The Conserved Intron Binding Protein EMB-4 Plays Differential Roles in Germline Small RNA Pathways of C. elegans

Proper regulation of the germline transcriptome is essential for the maintenance of fertility and survival of a species. In C. elegans, germline transcriptome homeostasis hinges on a complex repertoire of small RNA pathways that act in both activating and silencing capacities. Our understanding of how fundamental RNA processing steps intersect with these small RNA machineries in the germline remains relatively limited. Here, we link the conserved intron binding protein and splicing factor, EMB-4/AQR/IBP160 to two key 22G-RNA pathways in the C. elegans germline. EMB-4 associates with the Argonautes CSR-1 and HRDE-1, and is enriched at the genomic loci of CSR-1 and HRDE-1 target genes. Loss of emb-4 leads to distinct alterations in CSR-1 vs. HRDE-1 small RNA and mRNA transcriptomes. Our transcriptome-wide analysis shows that EMB-4 is enriched along pre-mRNAs of nearly 10,000 transcripts. For a subset of these genes, including mostly CSR-1 pathway targets, EMB-4 enriches for intronic, but not exonic, sequences. Together these data point to EMB-4 as a factor that may help to distinguish the targets of these two germline small RNA pathways. Overall design: For mRNA and small RNA-seq experiments, total RNA was extracted from N2 and emb-4(hc60) mutant worms harvested as young adults. EMB-4 IPs were performed on RNA extracted from gravid adult N2 worms.生殖系转录组的精准调控对于维持物种的生育能力与存续至关重要。在秀丽隐杆线虫（C. elegans）中，生殖系转录组稳态依赖于一套兼具激活与沉默功能的复杂小RNA通路网络。目前学界对于生殖系中基础RNA加工步骤与这类小RNA分子机器如何相互作用的认知仍相对有限。本研究将保守型内含子结合蛋白、剪接因子EMB-4/AQR/IBP160与秀丽隐杆线虫生殖系内的两条关键22G-RNA通路建立了关联。EMB-4可与Argonaute蛋白（Argonaute）CSR-1及HRDE-1结合，并在CSR-1与HRDE-1的靶基因基因组位点处富集。emb-4基因的缺失会导致CSR-1与HRDE-1对应的小RNA及mRNA转录组出现显著差异。我们的全转录组分析显示，EMB-4在近10000个转录本的前体mRNA（pre-mRNA）上均有富集。对于其中一部分基因（多数为CSR-1通路靶标），EMB-4仅在内含子序列而非外显子序列处富集。综合以上数据表明，EMB-4或可作为区分这两条生殖系小RNA通路靶标的关键因子。整体实验设计：为开展mRNA与小RNA测序实验，我们从同步化至年轻成虫阶段的野生型N2与emb-4(hc60)突变体线虫中提取总RNA。同时，从产卵期成年野生型N2线虫提取的RNA中开展EMB-4免疫沉淀（IP）实验。