The "PUF hub" is responsible for self-renewal of germline stem cells (GSCs)1. This hub consists of four PUF (Pumilio and FBF) RNA-binding proteins - FBF-1, FBF-2, PUF-3 & PUF-11 - and two novel proteins, LST-1 and SYGL-1. The molecular elucidation of both regulation and function of the PUF hub is essential to understand how GSCs are maintained in the face of challenges from physiology and the environment. GLP-1/Notch signaling activates
lst-1 and
sygl-1 transcription to launch and maintain hub activity2,3. We now find that point mutations at one or more of three LAG-1 binding sites (LBS) in the
sygl-1 promoter tune its response to niche signaling. Single LBS mutants substantially lower the number of
sygl-1 active transcription sites (ATS) and double mutants nearly eliminate them; importantly, the GSC pool shrinks in single mutants and is lost in double mutants, when assayed without LST-1. We also find that LST-1 has an unexpected role in
sygl-1 transcription:
sygl-1 ATS number increases in
lst-1(ø) mutants with a corresponding increase in SYGl-1 protein. The converse is not true:
lst-1 expression is unaffected in
sygl-1(ø) mutants. Therefore, LST-1, but not SYGL-1, affects transcription. The primary PUF hub function is regulation of target RNAs. We proposed earlier that each hub PUF protein forms a complex with either LST-1 or SYGL-1 to repress differentiation RNAs4. We have investigated this model. We first used co-immunoprecipitation (coIP) to explore the proposed battery of complexes. Though the matrix is only about half done, all coIPs so far support the model. We next identified "PUF-interaction sites" (PIS) in both LST-1 and SYGL-1. Intriguingly, each harbors two redundant PIS: single PIS mutants of either protein retain self-renewal activity but double mutants abolish it. We finally tethered LST-1 and SYGL-1 to a reporter RNA with the lambdaN-boxB system, and found that both lambdaN-LST-1 and lambdaN-SYGL-1 repressed a GFP::H2B reporter with boxB sites in its 3'UTR. These results provide strong evidence that PUF:LST-1 and PUF:SYGL-1 partnerships are essential for GSC maintenance and that LST-1 and SYGL-1 are RNA repressors. In sum, both hub regulation and function are intensively buffered, and our mutants provide new tools for analyses of the hub when poised in an intermediate state. 1 Haupt et al 2020 Genetics 214,147; 2 Kershner et al 2014 PNAS 111, 3739; 3 Lee et al 2016 eLife 5,
e18370; 4 Shin et al 2017 PLoS genetics 13,
e1007121.