Spatio-temporal m(i)RNA architecture and 3' UTR regulation in the C. elegans germline

In animal germlines, regulation of cell proliferation and differentiation is particularly important but poorly understood. Here, using a cryo-cut approach, we mapped RNA expression along the Caenorhabditis elegans germline and, using mutants, dissected gene regulatory mechanisms that control spatio-temporal expression. We detected, at near single-cell resolution, > 10,000 mRNAs, > 300 miRNAs and numerous unannotated miRNAs. Most RNAs were organized in distinct spatial patterns. Germline-specific miRNAs and their targets were co-localized. Moreover, we observed differential 3' UTR isoform usage for hundreds of mRNAs. In tumorous gld-2 gld-1 mutants, gene expression was strongly perturbed. In particular, differential 3' UTR usage was significantly impaired. We propose that PIE-1, a transcriptional repressor, functions to maintain spatial gene expression. Our data also suggest that cpsf-4 and fipp-1 control differential 3' UTR usage for hundreds of genes. Finally, we constructed a “virtual gonad” enabling “virtual in situ hybridizations” and access to all data (https://shiny.mdc-berlin.de/spacegerm/). Overall design: Investigation of the spatio-temporal gene expression in the Caenorhabditis elegans germline using a cryo-based approach and single cell protocols.在动物生殖系中，细胞增殖与分化的调控尤为关键，但目前相关机制仍不甚明确。本研究采用冰冻切割（cryo-cut）技术，绘制了秀丽隐杆线虫（Caenorhabditis elegans）生殖系的RNA空间表达谱，并借助突变体解析了调控时空表达的基因调控机制。本研究以接近单细胞的分辨率检测到超过10000种mRNA、300余种microRNA（miRNA）以及大量未注释的miRNA。多数RNA呈现出独特的空间分布模式，生殖系特异性miRNA与其靶基因存在共定位现象。此外，我们还观察到数百种mRNA存在不同的3'非翻译区（3' UTR）异构体使用模式。在致瘤性gld-2 gld-1突变体中，基因表达受到显著扰动，尤其是3' UTR异构体的差异使用模式遭到明显破坏。我们推测，转录抑制因子PIE-1的功能是维持基因的空间表达模式。我们的数据还表明，cpsf-4与fipp-1可调控数百个基因的3' UTR异构体差异使用模式。最后，我们构建了「虚拟性腺（virtual gonad）」，可实现「虚拟原位杂交（virtual in situ hybridizations）」并开放全部数据的访问权限（https://shiny.mdc-berlin.de/spacegerm/）。实验整体设计：基于冷冻技术与单细胞实验方案，探究秀丽隐杆线虫生殖系中的时空基因表达调控。