Translational partitioning of the SL RNA

描述（由申请方提供）：动质体目含有几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的反式剪接。这种RNA和剪接模式在人类中没有发现，代表了化疗的独特靶点。SL RNA共转录地接收m7 G帽，随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“帽4”。在布氏锥虫和塔氏利什曼原虫中，未甲基化的底物SL RNA在反式剪接之前在细胞内被运输，从而将潜在的竞争者引入到细胞溶质中用于翻译起始因子。本申请的基础是对我们关于SL RNA成熟的假设的新补充，该假设涉及防止eIF 4F在未剪接底物上形成起始复合物的“掩蔽”蛋白。一个统一的解释甲基化不足和损失的多核糖体形成的SL RNA突变体在L。通常表现出Cap 1表型的Tarentolae的特征在于，它们被翻译掩蔽因子识别，但不能被揭开。这些SL突变体不能去除翻译掩蔽是1）它们的5'末端对于随后的帽2-4甲基化是不可接近的，和2）一旦它们被反式剪接，它们仍然被限制性地掩蔽并且不从细胞核输出或不加载到胞质溶胶中的多核糖体上。在已知的帽结合蛋白中存在的歧视水平使我们在eIF 4F因子家族中寻找这种SL掩模。这些实验背后的假设是，未成熟的SL RNA必须从翻译机制中被掩蔽，以便不完整的5 '帽结构不会干扰蛋白质合成。在T. brucei提供了候选物用于检查;初步数据表明TbeIF 4 E-3符合SL掩模要求。本申请的具体目的是：1）验证TbeIF 4 E-3（主要利什曼原虫帽结合蛋白的同源物）和/或其他候选物的帽结合性质;和2）确定TbeIF 4 E-3 RNA底物和任何相关蛋白。这些研究将导致对动质体SL生物发生以及RNA加工和翻译之间的相互作用的透彻理解。公共卫生相关性：动质体目含有几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的反式剪接。这种RNA和剪接模式在人类中没有发现，代表了化疗的独特靶点。

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.