Variant-specific interaction of kinectin 1 with the multi–tRNA synthetase complex regulates ER sheet organization

Kinectin 1 (KTN1) is an integral endoplasmic reticulum (ER) sheet protein that functions in ER organization and translation elongation. Alternative splicing introduces sequence diversity into the cytoplasmic region of KTN1, but the functional relevance of these variants has remained unclear. Here we show that the multi–tRNA synthetase complex (MSC), composed of eight aminoacyl-tRNA synthetases and three nonenzymatic proteins, specifically interacts with KTN1 in a manner dependent on the alternative exon V2 and glutamine-tRNA synthetase (QARS). Using V2 exon-specific knockout cells and KTN1 knockout cells with variant-specific resucue, we found that the V2 exon is required for KTN1-mediated recruitment of the MSC to the ER, where it promotes the formation of rough ER stacks. These findings define a variant-specific role for KTN1 in anchoring the MSC to the ER, and suggest that this interaction underlies a noncanonical activity of the MSC in regulating ER sheet organization. Overall design: Ribo-seq together with RNA-seq (for normalization of Ribo-seq reads) of KTN1 knockout HeLa cells rescued by doxycycline(dox)-inducible one single variant of KTN1, either KTN1 V2 exon(+) or KTN1 V2 exon(-).

动联蛋白1（Kinectin 1, KTN1）是内质网（endoplasmic reticulum, ER）层状整合蛋白，参与内质网结构组织与翻译延伸过程。可变剪接可使KTN1的胞质区域产生序列多样性，但此类变体的功能相关性至今仍不明确。本研究证实，由8种氨酰-tRNA合成酶与3种非酶蛋白组成的多tRNA合成酶复合物（multi–tRNA synthetase complex, MSC），可通过依赖可变外显子V2与谷氨酰胺-tRNA合成酶（glutamine-tRNA synthetase, QARS）的方式特异性结合KTN1。通过构建V2外显子特异性敲除细胞与变体特异性回补的KTN1敲除细胞，我们发现V2外显子是KTN1介导MSC招募至内质网的必需元件，该招募过程可促进粗面内质网堆叠的形成。本研究明确了KTN1变体在将MSC锚定至内质网过程中的变体特异性功能，并提示该相互作用是MSC调控内质网层状组织的非经典活性的分子基础。实验整体设计：对KTN1敲除的HeLa细胞，采用强力霉素（doxycycline, dox）诱导型单个KTN1变体进行回补，分别为携带V2外显子的KTN1（KTN1 V2 exon(+)）与不携带V2外显子的KTN1（KTN1 V2 exon(-)）；同时结合核糖体测序（Ribo-seq）与RNA测序（RNA-seq，用于Ribo-seq读段的标准化分析）开展实验。