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

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

10.1101/2020.08.03.235127 ◽

2020 ◽

Author(s):

D Haskell ◽

A 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

ABSTRACTmicroRNAs (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 and the precise mechanisms of their coordination are not well understood. RNA binding proteins 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, while other KH domain genes exhibited functional 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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HRPK-1, a conserved KH-domain protein, modulates microRNA activity during Caenorhabditis elegans development

10.1101/569905 ◽

2019 ◽

Author(s):

Li Li ◽

Isana Veksler-Lublinsky ◽

Anna Y. Zinovyeva

Keyword(s):

Gene Expression ◽

Messenger Rna ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Genetic Interaction ◽

Transcriptional Level ◽

Regulate Gene Expression ◽

C Elegans ◽

Multiple Levels

AbstractmicroRNAs (miRNAs) are potent regulators of gene expression that function in diverse developmental and physiological processes. Argonaute proteins loaded with miRNAs form the miRNA Induced Silencing Complexes (miRISCs) that repress gene expression at the post-transcriptional level. miRISCs target genes through partial sequence complementarity between the miRNA and the target mRNA’s 3’ UTR. In addition to being targeted by miRNAs, these mRNAs are also extensively regulated by RNA-binding proteins (RBPs) through RNA processing, transport, stability, and translation regulation. While the degree to which RBPs and miRISCs functionally interact to regulate gene expression is likely extensive, we have only begun to unravel these functional interactions. An RNAi-based screen of putative ALG-1 Argonaute interactors has identified a role for a conserved RNA binding protein, HRPK-1, in modulating miRNA activity during C. elegans development. Here, we report the physical and genetic interaction between HRPK-1 and ALG-1/miRNAs. Specifically, we report the genetic and molecular characterizations of hrpk-1 and its role in C. elegans development and miRNA-mediated target repression. We show that loss of hrpk-1 causes numerous developmental defects and enhances the mutant phenotypes associated with reduction of miRNA activity, including those of lsy-6, mir-35-family, and let-7-family miRNAs. In addition to hrpk-1 genetic interaction with these miRNA families, hrpk-1 is required for efficient regulation of lsy-6 target cog-1. We report that hrpk-1 may play a role in miRNA processing but is not globally required for mature miRNA biogenesis or ALG-1/AIN-1 miRISC assembly and confirm HRPK-1 ability to co-precipitate with ALG-1. We suggest that HRPK-1 may functionally interact with miRNAs on multiple levels to enhance miRNA/miRISC gene regulatory activity and present several models for its activity.Author summarymicroRNAs are small non-coding RNAs that regulate gene expression at the post-transcriptional level. The core microRNA Induced Silencing Complex (miRISC), composed of Argonaute, mature microRNA, and GW182 protein effector, assembles on the target messenger RNA and inhibits translation or leads to messenger RNA degradation. RNA binding proteins interface with miRNA pathways on multiple levels to coordinate gene expression regulation. Here, we report identification and characterization of HRPK-1, a conserved RNA binding protein, as a physical and functional interactor of miRNAs. We confirm the physical interaction between HRPK-1, an hnRNPK homolog, and Argonaute ALG-1. We report characterizations of hrpk-1 role in development and its functional interactions with multiple miRNA families. We suggest that HRPK-1 promotes miRNA activity on multiple levels in part by contributing to miRNA processing and by coordinating with miRISC at the level of target RNAs. This work contributes to our understanding of how RNA binding proteins and auxiliary miRNA cofactors may interface with miRNA pathways to modulate miRNA gene regulatory activity.

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Functional Identification of MicroRNA-Centered Complexes in C. Elegans.

10.21203/rs.3.rs-1182303/v1 ◽

2022 ◽

Author(s):

Shilpa Hebbar ◽

Ganesh Panzade ◽

Ajay Vashisht ◽

James Wohlschlegel ◽

Isana Veksler-Lublinsky ◽

...

Keyword(s):

Gene Expression ◽

Binding Proteins ◽

Molecular Mechanisms ◽

Rna Binding ◽

Rna Binding Proteins ◽

Mass Spectrometry Analysis ◽

Functional Identification ◽

Regulate Gene Expression ◽

Mutant Phenotypes ◽

Regulate Gene

Abstract microRNAs (miRNAs) are crucial for normal development and physiology. To identify factors that might coordinate with miRNAs to regulate gene expression, we used 2’-O methylated oligonucleotides to precipitate Caenorhabditis elegans let-7, miR-58, and miR-2 miRNAs and the associated proteins. A total of 211 proteins were identified through mass-spectrometry analysis of miRNA co-precipitates, which included previously identified interactors of key miRNA pathway components. Gene ontology analysis of the identified interactors revealed an enrichment for RNA binding proteins, suggesting that we captured proteins that may be involved in mRNA lifecycle. To determine which miRNA interactors are important for miRNA activity, we used RNAi to deplete putative miRNA co-factors in animals with compromised miRNA activity and looked for alterations of the miRNA mutant phenotypes. Depletion of 25 of 39 tested genes modified the miRNA mutant phenotypes in three sensitized backgrounds. Modulators of miRNA phenotypes ranged from RNA binding proteins RBD-1 and CEY-1 to metabolic factors such as DLST-1 and ECH-5, among others. The observed functional interactions suggest widespread coordination of these proteins with miRNAs to ultimately regulate gene expression. This study provides a foundation for future investigations aimed at deciphering the molecular mechanisms of miRNA-mediated gene regulation.

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The Role of RNA-Binding Proteins in Vertebrate Neural Crest and Craniofacial Development

Journal of Developmental Biology ◽

10.3390/jdb9030034 ◽

2021 ◽

Vol 9 (3) ◽

pp. 34

Author(s):

Thomas E. Forman ◽

Brenna J. C. Dennison ◽

Katherine A. Fantauzzo

Keyword(s):

Gene Expression ◽

Neural Crest ◽

Binding Proteins ◽

Rna Binding ◽

Gene Expression Regulation ◽

Rna Binding Proteins ◽

Craniofacial Development ◽

Specific Sequence ◽

Altered Expression ◽

Binding Domains

Cranial neural crest (NC) cells delaminate from the neural folds in the forebrain to the hindbrain during mammalian embryogenesis and migrate into the frontonasal prominence and pharyngeal arches. These cells generate the bone and cartilage of the frontonasal skeleton, among other diverse derivatives. RNA-binding proteins (RBPs) have emerged as critical regulators of NC and craniofacial development in mammals. Conventional RBPs bind to specific sequence and/or structural motifs in a target RNA via one or more RNA-binding domains to regulate multiple aspects of RNA metabolism and ultimately affect gene expression. In this review, we discuss the roles of RBPs other than core spliceosome components during human and mouse NC and craniofacial development. Where applicable, we review data on these same RBPs from additional vertebrate species, including chicken, Xenopus and zebrafish models. Knockdown or ablation of several RBPs discussed here results in altered expression of transcripts encoding components of developmental signaling pathways, as well as reduced cell proliferation and/or increased cell death, indicating that these are common mechanisms contributing to the observed phenotypes. The study of these proteins offers a relatively untapped opportunity to provide significant insight into the mechanisms underlying gene expression regulation during craniofacial morphogenesis.

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CELF RNA binding proteins promote axon regeneration in C. elegans and mammals through alternative splicing of Syntaxins

eLife ◽

10.7554/elife.16072 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 13

Author(s):

Lizhen Chen ◽

Zhijie Liu ◽

Bing Zhou ◽

Chaoliang Wei ◽

Yu Zhou ◽

...

Keyword(s):

Gene Expression ◽

Transcriptional Regulation ◽

Alternative Splicing ◽

Binding Proteins ◽

Rna Binding ◽

Axon Regeneration ◽

Rna Binding Proteins ◽

Mrna Isoforms ◽

C Elegans ◽

Mrna Targets

Axon injury triggers dramatic changes in gene expression. While transcriptional regulation of injury-induced gene expression is widely studied, less is known about the roles of RNA binding proteins (RBPs) in post-transcriptional regulation during axon regeneration. In C. elegans the CELF (CUGBP and Etr-3 Like Factor) family RBP UNC-75 is required for axon regeneration. Using crosslinking immunoprecipitation coupled with deep sequencing (CLIP-seq) we identify a set of genes involved in synaptic transmission as mRNA targets of UNC-75. In particular, we show that UNC-75 regulates alternative splicing of two mRNA isoforms of the SNARE Syntaxin/unc-64. In C. elegans mutants lacking unc-75 or its targets, regenerating axons form growth cones, yet are deficient in extension. Extending these findings to mammalian axon regeneration, we show that mouse Celf2 expression is upregulated after peripheral nerve injury and that Celf2 mutant mice are defective in axon regeneration. Further, mRNAs for several Syntaxins show CELF2 dependent regulation. Our data delineate a post-transcriptional regulatory pathway with a conserved role in regenerative axon extension.

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Faculty Opinions recommendation of C. elegans RNA-binding proteins PUF-8 and MEX-3 function redundantly to promote germline stem cell mitosis.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.2916956.2587054 ◽

2010 ◽

Author(s):

Jane Hubbard

Keyword(s):

Stem Cell ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Germline Stem Cell ◽

C Elegans ◽

Cell Mitosis

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The Interplay of RNA Binding Proteins, Oxidative Stress and Mitochondrial Dysfunction in ALS

Antioxidants ◽

10.3390/antiox10040552 ◽

2021 ◽

Vol 10 (4) ◽

pp. 552

Author(s):

Jasmine Harley ◽

Benjamin E. Clarke ◽

Rickie Patani

Keyword(s):

Oxidative Stress ◽

Gene Expression ◽

Mitochondrial Dysfunction ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Binding Protein ◽

Rna Binding Protein ◽

Therapeutic Strategies ◽

Lateral Sclerosis

RNA binding proteins fulfil a wide number of roles in gene expression. Multiple mechanisms of RNA binding protein dysregulation have been implicated in the pathomechanisms of several neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). Oxidative stress and mitochondrial dysfunction also play important roles in these diseases. In this review, we highlight the mechanistic interplay between RNA binding protein dysregulation, oxidative stress and mitochondrial dysfunction in ALS. We also discuss different potential therapeutic strategies targeting these pathways.

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RNA-binding proteins regulate aldosterone homeostasis in human steroidogenic cells

10.1101/2021.02.19.431050 ◽

2021 ◽

Author(s):

Rui Fu ◽

Kimberly Wellman ◽

Amber Baldwin ◽

Juilee Rege ◽

Kathryn Walters ◽

...

Keyword(s):

Gene Expression ◽

Time Course ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Ex Vivo ◽

Human Cell Line ◽

Rna Decay ◽

Type I ◽

Aldosterone Production

ABSTRACTAngiotensin II (AngII) binds to the type I angiotensin receptor in the adrenal cortex to initiate a cascade of events leading to the production of aldosterone, a master regulator of blood pressure. Despite extensive characterization of the transcriptional and enzymatic control of adrenocortical steroidogenesis, there are still major gaps in our knowledge related to precise regulation of AII-induced gene expression kinetics. Specifically, we do not know the regulatory contribution of RNA-binding proteins (RBPs) and RNA decay, which can control the timing of stimulus-induced gene expression. To investigate this question, we performed a high-resolution RNA-seq time course of the AngII stimulation response and 4-thiouridine pulse labeling in a steroidogenic human cell line (H295R). We identified twelve temporally distinct gene expression responses that contained mRNA encoding proteins known to be important for various steps of aldosterone production, such as cAMP signaling components and steroidogenic enzymes. AngII response kinetics for many of these mRNAs revealed a coordinated increase in both synthesis and decay. These findings were validated in primary human adrenocortical cells stimulated ex vivo with AngII. Using a candidate siRNA screen, we identified a subset of RNA-binding protein and RNA decay factors that activate or repress AngII-stimulated aldosterone production. Among the repressors of aldosterone were BTG2, which promotes deadenylation and global RNA decay. BTG2 was induced in response to AngII stimulation and promoted the repression of mRNAs encoding pro-steroidogenic factors indicating the existence of an incoherent feedforward loop controlling aldosterone homeostasis. Together, these data support a model in which coordinated increases in transcription and regulated RNA decay facilitates the major transcriptomic changes required to implement a pro-steroidogenic gene expression program that is temporally restricted to prevent aldosterone overproduction.

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PUB1: a major yeast poly(A)+ RNA-binding protein

Molecular and Cellular Biology ◽

10.1128/mcb.13.10.6114-6123.1993 ◽

1993 ◽

Vol 13 (10) ◽

pp. 6114-6123

Author(s):

M J Matunis ◽

E L Matunis ◽

G Dreyfuss

Keyword(s):

Rna Polymerase Ii ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Uv Light ◽

Binding Protein ◽

Gene Replacement ◽

Yeast Saccharomyces Cerevisiae ◽

Binding Domains ◽

Development And Differentiation

The expression of RNA polymerase II transcripts can be regulated at the posttranscriptional level by RNA-binding proteins. Although extensively characterized in metazoans, relatively few RNA-binding proteins have been characterized in the yeast Saccharomyces cerevisiae. Three major proteins are cross-linked by UV light to poly(A)+ RNA in living S. cerevisiae cells. These are the 72-kDa poly(A)-binding protein and proteins of 60 and 50 kDa (S.A. Adam, T.Y. Nakagawa, M.S. Swanson, T. Woodruff, and G. Dreyfuss, Mol. Cell. Biol. 6:2932-2943, 1986). Here, we describe the 60-kDa protein, one of the major poly(A)+ RNA-binding proteins in S. cerevisiae. This protein, PUB1 [for poly(U)-binding protein 1], was purified by affinity chromatography on immobilized poly(rU), and specific monoclonal antibodies to it were produced. UV cross-linking demonstrated that PUB1 is bound to poly(A)+ RNA (mRNA or pre-mRNA) in living cells, and it was detected primarily in the cytoplasm by indirect immunofluorescence. The gene for PUB1 was cloned and sequenced, and the sequence was found to predict a 51-kDa protein with three ribonucleoprotein consensus RNA-binding domains and three glutamine- and asparagine-rich auxiliary domains. This overall structure is remarkably similar to the structures of the Drosophila melanogaster elav gene product, the human neuronal antigen HuD, and the cytolytic lymphocyte protein TIA-1. Each of these proteins has an important role in development and differentiation, potentially by affecting RNA processing. PUB1 was found to be nonessential in S. cerevisiae by gene replacement; however, further genetic analysis should reveal important features of this class of RNA-binding proteins.

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