RNA-Seq, NChip-Seq and RIP-Seq analysis of FUBP3 in T cells

The Far Upstream Element Binding Protein 3 (FUBP3) enhances HIV-1 transcription and regulates immune responses pathways in T cells:
FUBP3 (Far Upstream Element-Binding Protein 3) has emerged as a hit factor in HIV-1 transcription, yet its molecular function and mechanisms remained unexplored. In this study, we investigate the role of FUBP3 in HIV-1 transcriptional activation, focusing on its interactions with the viral transactivator protein Tat and TAR RNA. Using a combination of transcriptomic analyses, chromatin immunoprecipitation, and biochemical assays, we demonstrate that FUBP3 enhances HIV-1 transcription in a Tat-dependent manner. FUBP3 directly binds and stabilizes TAR RNA, exhibiting a strong preference for its secondary structure, which is essential for efficient Tat function. Knockdown of FUBP3 results in significant downregulation of HIV-1 transcription and alters the expression of numerous host genes involved in T cell activation and inflammation, underscoring its role in regulating immune responses. Our findings position FUBP3 as a key player in the HIV-1 lifecycle, enhancing our understanding of the interplay between viral and host factors. This study opens avenues for future research aimed at targeting FUBP3 for therapeutic intervention in HIV-1 infection, particularly in the context of latency and reactivation. By elucidating the multifaceted roles of FUBP3, we contribute valuable insights into its potential as a therapeutic target and its implications in oncogenic and inflammatory pathways.

RNA-Sequencing analysis of total RNA extracted from J-Lat 10.6, Jurkat and primary CD4+T cells upon FUBP3 depletion by shRNAmirs (shFUBP3 and shCD8B).
RIP-Seq analysis of FUBP3-IP using the EZ-Magna RIP Kit (Millipore, #17-701) in Jurkat-D6 cells.
Native ChIP-Seq (NChIP) analysis of RNAPII and FUBP3 in J-Lat 10.6 cells with TNFa stimulation or without stimulation (MOCK).

远上游元件结合蛋白3（FUBP3）增强HIV-1转录并调控T细胞中的免疫反应途径：FUBP3（Far Upstream Element-Binding Protein 3）已成为HIV-1转录中的一个关键因素，然而其分子功能和作用机制尚未得到充分研究。在本研究中，我们探讨了FUBP3在HIV-1转录激活中的作用，重点关注其与病毒反式激活蛋白Tat及TAR RNA的相互作用。通过转录组学分析、染色质免疫沉淀和生化实验相结合的方法，我们证明了FUBP3以Tat依赖的方式增强HIV-1的转录。FUBP3可直接结合并稳定TAR RNA，对TAR RNA的二级结构具有强烈的偏好性，这对于Tat的高效功能至关重要。FUBP3敲低导致HIV-1转录显著下调，并改变参与T细胞活化和炎症的宿主基因的表达，从而突显其在调节免疫反应中的作用。我们的研究将FUBP3定位为HIV-1生命周期中的关键参与者，加深了我们对于病毒与宿主因子之间相互作用的了解。本研究为未来针对FUBP3进行HIV-1感染治疗干预的研究开辟了道路，特别是在潜伏期和活化期的治疗背景下。通过阐明FUBP3的多重作用，我们为其作为治疗靶点的潜在价值及其在肿瘤发生和炎症途径中的影响提供了宝贵的见解。

J-Lat 10.6、Jurkat和原代CD4+T细胞中总RNA的RNA测序分析，通过shRNAmirs（shFUBP3和shCD8B）耗竭FUBP3。

使用Millipore的EZ-Magna RIP试剂盒（#17-701）对Jurkat-D6细胞中的FUBP3-IP进行RIP测序分析。

在TNFa刺激或未刺激（MOCK）的J-Lat 10.6细胞中进行的原生ChIP测序（NChIP）分析，分析RNAPII和FUBP3。