苷酸化RNA；MBT - 小鼠大脑总RNA；MLP - 小鼠肝脏聚腺苷酸化RNA；MLT - 小鼠肝脏总RNA；MHP - 小鼠心脏聚腺苷酸化RNA；MHT - 小鼠心脏总RNA；MTP - 小鼠睾丸聚腺苷酸化RNA；MTT - 小鼠睾丸总RNA