Alternative polyadenylation regulation in Trypanosoma brucei

人类非洲锥虫病(HAT)和相关的家畜疾病Nagana，是由感染 动质体寄生虫，布氏锥虫。这些疾病会造成毁灭性的健康和经济影响。 在撒哈拉以南非洲。包括布氏锥虫、克鲁兹锥虫和利什曼原虫在内的动质体虫展示了许多新奇的东西 生物学特性，如U插入/缺失编辑和mRNAs的通用反式剪接。因此， 了解这些寄生虫的基本生物学是发现独特生物学的基石 可能成为急需的新药物靶点的过程。一个新的动态体特征是 几乎完全缺乏对基因表达的转录控制。相反，这些寄生虫构成了 产生长的多顺反子转录本，通过5‘反式剪接和3’切割分解成单顺反子 和聚腺苷酸化(PA)。由此产生的专性转录后基因调控依赖于rna结合。 蛋白质(RBP)和RBP通过与顺式作用序列的结合来调节转录物的命运 元件通常位于mRNA3‘非翻译区(UTRs)。布氏毛滴虫转录组的研究 在许多转录本上显示了广泛的替代PA(APA)位点。APA很重要，因为使用不同的 PA位点可导致包含/排除调控转录本稳定性或翻译的关键3‘UTR元件 效率。尽管APA有可能深刻地影响基因表达，但关于APA 在动质体内控制APA的机制，构成了我们知识的一个主要缺口。我们发现 布鲁氏锥虫RBP，DRBD18的敲除导致了APA的实质性变化，使DRBD18成为第一个 已知的调节APA的锥体中的因子，并突出其控制包含性/排除性的潜力 不同的顺式调控3‘非编码区元件。DRBD18充满细胞与耗尽细胞的单细胞RNAseq分析 不同的生物信息学平台显示，由DRBD18介导的APA产生的不同的3‘UTR变体， 是比整体转录物丰度更重要的细胞特性驱动因素。这些发现支持了我们的 中心假设DRBD18通过控制裂解/PA机制对不同的 3‘非编码区，并以这种方式极大地影响细胞特性。为了检验这一假设，我们将(1)确定 DRBD18调控的3‘非编码区变异与细胞特性改变相关；(2)定义转录组范围 DRBD18介导的APA是否导致下游开放阅读框上的反式剪接位点改变或 它是否会导致新的反式剪接和多腺苷化基因间片段的产生，从而告知 其作用机制；(3)确定DRBD18在裂解/PA机制上的全转录效应 RNA结合。拟议的研究具有重要意义，因为它们将填补我们在以下方面的主要知识空白 MRNA加工的基本方面，RBP对APA的调节，以及APA在体内的功能后果 控制这种在医学和经济上具有重要意义的寄生虫的细胞间异质性。

Human African Trypanosomiasis (HAT), and the related livestock disease, nagana, are caused by infection of the kinetoplastid parasite, Trypanosoma brucei. These maladies cause devastating health and economic impacts in sub-Saharan Africa. Kinetoplastids, including T. brucei, T. cruzi and Leishmania spp., exhibit many novel biological features, such as U insertion/deletion editing and universal trans-splicing of mRNAs. Thus, understanding the basic biology of these parasites is a cornerstone on the path to discovery of unique biological processes that could potentially serve as much-needed new drug targets. One novel kinetoplastid feature is the almost complete lack of transcriptional control of gene expression. Instead, these parasites constitutively generate long polycistronic transcripts that are resolved into monocistrons by 5’ trans-splicing and 3’ cleavage and polyadenylation (PA). The resulting obligate posttranscriptional gene regulation relies on RNA binding proteins (RBPs), and RBP regulation of transcript fate is mediated by their binding to cis-acting sequence elements typically located in mRNA 3’ untranslated regions (UTRs). Transcriptome-wide studies in T. brucei revealed widespread alternative PA (APA) sites on many transcripts. APA is significant because use of different PA sites can lead to inclusion/exclusion of critical 3’UTR elements that regulate transcript stability or translation efficiency. Despite the potential for APA to profoundly affect gene expression, nothing is known regarding the mechanisms that control APA in kinetoplastids, constituting a major gap in our knowledge. We discovered that knockdown of the T. brucei RBP, DRBD18, leads to substantial changes in APA, marking DRBD18 as the first factor in trypanosomes known to modulate APA, and highlighting its potential to control inclusion/exclusion of distinct cis-regulatory 3’UTR elements. Single cell RNAseq in DRBD18-replete vs. -depleted cells analyzed using different bioinformatic platforms revealed that distinct 3’ UTR variants, generated by DRBD18-mediated APA, are more important drivers of cell identity than is overall transcript abundance. These findings underpin our central hypothesis that DRBD18 regulates APA by controlling access of the cleavage/PA machinery to distinct 3’ UTR sites and, in this manner, dramatically influences cell identity. To test this hypothesis, we will (1) Identify DRBD18-regulated 3’UTR variants associated with alterations in cell identity; (2) Define transcriptome-wide whether DRBD18-mediated APA results in altered trans-splice sites on downstream open reading frames or whether it leads to generation of new trans-spliced and polyadenylated intergenic fragments, thereby informing its mechanism of action; (3) Determine the transcriptome-wide effect of DRBD18 on cleavage/PA machinery RNA binding. The proposed studies are significant because they will fill major gaps in our knowledge regarding fundamental aspects of mRNA processing, RBP regulation of APA, and the functional consequences of APA in controlling cell-to-cell heterogeneity in this medically and economically important parasite.