Single-molecule dynamics of the P granule scaffold MEG-3 in the Caenorhabditis elegans zygote

During the asymmetric division of the Caenorhabditis elegans zygote, germ (P) granules are disassembled in the anterior cytoplasm and stabilized/assembled in the posterior cytoplasm, leading to their inheritance by the germline daughter cell. P granule segregation depends on MEG (maternal-effect germline defective)-3 and MEG-4, which are enriched in P granules and in the posterior cytoplasm surrounding P granules. Here we use single-molecule imaging and tracking to characterize the reaction/diffusion mechanisms that result in MEG-3::Halo segregation. We find that the anteriorly enriched RNA-binding proteins MEX (muscle excess)-5 and MEX-6 suppress the retention of MEG-3 in the anterior cytoplasm, leading to MEG-3 enrichment in the posterior. We provide evidence that MEX-5/6 may work in conjunction with PLK-1 kinase to suppress MEG-3 retention in the anterior. Surprisingly, we find that the retention of MEG-3::Halo in the posterior cytoplasm surrounding P granules does not appear to contribute significantly to the maintenance of P granule asymmetry. Rather, our findings suggest that the formation of the MEG-3 concentration gradient and the segregation of P granules are two parallel manifestations of MEG-3′s response to upstream polarity cues.

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The Dynamics of P Granule Liquid Droplets Are Regulated by the Caenorhabditis elegans Germline RNA Helicase GLH-1 via Its ATP Hydrolysis Cycle

Genetics ◽

10.1534/genetics.120.303052 ◽

2020 ◽

Vol 215 (2) ◽

pp. 421-434 ◽

Cited By ~ 1

Author(s):

Wenjun Chen ◽

Yabing Hu ◽

Charles F. Lang ◽

Jordan S. Brown ◽

Sierra Schwabach ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Cell Fate ◽

Rna Binding ◽

Atp Hydrolysis ◽

Rna Binding Proteins ◽

Rna Helicase ◽

Liquid Droplets ◽

P Granules ◽

Link Type ◽

Show Evidence

P granules are phase-separated liquid droplets that play important roles in the maintenance of germ cell fate in Caenorhabditis elegans. Both the localization and formation of P granules are highly dynamic, but mechanisms that regulate such processes remain poorly understood. Here, we show evidence that the VASA-like germline RNA helicase GLH-1 couples distinct steps of its ATPase hydrolysis cycle to control the formation and disassembly of P granules. In addition, we found that the phenylalanine-glycine-glycine repeats in GLH-1 promote its localization at the perinucleus. Proteomic analyses of the GLH-1 complex with a GLH-1 mutation that interferes with P granule disassembly revealed transient interactions of GLH-1 with several Argonautes and RNA-binding proteins. Finally, we found that defects in recruiting the P granule component PRG-1 to perinuclear foci in the adult germline correlate with the fertility defects observed in various GLH-1 mutants. Together, our results highlight the versatile roles of an RNA helicase in controlling the formation of liquid droplets in space and time.

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mRNA localization is linked to translation regulation in the Caenorhabditis elegans germ lineage

10.1101/2020.01.09.900498 ◽

2020 ◽

Cited By ~ 2

Author(s):

Dylan M. Parker ◽

Lindsay P. Winkenbach ◽

Samuel P. Boyson ◽

Matthew N. Saxton ◽

Camryn Daidone ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Rna Binding ◽

Rna Binding Proteins ◽

Regulatory Control ◽

Translational Repression ◽

Translation Regulation ◽

Germline Development ◽

P Granules ◽

C Elegans ◽

Early Embryos

AbstractCaenorhabditis elegans early embryos generate cell-specific transcriptomes despite lacking active transcription. This presents an opportunity to study mechanisms of post-transcriptional regulatory control. In seeking the mechanisms behind this patterning, we discovered that some cell-specific mRNAs accumulate non-homogenously within cells, localizing to membranes, P granules (associated with progenitor germ cells in the P lineage), and P-bodies (associated with RNA processing). Transcripts differed in their dependence on 3’UTRs and RNA Binding Proteins, suggesting diverse regulatory mechanisms. Notably, we found strong but imperfect correlations between low translational status and P granule localization within the progenitor germ lineage. By uncoupling these, we untangled a long-standing question: Are mRNAs directed to P granules for translational repression or do they accumulate there as a downstream step? We found translational repression preceded P granule localization and could occur independent of it. Further, disruption of translation was sufficient to send homogenously distributed mRNAs to P granules. Overall, we show transcripts important for germline development are directed to P granules by translational repression, and this, in turn, directs their accumulation in the progenitor germ lineage where their repression can ultimately be relieved.SummaryMaternally loaded mRNAs localize non-homogeneously within C. elegans early embryos correlating with their translational status and lineage-specific fates.

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Characterization and Functional Analysis of Polyadenylation Sites in Fast and Slow Muscles

BioMed Research International ◽

10.1155/2020/2626584 ◽

2020 ◽

Vol 2020 ◽

pp. 1-13

Author(s):

Lulu Deng ◽

Long Li ◽

Cheng Zou ◽

Chengchi Fang ◽

Changchun Li

Keyword(s):

Single Molecule ◽

Posttranscriptional Regulation ◽

Muscle Development ◽

Target Genes ◽

Rna Binding ◽

De Novo ◽

Rna Binding Proteins ◽

Fast And Slow Muscles ◽

Molecular Features ◽

Polyadenylation Sites

Many increasing documents have proved that alternative polyadenylation (APA) events with different polyadenylation sites (PAS) contribute to posttranscriptional regulation. However, little is known about the detailed molecular features of PASs and its role in porcine fast and slow skeletal muscles through microRNAs (miRNAs) and RNA binding proteins (RBPs). In this study, we combined single-molecule real-time sequencing and Illumina RNA-seq datasets to comprehensively analyze polyadenylation in pigs. We identified a total of 10,334 PASs, of which 8734 were characterized by reference genome annotation. 32.86% of PAS-associated genes were determined to have more than one PAS. Further analysis demonstrated that tissue-specific PASs between fast and slow muscles were enriched in skeletal muscle development pathways. In addition, we obtained 1407 target genes regulated by APA events through potential binding 69 miRNAs and 28 RBPs in variable 3′ UTR regions and some are involved in myofiber transformation. Furthermore, the de novo motif search confirmed that the most common usage of canonical motif AAUAAA and three types of PASs may be related to the strength of motifs. In summary, our results provide a useful annotation of PASs for pig transcriptome and suggest that APA may serve as a role in fast and slow muscle development under the regulation of miRNAs and RBPs.

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KH domain containing RNA-binding proteins coordinate with microRNAs to regulate Caenorhabditis elegans development

G3 Genes|Genome|Genetics ◽

10.1093/g3journal/jkab013 ◽

2021 ◽

Vol 11 (2) ◽

Author(s):

Dustin Haskell ◽

Anna Zinovyeva

Keyword(s):

Gene Expression ◽

Caenorhabditis Elegans ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Regulate Gene Expression ◽

C Elegans ◽

Binding Domains ◽

Kh Domain ◽

Regulate Gene

Abstract MicroRNAs (miRNAs) and RNA-binding proteins (RBPs) regulate gene expression at the post-transcriptional level, but the extent to which these key regulators of gene expression coordinate their activities and the precise mechanisms of this coordination are not well understood. RBPs often have recognizable RNA binding domains that correlate with specific protein function. Recently, several RBPs containing K homology (KH) RNA binding domains were shown to work with miRNAs to regulate gene expression, raising the possibility that KH domains may be important for coordinating with miRNA pathways in gene expression regulation. To ascertain whether additional KH domain proteins functionally interact with miRNAs during Caenorhabditis elegans development, we knocked down twenty-four genes encoding KH-domain proteins in several miRNA sensitized genetic backgrounds. Here, we report that a majority of the KH domain-containing genes genetically interact with multiple miRNAs and Argonaute alg-1. Interestingly, two KH domain genes, predicted splicing factors sfa-1 and asd-2, genetically interacted with all of the miRNA mutants tested, whereas other KH domain genes showed genetic interactions only with specific miRNAs. Our domain architecture and phylogenetic relationship analyses of the C. elegans KH domain-containing proteins revealed potential groups that may share both structure and function. Collectively, we show that many C. elegans KH domain RBPs functionally interact with miRNAs, suggesting direct or indirect coordination between these two classes of post-transcriptional gene expression regulators.

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Regulation of the mitosis/meiosis decision in the Caenorhabditis elegans germline

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2003.1333 ◽

2003 ◽

Vol 358 (1436) ◽

pp. 1359-1362 ◽

Cited By ~ 60

Author(s):

Sarah L. Crittenden ◽

Christian R. Eckmann ◽

Liaoteng Wang ◽

David S. Bernstein ◽

Marvin Wickens ◽

...

Keyword(s):

Stem Cells ◽

Caenorhabditis Elegans ◽

Rna Binding ◽

Rna Binding Proteins ◽

Germline Stem Cells ◽

Puf Proteins ◽

C Elegans ◽

Transcriptional Regulatory ◽

Mitotic Proliferation ◽

Multicellular Organisms

During the development of multicellular organisms, the processes of growth and differentiation are kept in balance to generate and maintain tissues and organs of the correct size, shape and cellular composition. We have investigated the molecular controls of growth and differentiation in the Caenorhabditis elegans germline. A single somatic cell, called the distal tip cell, promotes mitotic proliferation in the adjacent germline by GLP–1/Notch signalling. Within the germline, the decisions between mitosis and meiosis and between spermatogenesis and oogenesis are controlled by a group of conserved RNA regulators. FBF, a member of the PUF (for Pumilio and FBF) family of RNA–binding proteins, promotes mitosis by repressing gld–1 mRNA activity; the GLD–1, GLD–2, GLD–3 and NOS–3 proteins promote entry into meiosis by regulating mRNAs that remain unknown. The regulatory balance between opposing FBF and GLD activities is crucial for controlling the extent of germline proliferation. PUF proteins regulate germline stem cells in both Drosophila and C. elegans and are localized to germline stem cells of the mammalian testis. Therefore, this post–transcriptional regulatory switch may be an ancient mechanism for controlling maintenance of stem cells versus differentiation.

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Stochastic dynamics of single molecules across phase boundaries

10.1101/2021.04.27.441464 ◽

2021 ◽

Author(s):

Stefano Bo ◽

Lars Hubatsch ◽

Jonathan Bauermann ◽

Christoph A. Weber ◽

Frank Julicher

Keyword(s):

Phase Separation ◽

Phase Boundary ◽

Single Molecule ◽

Concentration Gradient ◽

Detailed Balance ◽

Single Molecules ◽

Thermal Fluctuations ◽

Phase Boundaries ◽

Single Molecule Dynamics ◽

Molecule Dynamics

We discuss the stochastic trajectories of single molecules in a phase-separated liquid, when a dense and a dilute phase coexist. Starting from a continuum theory of macroscopic phase separation we derive a stochastic Langevin equation for molecular trajectories that takes into account thermal fluctuations. We find that molecular trajectories can be described as diffusion with drift in an effective potential, which has a steep gradient at phase boundaries. We discuss how the physics of phase coexistence affects the statistics of molecular trajectories and in particular the statistics of displacements of molecules crossing a phase boundary. At thermodynamic equilibrium detailed balance imposes that the distributions of displacements crossing the phase boundary from the dense or from the dilute phase are the same. Our theory can be used to infer key phase separation parameters from the statistics of single-molecule trajectories. For simple Brownian motion, there is no drift in the presence of a concentration gradient. We show that interactions in the fluid give rise to an average drift velocity in concentration gradients. Interestingly, under non-equilibrium conditions, single molecules tend to drift uphill the concentration gradient. Thus, our work bridges between single-molecule dynamics and collective dynamics at macroscopic scales and provides a framework to study single-molecule dynamics in phase-separating systems.

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Single-molecule imaging of mRNA localization and regulation during the integrated stress response

10.1101/332502 ◽

2018 ◽

Cited By ~ 4

Author(s):

Johannes H. Wilbertz ◽

Franka Voigt ◽

Ivana Horvathova ◽

Gregory Roth ◽

Yinxiu Zhan ◽

...

Keyword(s):

Stress Response ◽

Single Molecule ◽

Rna Binding ◽

Rna Binding Proteins ◽

Mrna Localization ◽

Mrna Degradation ◽

Integrated Stress Response ◽

Cellular Space ◽

Processing Bodies ◽

Functional Consequence

AbstractBiological phase transitions form membrane-less organelles that generate distinct cellular environments. How molecules are partitioned between these compartments and the surrounding cellular space and the functional consequence of this localization is not well understood. Here, we report the localization of mRNA to stress granules(SGs) and processing bodies(PBs), which are distinct biomolecular condensates, and its effect on translation and mRNA degradation during the integrated stress response. Using single mRNA imaging in living human cells, we find that the interactions of mRNAs with SGs and PBs have different dynamics and that specific RNA binding proteins can anchor mRNAs within these compartments. During recovery from stress, mRNAs that were within SGs and PBs are translated and degraded at similar rates as their cytosolic counterparts.

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An autoregulation loop in fust-1 for circular RNA regulation in Caenorhabditis elegans

10.1101/2021.03.22.436400 ◽

2021 ◽

Author(s):

Dong Cao

Keyword(s):

Caenorhabditis Elegans ◽

Rna Binding ◽

Rna Binding Proteins ◽

Exon Skipping ◽

Circular Rna ◽

Circular Rnas ◽

Rna Regulation ◽

Fused In Sarcoma ◽

Specific Factors ◽

Isoform B

Circular RNAs (circRNAs) are always expressed tissue-specifically, suggestive of specific factors that regulate their biogenesis. Here, taking advantage of available mutation strains of RNA binding proteins (RBPs) in Caenorhabditis elegans, I performed a screening of circRNA regulation in thirteen conserved RBPs. Among them, loss of FUST-1, the homolog of FUS (Fused in Sarcoma), caused downregulation of multiple circRNAs. By rescue experiments, I confirmed FUST-1 as a circRNA regulator. Further, I showed that FUST-1 regulates circRNA formation without affecting the levels of the cognate linear mRNAs. When recognizing circRNA pre-mRNAs, FUST-1 can affect both exon-skipping and circRNA in the same genes. Moreover, I identified an autoregulation loop in fust-1, where FUST-1, isoform a promotes the skipping of exon 5 of its own pre-mRNA, which produces FUST-1, isoform b with different N-terminal sequences. FUST-1, isoform a is the functional isoform in circRNA regulation. Although FUST-1, isoform b has the same functional domains as isoform a, it cannot regulate either exon-skipping or circRNA formation.

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The Impact of Pro-Inflammatory Cytokines on Alternative Splicing Patterns in Human Islets

10.2337/figshare.16828255 ◽

2021 ◽

Author(s):

Wenting Wu ◽

Farooq Syed ◽

Edward Simpson ◽

Chih-Chun Lee ◽

Jing Liu ◽

...

Keyword(s):

Alternative Splicing ◽

Mhc Class Ii ◽

Single Molecule ◽

Rna Binding ◽

Rna Binding Proteins ◽

Ex Vivo ◽

Class Ii ◽

Human Islets ◽

Β Cell ◽

The Impact

Alternative splicing (AS) within the β cell has been proposed as one potential pathway that may exacerbate autoimmunity and unveil novel immunogenic epitopes in type 1 diabetes (T1D). We employed a computational strategy to prioritize pathogenic splicing events in human islets treated with IL-1β + IFN-γ as an <i>ex vivo</i> model of T1D and coupled this analysis with a k-mer based approach to predict RNA binding proteins involved in AS. In total, 969 AS events were identified in cytokine-treated islets, with the majority (44.8%) involving a skipped exon. ExonImpact identified 129 events predicted to impact protein structure. AS occurred with high frequency in MHC Class II-related mRNAs, and targeted qPCR validated reduced inclusion of Exon5 in the MHC Class II gene HLA-DMB. Single-molecule RNA FISH confirmed increased HLA-DMB splicing in pancreatic sections from human donors with established T1D and autoantibody positivity. Serine and Arginine Rich Splicing Factor 2 was implicated in 37.2% of potentially pathogenic events, including Exon5 exclusion in HLA-DMB. Together, these data suggest that dynamic control of AS plays a role in the β cell response to inflammatory signals during T1D evolution.

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