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. FBF-2 interacts with multiple protein partners via a key tyrosine, Y479. Here, we investigate the in vivo significance of partnerships using a Y479A mutant. Occupancy of the Y479A mutant protein increases or decreases at specific sites across the transcriptome, varying with RNAs. Germline development also changes in a specific fashion: Y479A abolishes one FBF-2 function-the sperm-to-oocyte cell fate switch. Y479A's effects on the regulation of one mRNA, gld-1, are critical to this fate change, though other network changes are also important. FBF-2 switches from repression to activation of gld-1 RNA, likely by distinct FBF-2 partnerships. The role of RNA-binding protein partnerships in governing RNA regulatory networks will likely extend broadly, as such partnerships pervade RNA controls in virtually all metazoan tissues and species.

SYGL-1 and LST-1 link niche signaling to PUF RNA repression for stem cell maintenance in Caenorhabditis elegans.

Central questions in regenerative biology include how stem cells are maintained and how they transition from self-renewal to differentiation. Germline stem cells (GSCs) in Caeno-rhabditis elegans provide a tractable in vivo model to address these questions. In this system, Notch signaling and PUF RNA binding proteins, FBF-1 and FBF-2 (collectively FBF), maintain a pool of GSCs in a naïve state. An open question has been how Notch signaling modulates FBF activity to promote stem cell self-renewal. Here we report that two Notch targets, SYGL-1 and LST-1, link niche signaling to FBF. We find that SYGL-1 and LST-1 proteins are cytoplasmic and normally restricted to the GSC pool region. Increasing the distribution of SYGL-1 expands the pool correspondingly, and vast overexpression of either SYGL-1 or LST-1 generates a germline tumor. Thus, SYGL-1 and LST-1 are each sufficient to drive "stemness" and their spatial restriction prevents tumor formation. Importantly, SYGL-1 and LST-1 can only drive tumor formation when FBF is present. Moreover, both proteins interact physically with FBF, and both are required to repress a signature FBF mRNA target. Together, our results support a model in which SYGL-1 and LST-1 form a repressive complex with FBF that is crucial for stem cell maintenance. We further propose that progression from a naïve stem cell state to a state primed for differentiation relies on loss of SYGL-1 and LST-1, which in turn relieves FBF target RNAs from repression. Broadly, our results provide new insights into the link between niche signaling and a downstream RNA regulatory network and how this circuitry governs the balance between self-renewal and differentiation.

The PUF binding landscape in metazoan germ cells.

PUF (Pumilio/FBF) proteins are RNA-binding proteins and conserved stem cell regulators. The Caenorhabditis elegans PUF proteins FBF-1 and FBF-2 (collectively FBF) regulate mRNAs in germ cells. Without FBF, adult germlines lose all stem cells. A major gap in our understanding of PUF proteins, including FBF, is a global view of their binding sites in their native context (i.e., their "binding landscape"). To understand the interactions underlying FBF function, we used iCLIP (individual-nucleotide resolution UV crosslinking and immunoprecipitation) to determine binding landscapes of C. elegans FBF-1 and FBF-2 in the germline tissue of intact animals. Multiple iCLIP peak-calling methods were compared to maximize identification of both established FBF binding sites and positive control target mRNAs in our iCLIP data. We discovered that FBF-1 and FBF-2 bind to RNAs through canonical as well as alternate motifs. We also analyzed crosslinking-induced mutations to map binding sites precisely and to identify key nucleotides that may be critical for FBF-RNA interactions. FBF-1 and FBF-2 can bind sites in the 5'UTR, coding region, or 3'UTR, but have a strong bias for the 3' end of transcripts. FBF-1 and FBF-2 have strongly overlapping target profiles, including mRNAs and noncoding RNAs. From a statistically robust list of 1404 common FBF targets, 847 were previously unknown, 154 were related to cell cycle regulation, three were lincRNAs, and 335 were shared with the human PUF protein PUM2.

RNA targets and specificity of Staufen, a double-stranded RNA-binding protein in Caenorhabditis elegans.

The Staufen family consists of proteins that possess double-stranded RNA-binding domains (dsRBDs). Staufen proteins of Drosophila and mammals regulate mRNA localization, translation, and decay. We report analysis of Staufen in Caenorhabditis elegans, which we have designated STAU-1. We focus on its biochemical properties, mRNA targets, and possible role in RNAi. We show that STAU-1 is expressed as mRNA and protein at all stages of C. elegans development. The wild-type, full-length protein, purified from bacteria, binds duplex RNA with high affinity in vitro. Purified, mutant proteins lacking single dsRBDs still bind RNA efficiently, demonstrating that no single domain is required for binding to duplex RNA (although dsRBD2 could not be tested). STAU-1 mRNA targets were identified via immunoprecipitation with specific anti-STAU-1 antibodies, followed by microarray analysis (RIP-Chip). These studies define a set of 418 likely STAU-1 mRNA targets. Finally, we demonstrate that stau-1 mutants enhance exogenous RNAi and that stau-1;eri-1 double mutants exhibit sterility and synthetic germ line defects.

A conserved PUF-Ago-eEF1A complex attenuates translation elongation.

PUF (Pumilio/FBF) RNA-binding proteins and Argonaute (Ago) miRNA-binding proteins regulate mRNAs post-transcriptionally, each acting through similar, yet distinct, mechanisms. Here, we report that PUF and Ago proteins can also function together in a complex with a core translation elongation factor, eEF1A, to repress translation elongation. Both nematode (Caenorhabditis elegans) and mammalian PUF-Ago-eEF1A complexes were identified, using coimmunoprecipitation and recombinant protein assays. Nematode CSR-1 (Ago) promoted repression of FBF (PUF) target mRNAs in in vivo assays, and the FBF-1-CSR-1 heterodimer inhibited EFT-3 (eEF1A) GTPase activity in vitro. Mammalian PUM2-Ago-eEF1A inhibited translation of nonadenylated and polyadenylated reporter mRNAs in vitro. This repression occurred after translation initiation and led to ribosome accumulation within the open reading frame, roughly at the site where the nascent polypeptide emerged from the ribosomal exit tunnel. Together, these data suggest that a conserved PUF-Ago-eEF1A complex attenuates translation elongation.