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Alternative mechanisms of different stages in eukaryotic translation

真核翻译不同阶段的替代机制

基本信息

批准号：
10408702
负责人：
CHRISTOPHER Ulrich Tristram HELLEN
金额：
$ 32.3万
依托单位：
SUNY DOWNSTATE MEDICAL CENTER
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-05-15 至 2024-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10408702
关键词：
5&apos Untranslated Regions Address Anticodon Binding Biochemical Biological Biophysics Cell physiology Cellular biology Codon Nucleotides Complex Cryoelectron Microscopy Data Development Dipeptides Disease Dissociation Drug Targeting Electron Transport Complex III Elements Endoribonucleases Ensure Eukaryotic Initiation Factors GTP Binding Gene Expression Gene Expression Regulation Genes Guanosine Triphosphate Guanosine Triphosphate Phosphohydrolases Homeostasis Hydrolysis In Vitro Individual Infection Inherited Initiator Codon Insecta Interferons Internal Ribosome Entry Site Intervention Leucine-Specific tRNA Malignant Neoplasms Mammalian Cell Mammals Mediating Messenger RNA Missense Mutation Molecular Molecular Analysis N-terminal Natural Immunity Neurodegenerative Disorders Open Reading Frames Pathogenesis Pathologic Pathway interactions Physiological Play Process Protein Biosynthesis Proteins RNA Regulation Ribosomal RNA Ribosomes Role Scanning Site Stress Structural Protein Structure Structure-Activity Relationship System Thrombocytopenia Transfer RNA Translation Initiation Translations Triplet Multiple Birth Untranslated Regions Viral biophysical techniques clinically relevant cricket paralysis virus eukaryotic initiation factor-5B improved influenza infection insight novel reconstitution recruit response tool translatome viral RNA

项目摘要

Initiation is the most complex, tightly regulated stage of eukaryotic protein synthesis. The process begins with formation of the 48S initiation complex (48S IC) at the initiation codon of mRNA. First, the 43S preinitiation complex (43S PIC) comprising the 40S ribosomal subunit, the eIF2•GTP•Met-tRNAMeti ternary complex and eukaryotic initiation factors eIF3, eIF1 and eIF1A binds to the cap-proximal region of mRNA in a step that is mediated by eIFs 4A, 4B and 4F, which cooperatively unwind the cap-proximal region, allowing for 43S PIC association. The 43S PIC then scans downstream to the initiation codon where it forms the 48S IC with the established codon-anticodon interaction. Scanning on structured mRNAs additionally requires the DExH-box protein DHX29 that binds directly to 40S subunits. eIFs 1 and 1A play key roles in ensuring the fidelity of initiation codon selection. Initiation codon recognition triggers dissociation of eIF1, eIF5-induced hydrolysis of eIF2-bound GTP and release of Pi. Subsequent joining of a 60S subunit is promoted by the translational GTPase eIF5B. Initiation on some viral mRNAs is mediated by an internal ribosome entry site (IRES). IRESs are highly structured RNA elements that promote 5’-end independent recruitment of the 40S subunit via non-canonical interactions with the 40S subunits and/or eIFs. Dysregulation of translation initiation is frequently observed in devastating diseases and is therefore becoming a focus for chemo-therapeutic intervention. Although the factors required for initiation have been identified, and their principal roles determined, important details concerning its molecular mechanism, regulation and alternative modes remain unknown. Characterization of these details is therefore a priority. We have reconstituted the entire translation cycle in vitro, which gives us the unique opportunity to address critical gaps in understanding of the mechanisms of mammalian initiation and the regulation of translation using biochemical and complementary biophysical and cell biology approaches. Aim 1 will concern characterization of the mechanisms by which DHX29 promotes scanning, eIF5B stabilizes Met-tRNAiMet on the 40S subunit and both factors influence initiation codon selection. In Aim 2, we will focus on investigating the mechanisms of physiologically important initiation with Leu-tRNALeu at CUG codons, and on non-AUG triplets during repeat-associated non-AUG (RAN) translation, which occurs on expansion repeats in mRNAs transcribed from genes that are responsible for severe neurodegenerative diseases. Aim 3 is devoted to elucidation of the molecular mechanism of initiation on the IRES located in the 5'UTR of Cricket paralysis virus RNA, which has a unique structure and that our preliminary data suggest can use novel mechanisms for initiation. Aim 4 concerns the cellular function and mechanism of action of Schlafen14, a novel endoribonuclease that binds 80S ribosomes and cleaves rRNA and ribosome-bound mRNAs. It is thus implicated in translational control, and likely influences this process in a previously undescribed manner.

起始是真核生物蛋白质合成中最复杂、最受严格调控的阶段。该过程开始于在mRNA的起始密码子处形成48 S起始复合物（48 S IC）。第一，43 S预启动复合物（43 S PIC），包括40 S核糖体亚基、eIF 2·GTP·Met-tRNAMeti三元复合物和真核生物起始因子eIF 3、eIF 1和eIF 1A在一个步骤中与mRNA的近帽区结合，由eIFs 4A、4 B和4F介导，其协同展开帽近端区域，允许43 S PIC 协会然后，43 S PIC向下游扫描至起始密码子，在那里它与起始密码子形成48 S IC。密码子-反密码子相互作用。结构化mRNA的扫描还需要DExH盒直接结合40 S亚基的蛋白DHX 29。eIFs 1和1A在确保初始化的精确性方面发挥着关键作用密码子选择起始密码子识别触发eIF 1的解离，eIF 5诱导的eIF 2结合的 GTP和Pi的释放。随后60 S亚基的连接由翻译的GTIF 5 B促进。一些病毒mRNA的起始由内部核糖体进入位点（IRES）介导。IRES是高度结构化的通过非典型相互作用促进40 S亚基的5 '端独立募集的RNA元件与40 S亚基和/或eIFs连接。翻译起始的失调经常被观察到，疾病，因此成为化学治疗干预的焦点。虽然所需的因素已经确定了起始，并确定了它们的主要作用，机制、监管和替代模式仍然未知。因此，这些细节的表征是要务我们已经在体外重建了整个翻译周期，这为我们提供了独特的机会，解决在理解哺乳动物启动和调节的机制方面的关键差距，使用生物化学和互补生物物理学和细胞生物学方法进行翻译。目标1关注通过DHX 29促进扫描的机制的表征，eIF 5 B稳定了细胞上的Met-tRNAiMet。 40 S亚基和这两个因素影响起始密码子的选择。在目标2中，我们将重点研究在CUG密码子和非AUG三联体上用Leu-tRNALeu进行生理学重要起始的机制在重复相关的非AUG（RAN）翻译过程中，这发生在转录的mRNA中的扩增重复序列上，导致严重神经退行性疾病的基因目标3致力于阐明在位于蟋蟀麻痹病毒RNA的5 'UTR的IRES上起始的分子机制，其具有独特的结构，我们的初步数据表明，它可以使用新的启动机制。目标4关注点 Schlafen 14的细胞功能和作用机制，一种新的结合80 S核糖体的核糖核酸内切酶并切割rRNA和核糖体结合的mRNA。因此，它涉及翻译控制，并可能影响这是一个以前没有描述过的过程。

项目成果

期刊论文数量（11）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

eIF5 and eIF5B together stimulate 48S initiation complex formation during ribosomal scanning.

DOI：
10.1093/nar/gku877
发表时间：
2014-10-29
期刊：
Nucleic acids research
影响因子：
14.9
作者：
Pisareva VP;Pisarev AV
通讯作者：
Pisarev AV

DHX29 and eIF3 cooperate in ribosomal scanning on structured mRNAs during translation initiation.

DOI：
10.1261/rna.057851.116
发表时间：
2016-12
期刊：
RNA (New York, N.Y.)
影响因子：
0
作者：
Pisareva VP;Pisarev AV
通讯作者：
Pisarev AV

DHX29 reduces leaky scanning through an upstream AUG codon regardless of its nucleotide context.

DOI：
10.1093/nar/gkw240
发表时间：
2016-05-19
期刊：
Nucleic acids research
影响因子：
14.9
作者：
Pisareva VP;Pisarev AV
通讯作者：
Pisarev AV

The structure and mechanism of action of a distinct class of dicistrovirus intergenic region IRESs.

DOI：
10.1093/nar/gkad569
发表时间：
2023-09-22
期刊：
NUCLEIC ACIDS RESEARCH
影响因子：
14.9
作者：
Abaeva, Irina S.;Young, Christina;Warsaba, Reid;Khan, Nadiyah;Tran, Lan Vy;Jan, Eric;Pestova, Tatyana, V;Hellen, Christopher U. T.
通讯作者：
Hellen, Christopher U. T.

Characterization of Novel Ribosome-Associated Endoribonuclease SLFN14 from Rabbit Reticulocytes.