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

Abstract

PUF RNA-binding proteins are conserved stem cell regulators. Four PUF proteins govern self-renewal of C. elegans 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(AmBm) mutant defective for PUF-interacting motifs does not complex with PUFs in nematodes. LST-1(AmBm) is used to explore the in vivo 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 in vivo. Comparison of PUF-LST-1 and Pumilio–Nanos reveals fundamental molecular differences, making PUF–LST-1 a distinct paradigm for PUF partnerships.

LST-1 is a bifunctional regulator that feeds back on Notch-dependent transcription to regulate C. elegans germline stem cells

“…All except lst-1(ø) harbor a C-terminal 3× V5 tag (inverted triangle). LST-1 (CΔ) deletes the C-terminal region ( amino acids 211 to 328); LST-1 (NΔ) carries a nonsense mutation early in LST-1L and makes only LST-1S, which lacks the N-terminal region unique to LST-1L (amino acids 1 to 94); LST-1 (ZnF) substitutes two key cysteines with serines (C260S and C263S, asterisks); LST-1(PIM), substitutes key amino acids for alanines in both PIM-A and PIM-B (L35A, K80A and L83A, asterisks) and abolishes LST-1 interactions with PUF proteins in both yeast and nematodes ( 15 , 16 ). ( B – I ) Boxplot and bar graph conventions as described in the Fig.…”

“…We also asked whether the LST-1 PIMs affect either sygl-1 or lst-1 transcription. PIMs were already established as essential for posttranscriptional regulation ( 15 , 16 ), but a transcriptional role had not been tested. We found that both sygl-1 ATS and lst-1 ATS were comparable in lst-1(+) and lst-1(PIM) mutants ( SI Appendix , Fig.…”

“…We then made strains carrying lag-1 FLAG , glp-1(gf ts) and either lst-1(+) V5 or lst-1(ZnF) V5 . The glp-1(gf ts) mutant was included to facilitate LST-1 IPs, as done previously ( 16 ). At permissive temperature (15 °C), most glp-1(gf ts) mutants have normal fertile germlines, but at restrictive temperature (25 °C), excess Notch signaling drives formation of germline tumors ( 23 ).…”

“…Both LST-1 and SYGL-1 are posttranscriptional regulators ( Fig. 1 B , Right ) ( 15 , 16 ). Each binds to a PUF (for Pumilio and FBF) RNA-binding protein via PUF-interacting motifs that reside within a stretch of intrinsically disordered regions ( Fig.…”

“…1 C ). The resultant LST-1/PUF and SYGL-1/PUF complexes repress RNAs and promote GSC self-renewal ( 15 – 17 ). Normally, PUF proteins are expressed throughout the progenitor zone, while their LST-1 and SYGL-1 partners are restricted to the distal progenitor zone ( Fig.…”