Institute of Molecular Physiology, Johannes Gutenberg University Mainz, 55128 Mainz, Germany.
Cells. 2024 Nov 8;13(22):1855. doi: 10.3390/cells13221855.
The protein SANS is a small multifunctional scaffold protein. It is involved in several different cellular processes, such as intracellular transport, in the cytoplasm, or splicing of pre-mRNA, in the cell nucleus. Here, we aimed to gain insight into the regulation of the subcellular localization and the nuclear-cytoplasmic shuttling of SANS and its paralog ANKS4B, not yet reported in the nucleus. We identified karyopherins mediating the nuclear import and export by screening the nuclear interactome of SANS. Sequence analyses predicted in silico evolutionarily conserved nuclear localization sequences (NLSs) and nuclear export sequences (NESs) in SANS, but only NESs in ANKS4B, which are suitable for karyopherin binding. Quantifying the nuclear-cytoplasmic localization of wild-type SANS and NLS/NES mutants, we experimentally confirmed in silico predicted NLS and NES functioning in the nuclear-cytoplasmic shuttling in situ in cells. The comparison of SANS and its paralog ANKS4B revealed substantial differences in the interaction with the nuclear splicing protein PRPF31 and in their nuclear localization. Finally, our results on pathogenic USH1G/SANS mutants suggest that the loss of NLSs and NESs and thereby the ability to control nuclear-cytoplasmic shuttling is disease-relevant.
SANS 蛋白是一种小型多功能支架蛋白。它参与了几种不同的细胞过程，如细胞质中的细胞内运输，或细胞核中前体 mRNA 的剪接。在这里，我们旨在深入了解 SANS 及其旁系同源物 ANKS4B 的亚细胞定位和核质穿梭的调节，目前尚未在核内报道。我们通过筛选 SANS 的核相互作用组来鉴定介导核输入和输出的核转运蛋白。序列分析预测了 SANS 中具有潜在核定位序列（NLS）和核输出序列（NES）的序列，但是在 ANKS4B 中仅预测了 NES，这些序列适合与核转运蛋白结合。通过定量野生型 SANS 和 NLS/NES 突变体的核质定位，我们在细胞内原位实验证实了 NLS 和 NES 对核质穿梭的功能。SANS 和其旁系同源物 ANKS4B 的比较显示，与核剪接蛋白 PRPF31 的相互作用以及它们的核定位存在显著差异。最后，我们对致病性 USH1G/SANS 突变体的研究结果表明，NLS 和 NES 的缺失以及控制核质穿梭的能力与疾病相关。
