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）和相关的牲畜疾病长纳氏病是由感染引起的动型寄生虫Brucei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。 Kinetoplastids，包括T. Brucei，T。Cruzi和Leishmania spp。生物学特征，例如U插入/缺失编辑和mRNA的通用式移动。那，了解这些寄生虫的基本生物学是发现独特生物学道路的基石可能会成为急需的新药靶标的过程。一个小说的动力质体功能是几乎完全缺乏对基因表达的转录控制。相反，这些寄生虫构成产生长的多重成绩单，通过5'移序和3'裂解分解为单科子。和聚腺苷酸化（PA）。由此产生的专性转录后基因调节依赖于RNA结合蛋白质（RBP）和转录本的RBP调节是由它们与顺式作用序列的结合介导的元素通常位于mRNA 3'非翻译区域（UTRS）中。 Brucei T. Brucei的整个转录组研究在许多成绩单上揭示了宽度替代PA（APA）位点。 APA很重要，因为使用不同 PA站点可以导致包含/排除关键的3'UTR元素，以调节转录本稳定性或翻译效率。尽管APA有可能深刻影响基因表达，但关于控制动力质体中APA的机制，在我们的知识上管理一个主要差距。我们发现了这一点 T. Brucei RBP的DRBD18的敲低导致APA发生实质性变化，将DRBD18标记为第一个已知可调节APA的锥虫中的因素，并突出了其控制包含/排除的潜力独特的顺式调节3'UTR元素。 DRBD18重生与使用使用的单元格中的单细胞RNASEQ使用使用的细胞进行分析不同的生物信息学平台表明，由DRBD18介导的APA生成的独特的3'UTR变体，比整体转录本丰度更重要。这些发现是我们的基础 DRBD18的中心假设通过控制裂解/PA机械的访问来调节APA 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）确定与细胞身份改变相关的DRBD18调节的3'UTR变体；（2）定义整个转录组 DRBD18介导的APA是在下游开放式阅读框架或它是否导致新的移植和聚腺苷酸化的基因间片段，从而告知它的作用机制；（3）确定DRBD18对裂解/PA机械的全转录组效应 RNA结合。拟议的研究很重要，因为它们将在我们的知识中填补有关 mRNA处理的基本方面，RBP的APA调节以及APA在中的功能后果在这种医学和经济上重要的寄生虫中控制细胞到细胞异质性。