Minor splicing factor 65K/RNPC3 interacts with ANKRD11 and mediates HDAC3-regulated histone deacetylation and transcription. Minor splicing factor 65K/RNPC3 interacts with ANKRD11 and mediates HDAC3-regulated histone deacetylation and transcription

Pre-mRNA splicing is coupled with DNA- and other RNA-processing machineries in the nucleus to form multilayer networks for accurate regulation of gene expression. However, those established couplings are major spliceosome-related, whether the minor spliceosome is involved remains unclear. Here, we first identified a direct interaction between a minor-specific protein 65K/RNPC3 and the histone deacetylase HDAC3 cofactor ANKRD11 through affinity co-purification using Drosophila lysate. In vivo assays revealed that the head of both DmAnkrd11Δ/Δ and Dm65KΔ/Δ mutants exhibited increased histone acetylation at H3K9 and H4K5, consistent with the high level of ANKRD11 in the central nervous systems. We then found that the 65K-ANKRD11 interaction is conserved in human, in which the HsANKRD11 middle-stretched domain mediates Hs65K association with HDAC3. CUT&Tag assays demonstrated that HDAC3 and Hs65K synergistically bind on chromatin, and the knockdown of ANKRD11 simultaneously decreased their bindings at the same chromatin locations and also increased nearby acetylation of H3K9. Sequencing of mRNAs revealed that the deficiency of HsANKRD11 or Hs65K caused changes in gene expression correlated with their chromatin-binding changes of HDAC3 and Hs65K and the levels of H3K9ac. This study provides a novel strategy for the regulation of histone deacetylation and gene expression through a minor spliceosome-specific component. Overall design: To investigate the cooperative function 65K/ANKRD11/HDAC3 complex in the regulation of histone acetylation and gene transcription, we established the Dm65K-deletion and DmAnkrd11-deletion Drosophila strain, RNA was from Proximal heads or adults from 100 flies (1 days old), then libraries was prepared for transcriptome sequencing. Comparative gene expression profiling analysis of RNA-seq data for WT, Dm65K-deletion and DmAnkrd11-deletion Drosophila strain. 293T cell lines in which each target gene has been knocked down by shRNA. Comparative gene expression profiling analysis of RNA-seq data for 293T cells and its KD derivatives (sh65K and shANKRD11). The CUT&Tag assay was performed as described (Kaya-Okur et al., 2019). CUT&Tag assay using 65K or HDAC3 antibody to find out binding sites in 293T cells or its KD derivatives (ctrl and shANKRD11). The ChIP assay was performed as described (Gorkin et al., 2020). Briefly, the HEK293T KD derivatives (ctrl, sh65K and shANKRD11) cells were cross-linked by 1% formaldehyde for 10 min and then the cell lysate was prepared by removing cytoplasm followed by sonication-based chromatin fragmentation. Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for Histone 3 lysine 9 acetylation DNAs were subjected to library preparation with the Scale ssDNA-seq Lib Prep Kit for Illumina V2 (#RK20228, Abclonal).

核内前体mRNA（pre-mRNA）剪接与DNA及其他RNA加工机器相互耦联，形成多层级调控网络以精准调控基因表达。然而，目前已报道的此类耦联均与主要剪接体相关，而次要剪接体（minor spliceosome）是否参与其中仍未明确。本研究首先通过果蝇（Drosophila）细胞裂解液的亲和共纯化（affinity co-purification）实验，鉴定出次要剪接体特异性蛋白65K/RNPC3与组蛋白去乙酰化酶HDAC3（histone deacetylase HDAC3）的辅因子ANKRD11之间存在直接相互作用。体内实验显示，DmAnkrd11Δ/Δ与Dm65KΔ/Δ突变体的头部组织中，H3K9及H4K5位点的组蛋白乙酰化水平均升高，这与ANKRD11在中枢神经系统中的高表达特征相符。随后研究发现，65K与ANKRD11的相互作用在人类中保守存在，其中人类ANKRD11（HsANKRD11）的中间伸展结构域介导人类65K（Hs65K）与HDAC3的结合。CUT&Tag实验证实，HDAC3与Hs65K可协同结合于染色质；而ANKRD11敲低会同时降低二者在相同染色质区域的结合，并提升邻近区域的H3K9乙酰化水平。mRNA测序结果显示，HsANKRD11或Hs65K的缺失会导致基因表达发生改变，且该变化与HDAC3、Hs65K的染色质结合水平变化及H3K9乙酰化（H3K9ac）水平相关。本研究通过次要剪接体特异性组分，为组蛋白去乙酰化与基因表达调控提供了全新的研究策略。
整体实验设计：为探究65K/ANKRD11/HDAC3复合物在组蛋白乙酰化与基因转录调控中的协同功能，本研究构建了Dm65K敲除与DmAnkrd11敲除的果蝇品系。实验材料取自1日龄成年果蝇的近端头部组织（共100只果蝇），随后提取RNA并构建文库用于转录组测序。对野生型、Dm65K敲除及DmAnkrd11敲除果蝇的RNA-seq数据进行比较基因表达谱分析。
针对293T细胞系，通过短发夹RNA（shRNA）分别敲低各靶基因，对野生型293T细胞及其敲低衍生株（sh65K与shANKRD11）的RNA-seq数据开展比较基因表达谱分析。
CUT&Tag实验按照已发表方法（Kaya-Okur等，2019）进行，使用65K或HDAC3抗体，在293T细胞及其敲低衍生株（对照组与shANKRD11组）中鉴定结合位点。
ChIP实验按照已发表方法（Gorkin等，2020）进行：简要流程为，将HEK293T敲低衍生株（对照组、sh65K与shANKRD11组）细胞用1%甲醛交联10分钟，随后去除细胞质组分并通过超声破碎实现染色质片段化；针对组蛋白H3赖氨酸9乙酰化的染色质免疫共沉淀测序（ChIP-seq）样本，采用Illumina平台的Scale ssDNA-seq Lib Prep Kit V2（货号#RK20228，Abclonal）进行文库构建。