Cytoplasmic Location of Translation Controls Protein Output [CLIP-seq]

The cytoplasm contains membrane-bound organelles and membraneless compartments. The TIS granule network is formed through assembly of the RNA-binding protein TIS11B together with its bound mRNAs and associates with a portion of the rough endoplasmic reticulum (ER) – one of the major sites of protein synthesis. Subcellular mRNA localization is widespread in polarized cells. However, it is largely unknown if mRNA localization is also widespread in non-polarized cells and if the location of protein synthesis has biological consequences. To identify compartment-specific cellular functions, we used fluorescent particle sorting to isolate TIS granules, the ER surface, and the cytosol and determined their mRNA contents. Overall design: iCLIP-sequencing in HEK293T cells transfected with GFP-TIS11B, three biological replicates.

细胞质包含膜结合细胞器（membrane-bound organelles）与无膜区室（membraneless compartments）。TIS颗粒网络（TIS granule network）由RNA结合蛋白TIS11B与其结合的mRNA组装形成，并与部分粗面内质网（rough endoplasmic reticulum，ER）——蛋白质合成的主要位点之一——相关联。亚细胞水平的mRNA定位在极化细胞中广泛存在。然而，目前尚不清楚亚细胞mRNA定位在非极化细胞中是否同样普遍，也不清楚蛋白质合成的位置是否具有生物学效应。为了鉴定区室特异性的细胞功能，我们采用荧光颗粒分选技术分离了TIS颗粒、内质网表面及细胞质，并测定了三者的mRNA组分。实验设计：对转染GFP-TIS11B的HEK293T细胞开展iCLIP测序（iCLIP-sequencing），共设置3次生物学重复。