Biology subjects

Wickens, M.

Publications and source records attributed to Wickens, M..

12 recordsLinked to original sources

bioRxiv2024

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

Like all canonical PUF proteins, C. elegans 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. Also unexpectedly, FBF-2 interactions with partner proteins can compensate for loss of RNA-binding. FBF-2 only loses biological activity when its RNA-binding and partner interactions are both defective. These findings highlight the complementary contributions of RNA-binding and protein partner interactions to activity of an RNA-binding protein.

developmental biology↗

bioRxiv2024

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-2 binding elements (FBEs) in its 3UTR. One LST-1 molecule links two FBF-2 molecules via motifs in the LST-1 intrinsically-disordered region; the gld-1 FBE pair includes a well-established canonical FBE and a newly-identified noncanonical FBE. Remarkably, this FBE pair drives both full RNA repression in GSCs and full RNA activation upon differentiation. Discovery of the LST-1-FBF-2 ternary complex, the gld-1 adjacent FBEs, and their in vivo significance predicts an expanded regulatory repertoire of different assemblies of PUF-partner complexes in nematode germline stem cells. It also suggests analogous PUF controls may await discovery in other biological contexts and organisms.

biochemistry↗

bioRxiv2023

Functional significance of PUF partnerships in C. elegans germline stem cells

PUF RNA-binding proteins are conserved stem cell regulators. Four PUF proteins govern self-renewal of C. elegans 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. Here, we investigate LST-1-PUF and SYGL-1-PUF partnerships and their molecular activities in their natural context - nematode stem cells. We confirm LST-1-PUF partnerships and their specificity to self-renewal PUFs by co-immunoprecipitation and show that an LST-1(AmBm) mutant defective for PUF-interacting motifs does not complex with PUFs in nematodes. LST-1(AmBm) is used to explore the functional significance of the LST-1-PUF partnership. Tethered LST-1 requires the partnership to repress expression of a reporter RNA, and LST-1 requires the partnership to co-immunoprecipitate with NTL-1/Not1 of the CCR4-NOT complex. We suggest that the partnership provides multiple molecular interactions that work together to form an effector complex on PUF target RNAs. Comparison of PUF-LST-1 and Pumilio-Nanos reveals fundamental molecular differences, making PUF-LST-1 a distinct paradigm for PUF partnerships. Summary statementPartnerships between PUF RNA-binding proteins and intrinsically disordered proteins are essential for stem cell maintenance and RNA repression.

developmental biology↗

bioRxiv2019

poly(UG)-tailed RNAs in Genome Protection and Epigenetic Inheritance

Mobile genetic elements threaten genome integrity in all organisms. MUT-2/RDE-3 is a ribonucleotidyltransferase required for transposon silencing and RNA interference (RNAi) in C. elegans. When tethered to RNAs in heterologous expression systems, RDE-3 can add long stretches of alternating non-templated uridine (U) and guanosine (G) ribonucleotides to the 3 termini of these RNAs (polyUG or pUG tails). Here, we show that, in its natural context in C. elegans, RDE-3 adds pUG tails to transposon RNAs, as well as to targets of RNAi. pUG tails with more than 16 perfectly alternating 3 U and G nucleotides convert otherwise inert RNA fragments into agents of gene silencing. pUG tails promote gene silencing by recruiting RNA-dependent RNA Polymerases (RdRPs), which use pUG-tailed RNAs as templates to synthesize small interfering RNAs (siRNAs). Cycles of pUG RNA-templated siRNA synthesis and siRNA-directed mRNA pUGylation underlie dsRNA-directed transgenerational epigenetic inheritance in the C. elegans germline. Our results show that pUG tails convert RNAs into transgenerational memories of past gene silencing events, which, we speculate, allow parents to inoculate progeny against the expression of unwanted or parasitic genetic elements.

molecular biology↗

bioRxiv2019

Records of RNA localization through covalent tagging

RNA movements and localization pervade biology, from embryonic development to disease. To identify RNAs at specific subcellular locations, we anchored a uridine-adding enzyme at those sites, which then marked RNAs in its vicinity with 3 terminal uridines. RNAs were tagged independent of their translation status, and included not only mRNAs, but also ncRNAs and ncRNA processing intermediates. A battery of RNAs, including the stress sensor, IRE1, were tagged at both ER and mitochondria, and reveal RNAs whose dual localization is conserved from yeast to human cells.

genomics↗

bioRxiv2019

A PUF hub drives self-renewal in C. elegans germline stem cells

Stem cell regulation relies on extrinsic signaling from a niche plus intrinsic factors that respond and drive self-renewal within stem cells. A priori, loss of niche signaling and loss of the intrinsic self-renewal factors might be expected to have equivalent stem cell defects. Yet this simple prediction has not been borne out for most stem cells, including C. elegans germline stem cells (GSCs). The central regulators of C. elegans GSCs include extrinsically-acting GLP-1/Notch signaling from the niche, intrinsically-acting RNA binding proteins in the PUF family, termed FBF-1 and FBF-2 (collectively FBF), and intrinsically-acting PUF partner proteins that are direct Notch targets. Abrogation of either GLP-1/Notch signaling or its targets yields an earlier and more severe GSC defect than loss of FBF-1 and FBF-2, suggesting that additional intrinsic regulators must exist. Here, we report that those missing regulators are two additional PUF proteins, PUF-3 and PUF-11. Remarkably, an fbf-1 fbf-2; puf-3 puf-11 quadruple null mutant has a GSC defect virtually identical to that of a glp-1/Notch null mutant. PUF-3 and PUF-11 both affect GSC maintenance; both are expressed in GSCs; and epistasis experiments place them at the same position as FBF within the network. Therefore, action of PUF-3 and PUF-11 explains the milder GSC defect in fbf-1 fbf-2 mutants. We conclude that a \"PUF hub\", comprising four PUF proteins and two PUF partners, constitutes the intrinsic self-renewal node of the C. elegans GSC RNA regulatory network. Discovery of this hub underscores the significance of PUF RNA-binding proteins as key regulators of stem cell maintenance.

developmental biology↗

bioRxiv2018

Towards identifying subnetworks from FBF binding landscapes in Caenorhabditis 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 Caenorhabditis elegans, FBF (fem-3 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. Specifically, we compared FBF iCLIP binding profiles in adults making sperm to those making oocytes. Normally, XX adults make oocytes. To generate XX adults making sperm, we used a fem-3(gf) mutant requiring growth at 25{degrees}; for comparison, wild-type oogenic hermaphrodites were also raised at 25{degrees}. Our FBF iCLIP data revealed FBF binding sites in 1522 RNAs from oogenic adults and 1704 RNAs from spermatogenic adults. More than half of these FBF targets were independent of germline gender. We next clustered RNAs by FBF-RNA complex frequencies and found four distinct blocks. Block I RNAs were enriched in spermatogenic germlines, and included validated target fog-3, while Block II and III RNAs were common to both genders, and Block IV RNAs were enriched in oogenic germlines. Block II (510 RNAs) included almost all validated FBF targets and was enriched for cell cycle regulators. Block III (21 RNAs) was enriched for RNA-binding proteins, including previously validated FBF targets gld-1 and htp-1. We suggest that Block I RNAs belong to the FBF network for spermatogenesis, and that Blocks II and III are associated with stem cell functions.

developmental biology↗

bioRxiv2018

Unbiased screen of RNA tailing enzymes at single-nucleotide resolution reveals a poly(UG) polymerase required for genome integrity and RNA silencing

Ribonucleotidyl transferases (rNTases) add non-templated ribonucleotides to diverse RNAs. We developed a screening strategy in S. cerevisiae to identify sequences added by candidate enzymes from different organisms at single-nucleotide resolution. The rNTase activities of 19 previously unexplored enzymes were determined. In addition to poly(A)- and poly(U)-adding enzymes, we identified a C-adding enzyme that is likely part of a two-enzyme system that adds CCA to tRNAs in a eukaryote; a nucleotidyl transferase that adds nucleotides to RNA without apparent nucleotide preference; and a poly(UG) polymerase, C. elegans MUT-2, which adds alternating U and G nucleotides to form poly(UG) tails. MUT-2 is known to be required for certain forms of RNA silencing, and mutations in the enzyme that are defective in silencing also fail to add poly(UG) tails in our assay. We propose that MUT-2 poly(UG) polymerase activity is required to promote genome integrity and RNA silencing.

molecular biology↗

bioRxiv2018

Nematode germ granule assembly is linked to mRNA repression

Cytoplasmic RNA-protein (RNP) granules have diverse biophysical properties, from liquid to solid, and play enigmatic roles in RNA metabolism. Nematode P-granules are paradigmatic liquid droplet granules and central to germ cell development. Here we analyze a key P-granule scaffolding protein, called PGL, to investigate the functional relationship between P-granule assembly and function. Using a protein-RNA tethering assay, we find that reporter mRNA expression is repressed when recruited to PGL granules. We determine the crystal structure of the PGL N-terminal region to 1.5 [A], discover its dimerization and identify key residues at the dimer interface. In vivo mutations of those interface residues prevent P-granule assembly, de-repress PGL-tethered mRNA and reduce fertility. Therefore, PGL dimerization lies at the heart of both P-granule assembly and function. Finally, we identify the P-granule-associated Argonaute WAGO-1 as crucial for repression of PGL-tethered mRNA. We conclude that P-granule function requires both assembly and localized regulators.

developmental biology↗

bioRxiv2017

Architecture and dynamics of overlapped RNA regulatory networks

A single protein can bind and regulate many mRNAs. Multiple proteins with similar specificities often bind and control overlapping sets of mRNAs. Yet, little is known about the architecture or dynamics of overlapped networks. We focused on three proteins with similar structures and related RNA-binding specificities - Puf3p, Puf4p, and Puf5p of S. cerevisiae. Using RNA Tagging, we identified a \"super-network\" comprised of four sub-networks: Puf3p, Puf4p, and Puf5p sub-networks, and one controlled by both Puf4p and Puf5p. The architecture of individual subnetworks, and thus the super-network, are determined by competition among particular PUF proteins to bind mRNAs, their affinities for binding elements, and the abundances of the proteins. The super-network responds dramatically: the remaining network can either expand or contract. These strikingly opposite outcomes are determined by an interplay between the relative abundance of the RNAs and proteins, and their affinities for one another. The diverse interplay between overlapping RNA-protein networks provides versatile opportunities for regulation and evolution.

molecular biology↗

bioRxiv2017

Post-Transcriptional Control of Coenzyme Q Biosynthesis Revealed by Transomic Analysis of the RNA-Binding Protein Puf3p

Coenzyme Q (CoQ) is a redox active lipid required for mitochondrial oxidative phosphorylation (OxPhos). How CoQ biosynthesis is coordinated with the biogenesis of OxPhos protein complexes is unclear. Here, we show that the Saccharomyces cerevisiae RNA-binding protein (RBP) Puf3p directly regulates CoQ biosynthesis. To establish the mechanism for this regulation, we employed a transomic strategy to identify mRNAs that not only bind Puf3p, but also are regulated by Puf3p in vivo. The CoQ biosynthesis enzyme Coq5p is a critical Put3p target: Puf3p regulates the level of Coq5p and prevents its toxicity, thereby enabling efficient CoQ production. In parallel, Puf3p represses a specific set of proteins involved in mitochondrial protein import, translation, and OxPhos complex assembly -- pathways essential to prime mitochondrial biogenesis. Our data reveal a mechanism for post-transcriptionally coordinating CoQ production with OxPhos biogenesis and, more broadly, demonstrate the power of transomics for defining genuine targets of RBPs.\n\nHIGHLIGHTSO_LIThe RNA binding protein (RBP) Puf3p regulates coenzyme Q (CoQ) biosynthesis\nC_LIO_LITransomic analysis of RNAs, proteins, lipids, and metabolites defines RBP targets\nC_LIO_LIPuf3p regulates the potentially toxic CoQ biosynthesis enzyme Coq5p\nC_LIO_LIPuf3p couples regulation of CoQ with a broader program for controlling mitochondria\nC_LI

biochemistry↗

bioRxiv2016

An RNA binding polymer specifies nematode sperm fate

Metazoan germ cells develop as sperm or oocytes, depending on chromosomal sex, extrinsic signaling from somatic tissue and intrinsic factors within the germ cells. Gamete fate regulatory networks have been analyzed in nematodes, flies and mammals, but only in C. elegans have terminal intrinsic regulators been identified, which include a Tob/BTG protein family member, FOG-3. Canonical Tob/BTG proteins function as monomeric adaptor proteins that link RNA binding proteins to deadenylases. To ask if FOG-3 functions similarly, we first determined its crystal structure. FOG-3 harbors a classical Tob/BTG fold, but unlike other Tob/BTG proteins, FOG-3 dimerizes and these FOG-3 dimers assemble into polymers. The importance of FOG-3 polymers to sperm fate specification was confirmed in vivo using CRISPR/Cas9 gene editing to create mutations designed to disrupt the polymer interface. The FOG-3 surface potential is highly basic, suggesting binding to nucleic acid. We find that FOG-3 binds RNA directly with a strong preference for 3UTRs of oogenic mRNAs. Our results reveal a divergent but striking molecular assembly for proteins with a Tob/BTG fold, make key advances in understanding the mechanism of sperm fate specification and highlight the potential for undiscovered protein polymers in biology.

developmental biology↗