in-situ-detection-ribonucleoprotein-complex-assembly-c-elegans

In Situ Detection of Ribonucleoprotein Complex Assembly in the <em>C. elegans</em> Germline using Proximity Ligation Assay

Understanding when and where protein-protein interactions (PPIs) occur is critical to understanding protein function in the cell and how broader processes ...

University of Montana

P-granules are conserved cytoplasmic organelles, similar to nuage, that are present in Caenorhabditis elegans germ cells. Based on the prevailing sterility phenotype of the component mutants, P-granules have been seen as regulators of germ cell development and function. Yet, specific germline defects resulting from P-granule failure vary, depending on which component(s) are inactivated, at which stage of development, as well as on the presence of stress factors during animal culture. This review discusses the unifying themes in many P-granule functions, with the main focus on their role as organizing centers nucleating RNA regulation in the germ cell cytoplasm.

The diverse functions of germline P-granules in Caenorhabditis elegans.

Genetic interaction screens are an important approach for understanding complex regulatory networks governing development. We used a genetic interaction screen to identify cofactors of FBF-1 and FBF-2, RNA-binding proteins that regulate germline stem cell proliferation in Caenorhabditis elegans. We found that components of splicing machinery contribute to FBF activity as splicing factor knockdowns enhance sterility of fbf-1 and fbf-2 single mutants. This sterility phenocopied multiple aspects of loss of fbf function, suggesting that splicing factors contribute to stem cell maintenance. However, previous reports indicate that splicing factors instead promote the opposite cell fate, namely, differentiation. We explain this discrepancy by proposing that splicing factors facilitate overall RNA regulation in the germline. Indeed, we find that loss of splicing factors produces synthetic phenotypes with a mutation in another RNA regulator, FOG-1, but not with a mutation in a gene unrelated to posttranscriptional regulation (dhc-1). We conclude that inefficient pre-mRNA splicing may interfere with multiple posttranscriptional regulatory events, which has to be considered when interpreting results of genetic interaction screens. 

Splicing Machinery Facilitates Post-Transcriptional Regulation by FBFs and Other RNA-Binding Proteins in Caenorhabditis elegans Germline.

Drosophila germ cell specification depends on localization of mRNAs required for patterning to the posterior of the oocyte during oogenesis. In a recent issue of Nature, Vourekas et al. (2016) suggest that Aubergine in complex with piRNAs may provide a low-specificity anchoring mechanism for posterior mRNAs.

mRNAs Hit a Sticky Wicket.

The KDM4 histone demethylases are conserved epigenetic regulators linked to development, spermatogenesis and tumorigenesis. However, how the KDM4 family targets specific chromatin regions is largely unknown. Here, an extensive histone peptide microarray analysis uncovers trimethyl-lysine histone-binding preferences among the closely related KDM4 double tudor domains (DTDs). KDM4A/B DTDs bind strongly to H3K23me3, a poorly understood histone modification recently shown to be enriched in meiotic chromatin of ciliates and nematodes. The 2.28 Å co-crystal structure of KDM4A-DTD in complex with H3K23me3 peptide reveals key intermolecular interactions for H3K23me3 recognition. Furthermore, analysis of the 2.56 Å KDM4B-DTD crystal structure pinpoints the underlying residues required for exclusive H3K23me3 specificity, an interaction supported by in vivo co-localization of KDM4B and H3K23me3 at heterochromatin in mammalian meiotic and newly postmeiotic spermatocytes. In vitro demethylation assays suggest H3K23me3 binding by KDM4B stimulates H3K36 demethylation. Together, these results provide a possible mechanism whereby H3K23me3-binding by KDM4B directs localized H3K36 demethylation during meiosis and spermatogenesis.

Reader domain specificity and lysine demethylase-4 family function.

PUF family translational repressors are conserved developmental regulators, but the molecular function provided by the regions flanking the PUF RNA-binding domain is unknown. In C. elegans, the PUF proteins FBF-1 and FBF-2 support germline progenitor maintenance by repressing production of meiotic proteins and use distinct mechanisms to repress their target mRNAs. We identify dynein light chain DLC-1 as an important regulator of FBF-2 function. DLC-1 directly binds to FBF-2 outside of the RNA-binding domain and promotes FBF-2 localization and function. By contrast, DLC-1 does not interact with FBF-1 and does not contribute to FBF-1 activity. Surprisingly, we find that the contribution of DLC-1 to FBF-2 activity is independent of the dynein motor. Our findings suggest that PUF protein localization and activity are mediated by sequences flanking the RNA-binding domain that bind specific molecular partners. Furthermore, these results identify a new role for DLC-1 in post-transcriptional regulation of gene expression.

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

The nematode Caenorhabditis elegans is well established as a system for characterization and discovery of molecular mechanisms mediating microbe-specific inducible innate immune responses to human pathogens. Coxiella burnetii is an obligate intracellular bacterium that causes a flu-like syndrome in humans (Q fever), as well as abortions in domesticated livestock, worldwide. Initially, when wild type C. elegans (N2 strain) was exposed to mCherry-expressing C. burnetii (CCB) a number of overt pathological manifestations resulted, including intestinal distension, deformed anal region and a decreased lifespan. However, nematodes fed autoclave-killed CCB did not exhibit these symptoms. Although vertebrates detect C. burnetii via TLRs, pathologies in tol-1(-) mutant nematodes were indistinguishable from N2, and indicate nematodes do not employ this orthologue for detection of C. burnetii. sek-1(-) MAP kinase mutant nematodes succumbed to infection faster, suggesting that this signaling pathway plays a role in immune activation, as previously shown for orthologues in vertebrates during a C. burnetii infection. C. elegans daf-2(-) mutants are hyper-immune and exhibited significantly reduced pathological consequences during challenge. Collectively, these results demonstrate the utility of C. elegans for studying the innate immune response against C. burnetii and could lead to discovery of novel methods for prevention and treatment of disease in humans and livestock.

Analysis of the Caenorhabditis elegans innate immune response to Coxiella burnetii.

Ribonucleoprotein complexes, which contain mRNAs and their regulator proteins, carry out post-transcriptional control of gene expression. The function of many RNA-binding proteins depends on their association with cofactors. Here, we use a genomic approach to identify transcripts associated with DLC-1, a protein previously identified as a cofactor of two unrelated RNA-binding proteins that act in the Caenorhabditis elegans germline. Among the 2732 potential DLC-1 targets, most are germline mRNAs associated with oogenesis. Removal of DLC-1 affects expression of its targets expressed in the oocytes, meg-1 and meg-3. We propose that DLC-1 acts as a cofactor for multiple ribonucleoprotein complexes, including the ones regulating gene expression during oogenesis.

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

Developmental transitions of germ cells are often regulated at the level of post-transcriptional control of gene expression. In the  germline, stem and progenitor cells exit the proliferative phase and enter meiotic differentiation to form gametes essential for fertility. The RNA binding protein GLD-1 is a cell fate regulator that promotes meiosis and germ cell differentiation during development by binding to and repressing translation of target messenger RNAs. Here, we discovered that some GLD-1 functions are promoted by binding to DLC-1, a small protein that functions as an allosteric regulator of multisubunit protein complexes. We found that DLC-1 is required to regulate a subset of GLD-1 target messenger RNAs and that DLC-1 binding GLD-1 prevents ectopic germ cell proliferation and facilitates gametogenesis  Additionally, our results reveal a new requirement for GLD-1 in the events of oogenesis leading to ovulation. DLC-1 contributes to GLD-1 function independent of its role as a light chain component of the dynein motor. Instead, we propose that DLC-1 promotes assembly of GLD-1 with other binding partners, which facilitates formation of regulatory ribonucleoprotein complexes and may direct GLD-1 target messenger RNA selectivity.

Dynein Light Chain DLC-1 Facilitates the Function of the Germline Cell Fate Regulator GLD-1 in .

Stem cell development depends on post-transcriptional regulation mediated by RNA-binding proteins (RBPs) (Zhang et al., 1997; Forbes and Lehmann, 1998; Okano et al., 2005; Ratti et al., 2006; Kwon et al., 2013). Pumilio and FBF (PUF) family RBPs are highly conserved post-transcriptional regulators that are critical for stem cell maintenance (Wickens et al., 2002; Quenault et al., 2011). The RNA-binding domains of PUF proteins recognize a family of related sequence motifs in the target mRNAs, yet individual PUF proteins have clearly distinct biological functions (Lu et al., 2009; Wang et al., 2018). The  germline is a simple and powerful model system for analyzing regulation of stem cell development. Studies in  uncovered specific physiological roles for PUFs expressed in the germline stem cells ranging from control of proliferation and differentiation to regulation of the sperm/oocyte decision. Importantly, recent studies started to illuminate the mechanisms behind PUF functional divergence. This review summarizes the many roles of PUF-8, FBF-1, and FBF-2 in germline stem and progenitor cells (SPCs) and discusses the factors accounting for their distinct biological functions. PUF proteins are conserved in evolution, and insights into PUF-mediated regulation provided by the  model system are likely relevant for other organisms.

Article	Year
The diverse functions of germline P-granules in Caenorhabditis elegans. Molecular reproduction and development\| PubMed ID: 23150384	2013
Splicing Machinery Facilitates Post-Transcriptional Regulation by FBFs and Other RNA-Binding Proteins in Caenorhabditis elegans Germline. G3 (Bethesda, Md.)\| PubMed ID: 26268245	2015
mRNAs Hit a Sticky Wicket. Developmental cell\| PubMed ID: 27046827	2016
Reader domain specificity and lysine demethylase-4 family function. Nature communications\| PubMed ID: 27841353	2016
Dynein light chain DLC-1 promotes localization and function of the PUF protein FBF-2 in germline progenitor cells. Development (Cambridge, England)\| PubMed ID: 27864381	2016
Analysis of the Caenorhabditis elegans innate immune response to Coxiella burnetii. Innate immunity\| PubMed ID: 27884946	2017
Caenorhabditis elegans DLC-1 associates with ribonucleoprotein complexes to promote mRNA regulation. FEBS letters\| PubMed ID: 30264890	2018
Dynein Light Chain DLC-1 Facilitates the Function of the Germline Cell Fate Regulator GLD-1 in . Genetics\| PubMed ID: 30509955	2019
Diverse Roles of PUF Proteins in Germline Stem and Progenitor Cell Development in . Frontiers in cell and developmental biology\| PubMed ID: 32117964	2020

Ekaterina Voronina

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