Noncanonical microRNA biogenesis and function in a gamma herpesvirus and mammals

描述(申请人提供)：microRNAs(MiRNAs)是~22个核苷酸(NT)普遍存在的基因调节因子，调节多种细胞途径，包括分化、增殖和凋亡，所有这些都对人类发育和疾病至关重要。越来越多的证据表明，各种miRNAs在癌细胞中异常表达，这突显了阐明miRNA生物发生机制的重要性。在疱疹病毒感染的绒猴T细胞中，病毒miRNAs直接在病毒小核糖核酸(SnRNAs)下游共转录。这是第一批鉴定出的SnRNA-miRNA嵌合体。它们被宿主整合者复合体切割，绕过了规范的微处理器切割。由此产生的miRNAs可能导致T细胞白血病和淋巴瘤，这些疾病是由人类免疫缺陷病毒感染引起的。我在哺乳动物中搜索SnRNA-miRNA嵌合体时，意外地发现了m7G封顶的前体(前)miRNAs，它们可以由Exportin-1(而不是典型的Exportin-5)输出，并且只产生一个成熟的miRNA。MIR-320来自m7G封顶的前体，可抑制乳腺上皮性肿瘤。该提案的目标是进一步描绘两条非规范的miRNA生物发生途径(目标1)，扩大可替代加工的miRNAs的谱系并确定它们的功能(目标2)。在K99阶段，将使用体外转录的HVS miRNA底物从核提取液中亲和纯化整合子复合体，并将检测重组整合子的活性(目标1a)。将开发一个体外RNA聚合酶II转录系统，以评估m7G封顶的前miRNAs(Aim 1b)32端的潜在转录终止。对于R00阶段的项目，正在设计一个siRNA/GFP系统来筛选影响miRNA生物发生的基因(目标1c)。将通过计算机研究和基因敲除研究在不同物种中寻找使用HVS miRNA或m7G封顶的前miRNA生物发生途径的miRNAs(目标2a)。为了确定可替代处理的miRNAs的mRNA靶标，我提出了一种方法，即ArgAerte-UV交联和免疫沉淀(CLIP)，然后将靶mRNAs与病毒miRNAs分子间连接，这两个mRNAs在ArgAerte(Aim 2b)中配对。针对人类免疫缺陷病毒miRNAs和以m7G为上限的前体衍生miRNAs确定的靶点将有助于阐明T细胞白血病/淋巴瘤和其他癌症的发病机制。破译这两条新的miRNA生物发生途径的细节，这两条途径不同于产生大多数细胞miRNAs的途径，将增强有希望的疗法，旨在调节特定的miRNAs来对抗相关人类疱疹病毒诱导的癌症和其他恶性肿瘤。

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.