LST-1 acts in trans with a conserved RNA-binding protein to maintain stem cells

Abstract

21 22 23 IMPACT STATEMENT 24 A key stem cell regulator partners with a broadly conserved PUF RNA-binding protein to drive self-25 renewal and maintain a stem cell pool.26 27 2 ABSTRACT 28Stem cell self-renewal is essential to development and tissue repair. The C. elegans LST-1 protein is a 29 pivotal regulator of self-renewal and oncogenic when misexpessed. Here we define regions within the 30 LST-1 protein that provide molecular insights into both its function and regulation. LST-1 self-renewal 31 activity resides within a predicted disordered region that harbors two KXXL motifs. These KXXL motifs 32 mediate LST-1 binding to FBF, a broadly conserved Pumilio/PUF RNA-binding protein that represses 33 differentiation. Point mutations of the KXXL motifs abrogate LST-1 self-renewal activity. Therefore, FBF 34 binding is essential to LST-1 function. A second distinct region regulates LST-1 spatial expression and 35 primarily affects LST-1 protein turnover. Upon loss of this regulatory region, LST-1 protein distribution 36 expands and drives formation of a larger than normal GSC pool. Thus, LST-1 promotes self-renewal as a 37 key FBF partner, and its spatial regulation helps determine size of the GSC pool.

A crystal structure of a collaborative RNA regulatory complex reveals mechanisms to refine target specificity

“…We therefore conclude that residues 55–105 are a minimal interacting fragment of LST-1. This site was also identified independently along with a second weaker interacting site at residues 32–35 (Haupt et al, 2019). We inadvertently disrupted the site at residues 32–35 when we removed residues 1–34 in our truncations.…”

“…We note that our LST-1 fragment containing residues 34–80 has the potential to interact with FBF-2 via residues 34 and 35. Both sites are important for LST-1 activity in vivo, and either site is sufficient for germline stem cell maintenance (Haupt et al, 2019).…”

A sensitized genetic screen to identify regulators ofC. elegansgermline stem cells

“…The lst-1 locus generates two RNA isoforms -one longer, called lst-1L, and one shorter, called lst-1S (Figure 2A; Table 4). Most lst-1 alleles available prior to this work were isolated in deletion screens (Kershner et al 2014) or engineered by CRISPR/Cas9 gene editing (Haupt et al 2019a). In addition, one allele from these screens was previously reported, the nonsense mutant lst-1(q826) (Shin et al 2017).…”

“…Downstream of GLP-1/Notch, a "PUF hub" is required for self-renewal (Figure 1B). This regulatory hub comprises four genes encoding PUF RNA binding proteins as well as two direct GLP-1/Notch target genes, lst-1 and sygl-1, that encode novel PUF interacting proteins (Crittenden et al 2002;Kershner et al 2014;Shin et al 2017;Haupt et al 2019aHaupt et al , 2019bQiu et al 2019).…”