The in vivo functional significance of PUF hub partnerships in C. elegans germline stem cells

PUF RNA-binding proteins are conserved stem cell regulators. Four PUF proteins govern self-renewal of Caenorhabditis elegans germline stem cells together with two intrinsically disordered proteins, LST-1 and SYGL-1. Based on yeast two-hybrid results, we previously 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(m)B(m)) mutant defective for PUF-interacting motifs does not complex with PUFs in nematodes. LST-1(A(m)B(m)) is used to explore the in vivo functional significance of the LST-1–PUF partnership. Tethered LST-1 requires this 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 in vivo. Comparison of LST-1–PUF and Nanos–Pumilio reveals fundamental molecular differences, making LST-1–PUF a distinct paradigm for PUF partnerships.