The RNA structurome in the asexual blood stages of malaria pathogen <i>plasmodium falciparum</i>

Plasmodium falciparum is a deadly human pathogen responsible for the devastating disease called malaria. In this study, we measured the differential accumulation of RNA secondary structures in coding and non-coding transcripts from the asexual developmental cycle in P. falciparum in human red blood cells. Our comprehensive analysis that combined high-throughput nuclease mapping of RNA structures by duplex RNA-seq, SHAPE-directed RNA structure validation, immunoaffinity purification and characterization of antisense RNAs collectively measured differentially base-paired RNA regions throughout the parasite’s asexual RBC cycle. Our mapping data not only aligned to a diverse pool of RNAs with known structures but also enabled us to identify new structural RNA regions in the malaria genome. On average, approximately 71% of the genes with secondary structures are found to be protein coding mRNAs. The mapping pattern of these base-paired RNAs corresponded to all regions of mRNAs, including the 5ʹ UTR, CDS and 3ʹ UTR as well as the start and stop codons. Histone family genes which are known to form secondary structures in their mRNAs and transcripts from genes which are important for transcriptional and post-transcriptional control, such as the unique plant-like transcription factor family, ApiAP2, DNA-/RNA-binding protein, Alba3 and proteins important for RBC invasion and malaria cytoadherence also showed strong accumulation of duplex RNA reads in various asexual stages in P. falciparum. Intriguingly, our study determined stage-specific, dynamic relationships between mRNA structural contents and translation efficiency in P. falciparum asexual blood stages, suggesting an essential role of RNA structural changes in malaria gene expression programs. Abbreviations: CDS: Coding Sequence; DNA: Deoxyribonucleic Acid; dsRNA: double-stranded RNA; IDC: Intra-erythrocytic Developmental Cycle (IDC); m6A: N6-methyladenosine; mRNA: Messenger RNA; ncRNA: Non-coding RNA; RBC: Red Blood cells; RBP: RNA-Binding Protein; REC: Relative Expression Counts; RNA-seq: RNA-sequencing; RNA: Ribonucleic Acid; RNP: Ribonucleoprotein; RPKM: Reads Per Kilobase of transcript Per Million; rRNA: Ribosomal RNA 16. RUFs: RNAs of Unknown Function; SHAPE: Selective 2’-hydroxyl acylation analysed by primer extension; snoRNA: Small Nucleolar RNA; snRNA: Small Nuclear RNA; SRP-RNA: Signal Recognition Particle RNA; ssRNA: (Single-stranded RNA); TE: Translation Efficiency; tRNA: transfer RNA; UTR: Untranslated Region恶性疟原虫(Plasmodium falciparum)是一种致命的人类病原体，可引发毁灭性的疟疾。本研究针对寄生于人类红细胞内的恶性疟原虫，对其无性发育周期中编码转录本与非编码转录本内RNA二级结构的差异积累水平进行了定量分析。本研究结合双链RNA测序(duplex RNA-seq)开展RNA结构的高通量核酸酶图谱分析、SHAPE指导的RNA结构验证、免疫亲和纯化及反义RNA表征，全面解析了该寄生虫红细胞无性周期内所有差异碱基配对的RNA区域。本研究绘制的图谱数据不仅与一批结构已明确的多样化RNA数据集匹配，还助力我们鉴定出疟疾基因组中全新的结构RNA区域。平均而言，约71%携带二级结构的基因为蛋白质编码信使RNA(messenger RNA, mRNA)。这些碱基配对RNA的图谱覆盖了mRNA的所有区域，包括5'非翻译区(Untranslated Region, UTR)、编码序列(Coding Sequence, CDS)、3'非翻译区(UTR)以及起始密码子与终止密码子。已知其mRNA内存在二级结构的组蛋白家族基因，以及在转录与转录后调控中发挥关键作用的基因转录本——例如独特的类植物转录因子家族ApiAP2、DNA/RNA结合蛋白Alba3，以及参与红细胞入侵和疟疾细胞黏附的关键蛋白——在恶性疟原虫的不同无性阶段中均表现出双链RNA测序读段的显著富集。值得注意的是，本研究明确了恶性疟原虫无性血液阶段中mRNA结构含量与翻译效率(Translation Efficiency, TE)之间存在阶段特异性的动态关联，这提示RNA结构变化在疟疾基因表达程序中发挥着不可或缺的作用。 缩写说明： CDS：编码序列(Coding Sequence, CDS) DNA：脱氧核糖核酸(Deoxyribonucleic Acid, DNA) dsRNA：双链RNA(double-stranded RNA, dsRNA) IDC：红细胞内发育周期(Intra-erythrocytic Developmental Cycle, IDC) m6A：N6-甲基腺嘌呤(N6-methyladenosine, m6A) mRNA：信使RNA(messenger RNA, mRNA) ncRNA：非编码RNA(Non-coding RNA, ncRNA) RBC：红细胞(Red Blood cells, RBC) RBP：RNA结合蛋白(RNA-Binding Protein, RBP) REC：相对表达计数(Relative Expression Counts, REC) RNA-seq：RNA测序(RNA-sequencing, RNA-seq) RNA：核糖核酸(Ribonucleic Acid, RNA) RNP：核糖核蛋白(Ribonucleoprotein, RNP) RPKM：每百万转录本每千碱基读段数(Reads Per Kilobase of transcript Per Million, RPKM) rRNA：核糖体RNA(Ribosomal RNA, rRNA) RUFs：功能未知RNA(RNAs of Unknown Function, RUFs) SHAPE：选择性2'-羟基酰化引物延伸分析(Selective 2’-hydroxyl acylation analysed by primer extension, SHAPE) snoRNA：核仁小RNA(Small Nucleolar RNA, snoRNA) snRNA：核小RNA(Small Nuclear RNA, snRNA) SRP-RNA：信号识别颗粒RNA(Signal Recognition Particle RNA, SRP-RNA) ssRNA：单链RNA(Single-stranded RNA, ssRNA) TE：翻译效率(Translation Efficiency, TE) tRNA：转运RNA(transfer RNA, tRNA) UTR：非翻译区(Untranslated Region, UTR)