A higher order PUF complex is central to regulation of C. elegans germline stem cells.

PUF RNA-binding proteins are broadly conserved stem cell regulators. Nematode PUF proteins maintain germline stem cells (GSCs) and, with key partner proteins, repress differentiation mRNAs, including gld-1. Here we report that PUF protein FBF-2 and its partner LST-1 form a ternary complex that represses gld-1 via a pair of adjacent FBF binding elements (FBEs) in its 3'UTR.

The PUF RNA-binding protein, FBF-2, maintains stem cells without binding to RNA.

Like all canonical PUF proteins, FBF-2 binds to specific RNAs via tripartite recognition motifs (TRMs). Here we report that an FBF-2 mutant protein that cannot bind to RNA, is nonetheless biologically active and maintains stem cells. This unexpected result challenges the conventional wisdom that RBPs must bind to RNAs to achieve biological activity.

A higher order PUF complex is central to regulation of germline stem cells.

PUF RNA-binding proteins are broadly conserved stem cell regulators. Nematode PUF proteins maintain germline stem cells (GSCs) and, with key partner proteins, repress differentiation mRNAs, including . Here we report that PUF protein FBF-2 and its partner LST-1 form a ternary complex that represses via a pair of adjacent FBF-2 binding elements (FBEs) in its 3ÚTR.

PUF partner interactions at a conserved interface shape the RNA-binding landscape and cell fate in Caenorhabditis elegans.

Protein-RNA regulatory networks underpin much of biology. C. elegans FBF-2, a PUF-RNA-binding protein, binds over 1,000 RNAs to govern stem cells and differentiation.

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.

Functional significance of PUF partnerships in germline stem cells.

Unlabelled: 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.

Toward Identifying Subnetworks from FBF Binding Landscapes in Spermatogenic or Oogenic Germlines.

Metazoan PUF (Pumilio and FBF) RNA-binding proteins regulate various biological processes, but a common theme across phylogeny is stem cell regulation. In , FBF ( Binding Factor) maintains germline stem cells regardless of which gamete is made, but FBF also functions in the process of spermatogenesis. We have begun to "disentangle" these biological roles by asking which FBF targets are gamete-independent, as expected for stem cells, and which are gamete-specific.

A Novel Recombinant DNA System for High Efficiency Affinity Purification of Proteins in Saccharomyces cerevisiae.

Isolation of endogenous proteins from Saccharomyces cerevisiae has been facilitated by inserting encoding polypeptide affinity tags at the C-termini of chromosomal open reading frames (ORFs) using homologous recombination of DNA fragments. Tagged protein isolation is limited by a number of factors, including high cost of affinity resins for bulk isolation and low concentration of ligands on the resin surface, leading to low isolation efficiencies and trapping of contaminants. To address this, we have created a recombinant "CelTag" DNA construct from which PCR fragments can be created to easily tag C-termini of S.