Alternative polyadenylation regulation in Trypanosoma brucei

人类非洲锥虫病（HAT）和相关的牲畜疾病，长角线虫病，是由感染 动质体寄生虫布氏锥虫这些疾病对健康和经济造成毁灭性的影响 于撒哈拉以南非洲的动质体包括T. brucei、T.克氏杆菌和利什曼原虫属，展出许多新奇的 生物学特征，如U插入/缺失编辑和mRNA的通用反式剪接。因此，在本发明中， 了解这些寄生虫的基本生物学是发现独特生物学的基石。 这些过程可能成为急需的新药物靶点。一个新的动质体特征是 几乎完全缺乏基因表达的转录控制。相反，这些寄生虫 产生长的多顺反子转录物，其通过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（RBP），RBP调节转录命运是通过它们与顺式作用序列的结合介导的 通常位于mRNA 3'非翻译区（UTR）的元件。T.布氏 揭示了广泛的替代PA（阿帕）网站上的许多成绩单。阿帕是重要的，因为使用不同的 PA位点可导致包含/排除调节转录物稳定性或翻译的关键3 'UTR元件 效率尽管阿帕有可能深刻地影响基因表达，但关于APA的作用还不清楚。 在动质体中控制阿帕的机制，构成了我们知识的一个主要空白。我们发现 T. Brucei RBP，DRBD 18，导致阿帕的实质性变化，标志着DRBD 18是第一个 已知调节阿帕的锥虫因子，并强调其控制包括/排除 不同的顺式调节3 'UTR元件。使用DRBD 18分析的DRBD 18-充满细胞与-耗尽细胞中的单细胞RNAseq 不同的生物信息学平台揭示了不同的3' UTR变体，由DRBD 18介导的阿帕产生， 是比整体转录丰度更重要的细胞身份驱动因素。这些发现支持了我们的 DRBD 18通过控制切割/PA机制进入不同的细胞来调节阿帕的中心假设， 3' UTR位点，并且以这种方式显著影响细胞身份。为了验证这一假设，我们将（1）确定 DRBD 18调节的与细胞身份改变相关的3 'UTR变体;（2）定义转录组范围 DRBD 18介导的阿帕是否导致下游开放阅读框上的反式剪接位点改变， 它是否导致产生新的反式剪接和多聚腺苷酸化的基因间片段，从而告知 其作用机制;（3）确定DRBD 18对切割/PA机制的转录组范围的影响 RNA结合。拟议的研究是重要的，因为它们将填补我们知识的主要空白， mRNA加工的基本方面，阿帕的RBP调节，以及阿帕在 控制这种医学和经济上重要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.