Alternative polyadenylation regulation in Trypanosoma brucei

布氏锥虫的替代多腺苷酸化调控

基本信息

批准号：
10584834
负责人：
Laurie K. Read
金额：
$ 24.03万
依托单位：
STATE UNIVERSITY OF NEW YORK AT BUFFALO
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-11-28 至 2024-10-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10584834
关键词：
3&apos Untranslated Regions 5&apos Untranslated Regions Adverse effects Affect Africa South of the Sahara African Trypanosomiasis Binding Binding Proteins Binding Sites Bioinformatics Biological Biological Process Biology Cells Cis-Acting Sequence Complement Data Set Development Disease Economics Elements Epidemic Exclusion Exhibits Gene Expression Gene Expression Regulation Generations Genes Genetic Transcription Goals Health Heterogeneity Human Infection Insecta Knowledge Leishmania Link Livestock Mediating Medical Messenger RNA Open Reading Frames Oral Parasites Pathway interactions Pharmaceutical Preparations Pharmacotherapy Phenotype Poly A Polyadenylation Population Positioning Attribute Post-Transcriptional Regulation Proteins RNA RNA Binding RNA Interference RNA-Binding Proteins Regulation Role Site Spliced Leader Sequences Testing Trans-Splicing Transcript Transcriptional Regulation Translations Trypanosoma Trypanosoma brucei brucei Trypanosoma cruzi Variant economic impact fexinidazole insertion/deletion mutation insight knock-down nagana nanopore neglect new therapeutic target novel posttranscriptional prevent single-cell RNA sequencing transcriptome transcriptomics vector-borne

项目摘要

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.

人类非洲锥虫病 (HAT) 以及相关的牲畜疾病 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。撒哈拉以南非洲的动质体，包括 T. brucei、T. cruzi 和 Leishmania spp.，表现出许多新颖的特征。生物学特征，例如 mRNA 的 U 插入/删除编辑和通用转拼。了解这些寄生虫的基本生物学是发现独特生物途径的基石一个新的动质体特征是可能成为急需的新药物靶标的过程。相反，这些寄生虫几乎完全缺乏基因表达的转录控制。生成长的多顺反子转录本，通过 5' 反式剪接和 3' 切割分解为单顺反子和聚腺苷酸化 (PA) 所产生的专性转录后基因调控依赖于 RNA 结合。蛋白质（RBP），转录命运的 RBP 调节是通过它们与顺式作用序列的结合介导的 T. brucei 的转录组研究中通常位于 mRNA 3' 非翻译区 (UTR) 的元件。揭示许多 APA 上广泛存在的替代 PA (APA) 位点具有重要意义，因为使用了不同的 APA。 PA 位点可导致包含/排除调节转录稳定性或翻译的关键 3'UTR 元件尽管 APA 有可能深刻影响基因表达，但我们对它的效率还一无所知。动质体中控制 APA 的机制，构成了我们知识的主要空白。 T. brucei RBP DRBD18 的敲低导致 APA 发生重大变化，这标志着 DRBD18 成为第一个已知调节 APA 的锥虫因素，并强调其控制包含/排除的潜力使用 DRBD18 充满与耗尽的细胞分析不同的顺式调节 3’UTR 元件。不同的生物信息平台揭示了由 DRBD18 介导的 APA 产生的不同 3’UTR 变体，这些发现是细胞身份比整体转录丰度更重要的驱动因素。中心假设是 DRBD18 通过控制裂解/PA 机制对不同的 3’ UTR 位点并以这种方式极大地影响细胞身份。为了检验这一假设，我们将 (1) 识别。 DRBD18 调节的 3'UTR 变异与细胞身份的改变相关（2）定义全转录组； DRBD18 介导的 APA 是否会导致下游开放阅读框上的反式剪接位点发生改变或是否会导致新的反式剪接和多腺苷酸化基因间片段的产生，从而告知 (3) 确定 DRBD18 对切割/PA 机制的全转录组影响 RNA 结合的研究意义重大，因为它们将填补我们在这方面的知识空白。 mRNA 加工的基本方面、APA 的 RBP 调节以及 APA 的功能后果控制这种在医学和经济上都很重要的寄生虫的细胞间异质性。