Ferdous Ahlan S, Costa Dos Santos Stephany J, Kanzler Charlotte R, Shin Heaji, Carrick Brian H, Crittenden Sarah L, Wickens Marvin, Kimble Judith
Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
bioRxiv. 2023 Feb 16:2023.02.15.528708. doi: 10.1101/2023.02.15.528708.
PUF RNA-binding proteins are conserved stem cell regulators. Four PUF proteins govern self-renewal of germline stem cells together with two intrinsically disordered proteins, LST-1 and SYGL-1. Based on yeast two-hybrid results, we proposed a composite self-renewal hub in the stem cell regulatory network, with eight PUF partnerships and extensive redundancy. Here, we investigate LST-1-PUF and SYGL-1-PUF partnerships and their molecular activities in their natural context - nematode stem cells. We confirm LST-1-PUF partnerships and their specificity to self-renewal PUFs by co-immunoprecipitation and show that an LST-1(A B ) mutant defective for PUF-interacting motifs does not complex with PUFs in nematodes. LST-1(A B ) is used to explore the functional significance of the LST-1-PUF partnership. Tethered LST-1 requires the partnership to repress expression of a reporter RNA, and LST-1 requires the partnership to co-immunoprecipitate with NTL-1/Not1 of the CCR4-NOT complex. We suggest that the partnership provides multiple molecular interactions that work together to form an effector complex on PUF target RNAs. Comparison of PUF-LST-1 and Pumilio-Nanos reveals fundamental molecular differences, making PUF-LST-1 a distinct paradigm for PUF partnerships.
Partnerships between PUF RNA-binding proteins and intrinsically disordered proteins are essential for stem cell maintenance and RNA repression.
PUF RNA结合蛋白是保守的干细胞调节因子。四种PUF蛋白与两种内在无序蛋白LST-1和SYGL-1共同调控生殖系干细胞的自我更新。基于酵母双杂交结果，我们在干细胞调节网络中提出了一个复合自我更新枢纽，其中有八个PUF伙伴关系且具有广泛的冗余性。在这里，我们研究了线虫干细胞自然环境中LST-1-PUF和SYGL-1-PUF伙伴关系及其分子活性。我们通过免疫共沉淀证实了LST-1-PUF伙伴关系及其对自我更新PUF的特异性，并表明一个在PUF相互作用基序上有缺陷的LST-1（A B ）突变体在秀丽隐杆线虫中不与PUF形成复合物。LST-1（A B ）被用于探究LST-1-PUF伙伴关系的功能意义。被拴系的LST-1需要这种伙伴关系来抑制报告RNA的表达，并且LST-1需要这种伙伴关系来与CCR4-NOT复合物的NTL-1/Not1进行免疫共沉淀。我们认为这种伙伴关系提供了多种分子相互作用，它们共同作用在PUF靶RNA上形成一个效应复合物。PUF-LST-1和Pumilio-Nanos的比较揭示了基本的分子差异，使PUF-LST-1成为PUF伙伴关系的一个独特范例。
PUF RNA结合蛋白与内在无序蛋白之间的伙伴关系对于干细胞维持和RNA抑制至关重要。
