Noncanonical microRNA biogenesis and function in a gamma herpesvirus and mammals

描述（由申请人提供）：MicroRNAs （miRNAs）是约22个核苷酸（nt）普遍存在的基因调节剂，可调节多种细胞通路，包括分化、增殖和凋亡，对人类发育和疾病都至关重要。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。在猴猴疱疹病毒（HVS）感染的狨猴T细胞中，病毒mirna直接在病毒小核rna （snrna）的下游共转录。这是第一个被鉴定的snRNA-miRNA嵌合体。它们被宿主积分器复合体分开，绕过规范的微处理器切割。由此产生的mirna可能有助于t细胞白血病和淋巴瘤，由HVS感染引起的疾病。我在哺乳动物中寻找snRNA-miRNA嵌合体，意外地发现了m7g -cap前体（pre-）miRNA，它们由Exportin-1输出（而不是规范的Exportin-5），只产生一个成熟的miRNA。MiR-320来源于m7g -cap前体并抑制乳腺上皮性肿瘤。本提案的目标是进一步描述两种非规范miRNA生物发生途径（目标1），扩大可替代加工miRNA的曲目并确定其功能（目标2）。在K99期，将使用体外转录的HVS miRNA底物从核提取物中亲和纯化Integrator复合物，并检测重组Integrator的活性（Aim 1a）。将开发一种体外RNA聚合酶II转录系统，以评估m7G覆盖的pre-miRNAs的32端潜在的转录终止（Aim 1b）。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（Aim 1c）。使用HVS miRNA或m7G-capped pre- miRNA生物发生途径的miRNA将通过硅化和敲除研究在不同物种中寻找（Aim 2a）。为了鉴定替代加工mirna的mRNA靶标，我提出了一种方法，包括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 mirna和m7G-capped前体衍生mirna的靶标将揭示t细胞白血病/淋巴瘤和其他癌症的潜在机制。破译这两种不同于大多数细胞miRNA产生途径的新型miRNA生物发生途径的细节，将有助于设计有希望的治疗方法来调节特定的miRNA，以对抗由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.