The PUF binding landscape in metazoan germ cells

Abstract

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.

Dynein light chain DLC-1 promotes localization and function of the PUF protein FBF-2 in germline progenitor cells

“…In the absence of both FBF-1 and FBF-2, all cells in the mitotic zone precociously enter meiosis after the L4 stage of development when maintained at 20°C (Crittenden et al, 2002), but are maintained in a mitotic state if grown at 25°C (Merritt and Seydoux, 2010). FBF-1 and FBF-2 recognize the same motif present in the 3′UTR of their target mRNAs in vitro and form complexes with largely the same mRNAs in vivo Merritt and Seydoux, 2010;Prasad et al, 2016). Despite high similarity between FBF-1 and FBF-2 (89% identity at the amino acid level) and apparent redundancy in their control of the switch from spermatogenesis to oogenesis, single fbf-1 and fbf-2 mutants have distinct phenotypes, suggesting that these genes play unique roles (Lamont et al, 2004).…”

Caenorhabditis elegans DLC‐1 associates with ribonucleoprotein complexes to promote mRNA regulation

“…We observed significant (hypergeometric distribution P < 0.05) overlap of DLC‐1‐associated mRNAs with the targets of FBF‐1 ( P < 10 −59 ), GLD‐2 ( P < 10 −7 ), LIN‐41 ( P < 0.01), and OMA‐1 ( P = 0.02; Table ). The significant overlap with FBF‐1 targets was expected since the targets of FBF‐1 and FBF‐2 are highly similar .…”

“…Indeed, within the 10 most significant recovered motifs, we found two motifs similar to those recognized by PUF family RNA-binding proteins that include FBF-2 ( Fig. 3B, C; [24,53]). The top-scoring motif is a lowaffinity OMA-1-binding site (UA[a/u]-rich repeats, Fig.…”

“…Despite similar protein expression patterns and related function, mRNAs encoding the MEG proteins might be differentially regulated as they have been recovered in association with distinct RNA‐binding proteins. meg‐1 mRNA was found in complexes with GLD‐1, GLD‐2, LIN‐41, RNP‐8, and FBF‐1 and its expression is regulated by LIN‐41, OMA‐1, and OMA‐2 . meg‐3 mRNA has not been identified in complex with germline RBPs so far, and meg‐4 mRNA was recovered with GLD‐2 and RNP‐8 .…”

Quantifying RNA binding sites transcriptome-wide using DO-RIP-seq

“…Despite their different methodologies, RIP and CLIP can produce complementary data when used to study the same RBPs under similar conditions (van der Brug et al 2008;Sanford et al 2009;Kershner and Kimble 2010;Jungkamp et al 2011;Mukherjee et al 2011;Brooks et al 2015;Hansen et al 2015;Ennajdaoui et al 2016;Prasad et al 2016;Uren et al 2016). Therefore, our goal has been to combine aspects of both techniques to produce a single procedure that quantifies RBP-RNA interactions at the whole-transcript level and at the binding site level.…”