Translational partitioning of the SL RNA

SL RNA 的翻译分配

• 批准号: 7472824
• 负责人: DAVID A CAMPBELL
• 金额: $ 23.1万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7472824
• 关键词: Binding; Binding Proteins; Biogenesis; Biological Assay; Cell Nucleus; Cells; Cloning; Complementary DNA; Complex; Cytosol; Data; Discrimination; EIF4EL3 gene; Eukaryota; Exhibits; Family; Gene Expression; Homologous Gene; Human; Kinetoplastida; Lead; Leishmania; Leishmania major; Masks; Mass Spectrum Analysis; Medical; Messenger RNA; Methylation; Mutate; Nature; Nuclear; Nucleotides; Organism; Pathway interactions; Peptide Initiation Factors; Phenotype; Play; Polyribosomes; Population; Primer Extension; Process; Property; Protein Biosynthesis; Proteins; RNA; RNA Cap-Binding Proteins; RNA Caps; RNA Processing; RNA Splicing; RNA-Binding Proteins; Small Nuclear RNA; Specificity; Spliced Leader RNA; Spliced Leader Sequences; Structure; System; Touch sensation; Trans-Splicing; Translation Initiation; Translations; Trypanosoma brucei brucei; Work; base; chemotherapy; in vivo; insight; interest; mRNA Stability; mutant; novel; pathogen; preference; prevent; public health relevance; research study; sugar; trafficking

DESCRIPTION (provided by applicant): The Order Kinetoplastida contains several pathogens of medical importance. These organisms employ an unusual mechanism of gene expression that requires the trans-splicing of every nuclear mRNA with the 39-nt spliced leader (SL) RNA. This RNA and mode of splicing is not found in humans and represents a unique target for chemotherapy. The SL RNA receives a m7G cap co-transcriptionally, followed by the addition of seven methylations on the sugar and/or base moieties of the first four nucleotides, constituting the unique 'cap 4'. Undermethylated substrate SL RNA is trafficked intracellularly prior to trans-splicing in Trypanosoma brucei and Leishmania tarentolae, introducing potential competitors into the cytosol for translation initiation factors. The basis of this application is a new addition to our hypothesis on the maturation of SL RNA involving a 'mask' protein that prevents eIF4F translation-initiation complex formation on the unspliced substrate. A unifying explanation for the undermethylation and loss of polysome formation for SL RNA mutants in L. tarentolae, which generally exhibit the cap 1 phenotype, is that they are recognized by the translational masking factor, but cannot be unmasked. The inability of these SL mutants to remove the translational mask is that 1) their 5' ends are not accessable for subsequent cap 2-4 methylations and 2) once they are trans-spliced, they are still translationally masked and either not exported from the nucleus or not loaded onto polysomes in the cytosol. The level of discrimination that exists among known cap-binding proteins has led us to search for this SL mask in the families of eIF4F factors. The hypothesis underlying these experiments is that immature SL RNA must be masked from the translation machinery so that the incomplete 5'-cap structure does not interfere with protein synthesis. The presence of six potential homologs of the cytosolic cap-binding protein eIF4E and six of associated translation initiation factor eIF4G in T. brucei provided candidates for examination; preliminary data indicates that TbeIF4E-3 fits the SL mask requirements. The specific aims of this application are: 1) To validate the cap-binding properties of TbeIF4E-3, a homolog of the Leishmania major cap 0-binding protein, and/or other candidates; and 2) To determine the TbeIF4E-3 RNA substrate(s) and any associated proteins. These studies will lead to a thorough understanding of kinetoplastid SL biogenesis and the interplay between RNA processing and translation. PUBLIC HEALTH RELEVANCE: The Order Kinetoplastida contains several pathogens of medical importance. These organisms employ an unusual mechanism of gene expression that requires the transsplicing of every nuclear mRNA with the 39-nt spliced leader (SL) RNA. This RNA and mode of splicing is not found in humans and represents a unique target for chemotherapy.

描述（由申请人提供）：动质体目含有多种具有医学重要性的病原体。这些生物体采用一种不寻常的基因表达机制，需要每个核 mRNA 与 39 nt 剪接前导 (SL) RNA 进行反式拼接。这种 RNA 和剪接模式在人类中未发现，是化疗的独特靶点。 SL RNA 共转录时接收 m7G cap，然后在前四个核苷酸的糖和/或碱基部分上添加七个甲基化，构成独特的“cap 4”。在布氏锥虫和塔伦托利什曼原虫中，甲基化底物 SL RNA 在转剪之前在细胞内运输，将潜在的竞争者引入细胞质中以获得翻译起始因子。该应用的基础是我们关于 SL RNA 成熟的假设的新补充，该假设涉及“掩码”蛋白，该蛋白可阻止 eIF4F 翻译起始复合物在未剪接的底物上形成。 L. tarentolae 中 SL RNA 突变体（通常表现出 cap 1 表型）的甲基化不足和多核糖体形成丧失的统一解释是，它们被翻译掩蔽因子识别，但无法被揭露。这些 SL 突变体无法去除翻译掩码，因为 1) 它们的 5' 末端无法进行后续的帽 2-4 甲基化，2) 一旦它们被转剪，它们仍然被翻译掩码，并且要么不从细胞核输出，要么不加载到胞质溶胶中的多聚核糖体上。已知帽结合蛋白之间存在的区分程度促使我们在 eIF4F 因子家族中寻找这种 SL 掩码。这些实验的假设是，未成熟的 SL RNA 必须被翻译机制屏蔽，以便不完整的 5'-帽结构不会干扰蛋白质合成。 T. brucei 中胞浆帽结合蛋白 eIF4E 的六种潜在同源物和六种相关翻译起始因子 eIF4G 的存在为检查提供了候选物；初步数据表明TbeIF4E-3符合SL掩模要求。本申请的具体目的是： 1) 验证 TbeIF4E-3（利什曼原虫大帽 0 结合蛋白的同源物）和/或其他候选物的帽结合特性； 2) 确定 TbeIF4E-3 RNA 底物和任何相关蛋白。这些研究将带来对动质体 SL 生物发生以及 RNA 加工和翻译之间相互作用的全面了解。公共卫生相关性：动质体含有多种具有医学重要性的病原体。这些生物体采用一种不寻常的基因表达机制，需要每个核 mRNA 与 39 nt 剪接前导 (SL) RNA 进行转拼。这种 RNA 和剪接模式在人类中未发现，是化疗的独特靶点。