Characterization of the transcriptome of Haloferax volcanii, grown under four different conditions, with mixed RNA-Seq
Characterization of the transcriptome of Haloferax volcanii, grown under four different conditions, with mixed RNA-Seq
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DOI：
10.1371/journal.pone.0215986
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发表时间：
2019-04-30
期刊：
影响因子：
3.7
通讯作者：
Soppa, Joerg
中科院分区：
文献类型：
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作者：
Laass, Sebastian;Monzon, Vivian A.;Soppa, Joerg
海洋古生菌是一种广为人知的模式物种。小规模的RNA组学和生物信息学预测被用来识别小的非编码RNA(SRNAs)，缺失突变揭示了sRNAs具有重要的调节功能。最近的一项dRNA-Seq研究被用来描述初级转录组。出人意料的是，研究发现，在最佳条件下，火山杆菌含有的非编码sRNA多于编码蛋白质的mRNAs。然而，dRNA-Seq方法不包含任何长度信息。因此，使用混合RNA-Seq方法来确定转录本的长度，并鉴定在最佳条件下不存在的其他转录本。总共获得了5000万个长度为150个核苷酸的成对末端读数。共检测到1861个蛋白质编码RNA(CdRNAs)，编码3092个蛋白质。与之前的dRNA-Seq研究相比，这几乎是cdRNAs覆盖率的两倍。大约2/3的cdRNAs是单顺反子的，1/3的cdRNAs包含一个以上的基因。此外，还鉴定了1635个非编码sRNA。非编码RNA中顺式反义RNA(AsRNAs)比例最高。对长度分布的分析表明，sRNA的平均长度约为150个核苷酸。根据RNA-Seq和dRNA-Seq的结果，通过Northern印迹分析选择基因来例证火山杆菌转录组的特征，例如：1)基因簇的转录模式可以是简单的，但也是非常复杂的，2)在所分析的四个条件下，许多转录本的表达水平不同，3)一些基因转录成不同长度的RNA异构体，可以进行差异调控，4)存在非常长的5‘-UTRs和非常长的3’-UTRs的转录本，以及5)大约30%的cdRNAs具有重叠的3‘-末端，这表明，与asRNAs一起，H.Volcanii充分利用了正反义间的相互作用。综上所述，这项RNA-Seq研究，以及之前的dRNA-Seq研究，使人们能够对H.Volcanii转录组进行前所未有的观察。
Haloferax volcanii is a well-established model species for haloarchaea. Small scale RNomics and bioinformatics predictions were used to identify small non-coding RNAs (sRNAs), and deletion mutants revealed that sRNAs have important regulatory functions. A recent dRNA-Seq study was used to characterize the primary transcriptome. Unexpectedly, it was revealed that, under optimal conditions, H. volcanii contains more non-coding sRNAs than protein-encoding mRNAs. However, the dRNA-Seq approach did not contain any length information. Therefore, a mixed RNA-Seq approach was used to determine transcript length and to identify additional transcripts, which are not present under optimal conditions. In total, 50 million paired end reads of 150 nt length were obtained. 1861 protein-coding RNAs (cdRNAs) were detected, which encoded 3092 proteins. This nearly doubled the coverage of cdRNAs, compared to the previous dRNA-Seq study. About 2/3 of the cdRNAs were monocistronic, and 1/3 covered more than one gene. In addition, 1635 non-coding sRNAs were identified. The highest fraction of non-coding RNAs were cis antisense RNAs (asRNAs). Analysis of the length distribution revealed that sRNAs have a median length of about 150 nt. Based on the RNA-Seq and dRNA-Seq results, genes were chosen to exemplify characteristics of the H. volcanii transcriptome by Northern blot analyses, e.g. 1) the transcript patterns of gene clusters can be straightforward, but also very complex, 2) many transcripts differ in expression level under the four analyzed conditions, 3) some genes are transcribed into RNA isoforms of different length, which can be differentially regulated, 4) transcripts with very long 5'-UTRs and with very long 3'-UTRs exist, and 5) about 30% of all cdRNAs have overlapping 3'-ends, which indicates, together with the asRNAs, that H. volcanii makes ample use of sense-antisense interactions. Taken together, this RNA-Seq study, together with a previous dRNA-Seq study, enabled an unprecedented view on the H. volcanii transcriptome.