Nuclear RNA cap-chaperones eIF4E and NCBP2 elicit distinct fates for 1000s of RNAs uncovering an unexpected regulatory point in gene expression cells [ribodeplete]

Nuclear mRNA processing occurs in a stepwise manner to generate the protein blueprints required for cellular function. The addition of the methyl-7-guanosine (m7G) cap on the 5'end of mRNAs is conserved in humans, plants and fungi. The m7G cap recruits the nuclear cap-binding protein NCBP2 co-/peri-transcriptionally where it mediates interactions between capped-RNAs and the processing machinery. Convention posits that NCBP2 is the sole cap-binding protein in the nucleus positioning cap-chaperoning as an important constitutive, housekeeping function. However, the eukaryotic translation initiation factor eIF4E is also found in the nucleus across Kingdoms. Both eIF4E and NCBP2 employ highly similar structural strategies to directly bind the m7G cap. The presence of two cap-binding factors in the nucleus could arise to provide redundancy to ensure gene expression security or it could provide the basis for tandem regulation of selected subsets of capped-mRNAs driving distinct gene expression programmes. To dissect these possibilities, we compared the spatial localizations, interactomes and impacts on gene expression of eIF4E and NCBP2. We found that like NCBP2, eIF4E physically and spatially associated with active sites of transcription and splicing machinery. However, contrary to expectations, eIF4E and NCBP2 drove distinct transcription and splicing signatures impacting ~1000 transcripts which in turn elicit different biological programmes. RNAs segregated with specific cap-binding proteins with only an overlap of only ~130 transcripts between eIF4E and NCBP2. Clearly, the cap-interaction was not sufficient to elicit sensitivity to these cap-binding proteins given the absence of generalized impacts on the transcriptome. We uncovered the molecular mechanism for cap-chaperone selectivity which lay in differences in conserved sequences motifs within introns of selected mRNAs, their mainly distinct spatial localizations, and differences in the capacity to alter splice factor production. Relevant to the 130 common targets, a fraction of eIF4E and NCBP2 did interact and spatially overlap. Despite their common m7G cap-binding activity, eIF4E and NCBP2 elicit substantively different impacts on gene expression. In contrast to the conventional paradigm, our data support a model whereby cap-chaperones direct processing of distinct subsets of mRNAs thereby eliciting diverse biological programmes. Differential programming by cap-chaperones reveals an unexpected regulatory point in gene expression.. Overall design: RNAseq profiling of triplicata of two different clones of U2-OS cells overexpressing the cap binding protein NCBP2. The comparison is made with three different clones obtained identically but with an empty vector.

细胞核mRNA加工以分步方式进行，以生成细胞功能所需的蛋白质蓝图。mRNA 5'端的7-甲基鸟苷（methyl-7-guanosine, m7G）帽修饰在人类、植物和真菌中均保守存在。m7G帽会在共转录及转录后早期招募细胞核帽结合蛋白NCBP2，介导带帽RNA与加工机器之间的相互作用。传统观点认为NCBP2是细胞核中唯一的帽结合蛋白，将帽伴侣功能定为重要的组成型持家功能。然而，在各生物界的细胞核中均发现了真核翻译起始因子eIF4E。eIF4E与NCBP2均采用高度相似的结构策略直接结合m7G帽。细胞核中存在两种帽结合因子，可能是为了提供冗余以保障基因表达的稳定性，也可能为选择性带帽RNA亚群的串联调控提供基础，从而驱动不同的基因表达程序。为解析这两种可能性，我们比较了eIF4E与NCBP2的空间定位、相互作用组以及对基因表达的影响。研究发现，与NCBP2类似，eIF4E在物理空间上与转录和剪接机器的活性位点相结合。但与预期相反，eIF4E与NCBP2诱导了截然不同的转录和剪接特征，分别影响约1000个转录本，进而引发不同的生物学程序。RNA与特定帽结合蛋白存在特异性关联，eIF4E与NCBP2的共同靶转录本仅约130个。显然，仅帽结合相互作用不足以使转录本对这两种帽结合蛋白产生敏感性，因为二者并未对转录组产生广泛影响。我们揭示了帽伴侣选择性的分子机制，其关键在于选择性mRNA内含子中保守序列基序的差异、二者主要不同的空间定位，以及改变剪接因子表达的能力差异。针对这130个共同靶标，确实有一部分eIF4E与NCBP2发生相互作用且空间定位存在重叠。尽管二者均具有m7G帽结合活性，eIF4E与NCBP2对基因表达却产生了实质性不同的影响。与传统范式相反，我们的数据支持如下模型：帽伴侣蛋白定向调控不同mRNA亚群的加工过程，从而引发多样化的生物学程序。帽伴侣蛋白介导的差异化编程揭示了基因表达中一个此前未被发现的调控节点。实验整体设计：对过表达帽结合蛋白NCBP2的两株不同U2-OS细胞克隆进行三次生物学重复的RNA测序分析，对照组为采用相同方法构建、转染空载体的三株不同克隆。