Noncanonical microRNA biogenesis and function in a gamma herpesvirus and mammals

γ 疱疹病毒和哺乳动物中的非典型 microRNA 生物发生和功能

• 批准号: 9341144
• 负责人: MINGYI XIE
• 金额: $ 24.65万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-19 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9341144
• 关键词: 7-methylguanosine; Adenosine; Affect; Affinity; Apoptosis; Biogenesis; Bioinformatics; Biological Assay; Bypass; Callithrix; Cell physiology; Cells; Chimera organism; Cleaved cell; Complex; Computer Simulation; Data; Development; Disease; ETS2 gene; Epithelial; Gene Expression Regulation; Gene Targeting; Genes; Genetic Transcription; Herpesviridae; Human; Human Development; Immunoprecipitation; In Vitro; Infection; Laboratories; Ligation; Light; Malignant Neoplasms; Mammalian Cell; Mammals; Mammary Neoplasms; Messenger RNA; Methods; MicroRNAs; Microprocessor; Modeling; Monkeys; Nuclear; Nuclear Extract; Nucleotides; Oncogenic; Organism; PTEN gene; Participant; Pathway interactions; Phase; Play; Polymerase; Primates; Process; Production; RNA Caps; RNA Polymerase II; Recombinants; Regulator Genes; Reporter; Reporting; Ribonucleoproteins; Role; Saimiriine Herpesvirus 2; Small Interfering RNA; Small Nuclear RNA; Small RNA; System; T-Cell Leukemia; T-Lymphocyte; Therapeutic; Transcript; Transcription Initiation Site; Tumor Suppressor Proteins; Viral; Viral Genes; base; cancer cell; combat; crosslink; design; exportin 1 protein; exportin 5; gammaherpesvirus; gene repression; genome-wide; human disease; in vitro activity; in vivo; knock-down; leukemia/lymphoma; novel; novel therapeutics; promoter; public health relevance; reconstitution; small hairpin RNA; therapy design; transcription termination

DESCRIPTION (provided by applicant): MicroRNAs (miRNAs) are ~22 nucleotide (nt) ubiquitous gene regulators that modulate diverse cellular pathways including differentiation, proliferation and apoptosis, all critical to human development and disease. Accumulating evidence suggests that various miRNAs are aberrantly expressed in cancer cells, underscoring the importance of elucidating miRNA biogenesis mechanisms. In Herpesvirus saimiri (HVS) infected marmoset T cells, viral miRNAs are co-transcribed immediately downstream of viral small nuclear RNAs (snRNAs). These were the first snRNA-miRNA chimeras to be identified. They are cleaved apart by the host Integrator complex, bypassing canonical Microprocessor cleavage. The resulting miRNAs may contribute to T-cell leukemias and lymphomas, diseases caused by HVS infection. My search for snRNA-miRNA chimeras in mammals led to the unexpected discovery of m7G-capped precursor (pre-)miRNAs, which are alternatively exported by Exportin-1 (rather than the canonical Exportin-5) and generate only a single mature miRNA. MiR-320 is derived from a m7G-capped precursor and suppresses epithelial mammary tumors. The objectives of this proposal are to further delineate the two noncanonical miRNA biogenesis pathways (Aim 1), expand the repertoire of alternatively-processed miRNAs and identify their functions (Aim 2). In the K99 phase, Integrator complex will be affinity purified from nuclear extract using in vitro transcribed HVS miRNA substrate and recombinant Integrator will be assayed for activity (Aim 1a). An in vitro RNA polymerase II transcription system will be developed to assess potential transcription termination at the 32 end of m7G- capped pre-miRNAs (Aim 1b). For the projects in R00 phase, an siRNA/GFP system is being devised to screen for genes that affect miRNA biogenesis (Aim 1c). MiRNAs using the HVS miRNA or m7G-capped pre- miRNA biogenesis pathways will be sought in different species through in silico and knockdown studies (Aim 2a). To identify mRNA targets of the alternatively-processed miRNAs, I propose a method involving Argonaute- UV crosslinking and immunoprecipitation (CLIP) followed by intermolecular ligation between target mRNAs and viral miRNAs, which are paired within Argonaute (Aim 2b). Identified targets for HVS miRNAs and m7G-capped precursor-derived miRNAs will shed light on mechanisms underlying T-cell leukemias/lymphomas and other cancers. Deciphering the details of these two novel miRNA biogenesis pathways, which are distinct from the pathway that generates most cellular miRNAs, will potentiate promising therapeutics designed to modulate specific miRNAs to combat cancer and other malignancies induced by related human Herpesviruses.

描述（由申请人提供）：微小RNA（miRNA）是~22个核苷酸（nt）的普遍存在的基因调节因子，其调节多种细胞途径，包括分化、增殖和凋亡，所有这些对人类发育和疾病至关重要。越来越多的证据表明，各种miRNA在癌细胞中异常表达，强调了阐明miRNA生物发生机制的重要性。在猴疱疹病毒（HVS）感染的绒猴T细胞中，病毒miRNA在病毒小核RNA（snRNA）的下游立即共转录。这是第一个被鉴定的snRNA-miRNA嵌合体。它们被宿主整合子复合物切割开，绕过典型的微处理器切割。由此产生的miRNA可能导致T细胞白血病和淋巴瘤，这些疾病是由HVS感染引起的。我在哺乳动物中对snRNA-miRNA嵌合体的研究导致了m7 G加帽的前体（前）miRNA的意外发现，这些前体（前）miRNA可以通过Exportin-1（而不是典型的Exportin-5）输出，并且只产生单一的成熟miRNA。miR-320来源于m7 G加帽的前体，并抑制上皮性乳腺肿瘤。该提案的目的是进一步描述两个非经典的miRNA生物合成途径（目标1），扩大替代加工的miRNA的库并确定其功能（目标2）。在K99阶段，使用体外转录的HVS miRNA底物从核提取物中亲和纯化整合子复合物，并测定重组整合子的活性（目的1a）。将开发体外RNA聚合酶II转录系统以评估在m7 G加帽的pre-miRNA的32末端的潜在转录终止（Aim 1b）。对于R 00阶段的项目，正在设计siRNA/GFP系统来筛选影响miRNA生物发生的基因（Aim 1c）。将通过计算机模拟和敲除研究在不同物种中寻找使用HVS miRNA或m7 G加帽的前miRNA生物发生途径的miRNA（目的2a）。为了鉴定交替加工的miRNA的mRNA靶标，我提出了一种方法，该方法涉及Argonaute- UV交联和免疫沉淀（CLIP），然后在Argonaute内配对的靶mRNA和病毒miRNA之间进行分子间连接（Aim 2b）。HVS miRNAs和m7 G加帽的EGFR衍生的miRNAs的鉴定靶点将揭示T细胞白血病/淋巴瘤和其他癌症的潜在机制。破译这两种新的miRNA生物发生途径的细节，这是不同于产生大多数细胞miRNA的途径，将加强有前途的治疗设计，以调节特定的miRNA，以打击癌症和其他恶性肿瘤诱导的相关人类疱疹病毒。