Translational partitioning of the SL RNA

描述(由申请人提供)：动殖目包含几种具有重要医学意义的病原体。这些生物采用了一种不寻常的基因表达机制，需要将每个核mRNA与39-NT剪接前导(SL)RNA反式剪接。这种RNA和剪接模式在人类中没有发现，是化疗的独特靶点。SL RNA共转录得到一个m7G帽，随后在前四个核苷酸的糖和/或碱基上增加了七个甲基化，构成了唯一的“帽4”。在布氏锥虫和塔氏利什曼原虫中，低甲基化底物SL RNA在反式剪接之前被细胞内运输，将潜在的竞争对手引入细胞质中，以获得翻译起始因子。这一应用的基础是对我们关于SL RNA成熟的假设的新补充，涉及到一种防止eIF4F翻译起始复合体在未剪接底物上形成的“掩膜”蛋白。对于L.tarentolae中SL RNA突变体甲基化不足和多聚体形成的丢失，一个统一的解释是，它们被翻译掩蔽因子识别，但不能被揭开。这些SL突变体无法去除翻译掩膜的原因是：1)它们的5‘端不能用于随后的2-4帽甲基化；2)一旦它们被反式剪接，它们仍然被翻译掩蔽，或者不能从细胞核输出，或者不能加载到细胞质中的多聚体上。已知的帽子结合蛋白中存在的歧视水平使我们在eIF4F因子家族中寻找这种SL面具。这些实验背后的假设是，不成熟的SL RNA必须从翻译机制中被屏蔽，这样不完整的5‘-帽结构才不会干扰蛋白质的合成。布鲁氏毛滴虫中存在6个胞质帽结合蛋白eIF4E和6个相关翻译起始因子eIF4G的潜在同源物，初步数据表明TbeIF4E-3符合SL掩码要求。本申请的具体目的是：1)验证TbeIF4E-3的帽结合特性，TbeIF4E-3是利什曼原虫主要帽0结合蛋白的同源物，和/或其他候选蛋白；2)确定TbeIF4E-3RNA底物(S)和任何相关蛋白。这些研究将有助于深入了解动质SL的生物发生以及RNA加工和翻译之间的相互作用。与公共卫生相关：动殖目包含几种具有重要医学意义的病原体。这些生物采用了一种不寻常的基因表达机制，需要将每个核mRNA与39-NT剪接前导(SL)RNA反式剪接。这种RNA和剪接模式在人类中没有发现，是化疗的独特靶点。

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.