Drosophila Suppressor of Sable Protein [Su(s)] Promotes Degradation of Aberrant and Transposon-Derived RNAs

ABSTRACT RNA-binding proteins act at various stages of gene expression to regulate and fine-tune patterns of mRNA accumulation. One protein in this class is Drosophila Su(s), a nuclear protein that has been previously shown to inhibit the accumulation of mutant transcripts by an unknown mechanism. Here, we have identified several additional RNAs that are downregulated by Su(s). These Su(s) targets include cryptic wild-type transcripts from the developmentally regulated Sgs4 and ng1 genes, noncoding RNAs derived from tandemly repeated αβ/αγ elements within an Hsp70 locus, and aberrant transcripts induced by Hsp70 promoter transgenes inserted at ectopic sites. We used the αβ RNAs to investigate the mechanism of Su(s) function and obtained evidence that these transcripts are degraded by the nuclear exosome and that Su(s) promotes this process. Furthermore, we showed that the RNA binding domains of Su(s) are important for this effect and mapped the sequences involved to a 267-nucleotide region of an αβ element. Taken together, these results suggest that Su(s) binds to certain nascent transcripts and stimulates their degradation by the nuclear exosome.

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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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Investigating the Roles of RNA Binding Protein Combinations in Neuronal Function and Organismal Behavior

10.25172/jour.4.1.7 ◽

2019 ◽

Vol 4 (Spring 2019) ◽

Author(s):

Alexa Vandenburg

Keyword(s):

Binding Sites ◽

Neuronal Development ◽

Rna Binding ◽

Rna Binding Proteins ◽

Binding Protein ◽

Rna Binding Protein ◽

Wild Type ◽

C Elegans ◽

Neuronal Gene ◽

Touch Response

The Norris lab recently identified two RNA binding proteins required for proper neuron-specific splicing. The lab conducted touch- response behavioral assays to assess the function of these proteins in touch-sensing neurons. After isolating C. elegans worms with specific phenotypes, the lab used automated computer tracking and video analysis to record the worms’ behavior. The behavior of mutant worms differed from that of wild-type worms. The Norris lab also discovered two possible RNA binding protein sites in SAD-1, a neuronal gene implicated in the neuronal development of C. elegans1. These two binding sites may control the splicing of SAD-1. The lab transferred mutated DNA into the genome of wild-type worms by injecting a mutated plasmid. The newly transformed worms fluoresced green, indicating that the two binding sites control SAD-1 splicing.

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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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The structural basis for RNA selectivity by the IMP family of RNA-binding proteins

Nature Communications ◽

10.1038/s41467-019-12193-7 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 4

Author(s):

Jeetayu Biswas ◽

Vivek L. Patel ◽

Varun Bhaskar ◽

Jeffrey A. Chao ◽

Robert H. Singer ◽

...

Keyword(s):

Neural Development ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Rna Stability ◽

Underlying Mechanism ◽

Structural Basis ◽

General Mechanism ◽

Binding Domains ◽

Variable Loops

Abstract The IGF2 mRNA-binding proteins (ZBP1/IMP1, IMP2, IMP3) are highly conserved post-transcriptional regulators of RNA stability, localization and translation. They play important roles in cell migration, neural development, metabolism and cancer cell survival. The knockout phenotypes of individual IMP proteins suggest that each family member regulates a unique pool of RNAs, yet evidence and an underlying mechanism for this is lacking. Here, we combine systematic evolution of ligands by exponential enrichment (SELEX) and NMR spectroscopy to demonstrate that the major RNA-binding domains of the two most distantly related IMPs (ZBP1 and IMP2) bind to different consensus sequences and regulate targets consistent with their knockout phenotypes and roles in disease. We find that the targeting specificity of each IMP is determined by few amino acids in their variable loops. As variable loops often differ amongst KH domain paralogs, we hypothesize that this is a general mechanism for evolving specificity and regulation of the transcriptome.

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Zis: a developmentally regulated gene expressed in juxtaglomerular cells

AJP Renal Physiology ◽

10.1152/ajprenal.1997.273.5.f731 ◽

1997 ◽

Vol 273 (5) ◽

pp. F731-F738 ◽

Cited By ~ 10

Author(s):

Elena A. Karginova ◽

Ellen Steward Pentz ◽

Irina G. Kazakova ◽

Victoria F. Norwood ◽

Robert M. Carey ◽

...

Keyword(s):

Cell Culture ◽

Rna Binding ◽

Rna Binding Proteins ◽

Renin Angiotensin System ◽

Organ Distribution ◽

Regulation Of Transcription ◽

Main Function ◽

Developmentally Regulated ◽

Arterial Blood ◽

Renin Mrna

Renal juxtaglomerular (JG) cells are specialized myoepithelioid cells located in the afferent arteriole at the entrance to the glomerulus. Their main function and distinctive feature is the synthesis and release of renin, the key hormone-enzyme of the renin-angiotensin system that regulates arterial blood pressure. Despite their relevance to health and disease, not much is known about factors that confer and/or maintain JG cell identity. To identify genes uniquely expressed in JG cells, we used a cell culture model and RNA differential display. JG cells cultured for 2 days express renin and renin mRNA, but after 10 days in culture they no longer contain or release renin and renin mRNA is reduced 700-fold. We report one cDNA differentially expressed in the 2-day JG cell culture that detects a 2.6-kb mRNA expressed at higher levels in newborn than adult kidney. Screening a 2-day culture JG cell cDNA library yielded clones representing differentially spliced transcripts. These cDNAs encode one unique protein (Zis) containing zinc fingers and domains characteristic of splicing factors and RNA binding proteins. Northern blot analysis confirmed Zis mRNA expression in differentiated JG cells, and identified an additional unique 1.5-kb transcript. The Zis transcripts are developmentally regulated in kidney and a number of other organs. The features of the Zis protein and its organ distribution suggest a possible role in regulation of transcription and/or splicing, both important steps for controlling developmentally expressed genes.

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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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A nuclear localization domain in the hnRNP A1 protein.

The Journal of Cell Biology ◽

10.1083/jcb.129.3.551 ◽

1995 ◽

Vol 129 (3) ◽

pp. 551-560 ◽

Cited By ~ 343

Author(s):

H Siomi ◽

G Dreyfuss

Keyword(s):

Nuclear Localization ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Binding Motif ◽

Hnrnp A1 ◽

Binding Domains ◽

Rich Domain ◽

Localization Domain ◽

Hnrnp Proteins

The heterogeneous nuclear RNP (hnRNP) A1 protein is one of the major pre-mRNA/mRNA binding proteins in eukaryotic cells and one of the most abundant proteins in the nucleus. It is localized to the nucleoplasm and it also shuttles between the nucleus and the cytoplasm. The amino acid sequence of A1 contains two RNP motif RNA-binding domains (RBDs) at the amino terminus and a glycine-rich domain at the carboxyl terminus. This configuration, designated 2x RBD-Gly, is representative of perhaps the largest family of hnRNP proteins. Unlike most nuclear proteins characterized so far, A1 (and most 2x RBD-Gly proteins) does not contain a recognizable nuclear localization signal (NLS). We have found that a segment of ca. 40 amino acids near the carboxyl end of the protein (designated M9) is necessary and sufficient for nuclear localization; attaching this segment to the bacterial protein beta-galactosidase or to pyruvate kinase completely localized these otherwise cytoplasmic proteins to the nucleus. The RBDs and another RNA binding motif found in the glycine-rich domain, the RGG box, are not required for A1 nuclear localization. M9 is a novel type of nuclear localization domain as it does not contain sequences similar to classical basic-type NLS. Interestingly, sequences similar to M9 are found in other nuclear RNA-binding proteins including hnRNP A2.

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The CELF Family of RNA Binding Proteins Is Implicated in Cell-Specific and Developmentally Regulated Alternative Splicing

Molecular and Cellular Biology ◽

10.1128/mcb.21.4.1285-1296.2001 ◽

2001 ◽

Vol 21 (4) ◽

pp. 1285-1296 ◽

Cited By ~ 254

Author(s):

Andrea N. Ladd ◽

Nicolas Charlet-B. ◽

Thomas A. Cooper

Keyword(s):

Alternative Splicing ◽

Heart Development ◽

Binding Proteins ◽

Rna Binding ◽

Striated Muscle ◽

Rna Binding Proteins ◽

Binding Protein ◽

Protein Isoforms ◽

Developmentally Regulated ◽

Exon Inclusion

ABSTRACT Alternative splicing of cardiac troponin T (cTNT) exon 5 undergoes a developmentally regulated switch such that exon inclusion predominates in embryonic, but not adult, striated muscle. We previously described four muscle-specific splicing enhancers (MSEs) within introns flanking exon 5 in chicken cTNT that are both necessary and sufficient for exon inclusion in embryonic muscle. We also demonstrated that CUG-binding protein (CUG-BP) binds a conserved CUG motif within a human cTNT MSE and positively regulates MSE-dependent exon inclusion. Here we report that CUG-BP is one of a novel family of developmentally regulated RNA binding proteins that includes embryonically lethal abnormal vision-type RNA binding protein 3 (ETR-3). This family, which we call CELF proteins for CUG-BP- and ETR-3-like factors, specifically bound MSE-containing RNAs in vitro and activated MSE-dependent exon inclusion of cTNT minigenes in vivo. The expression of two CELF proteins is highly restricted to brain. CUG-BP, ETR-3, and CELF4 are more broadly expressed, and expression is developmentally regulated in striated muscle and brain. Changes in the level of expression and isoforms of ETR-3 in two different developmental systems correlated with regulated changes in cTNT splicing. A switch from cTNT exon skipping to inclusion tightly correlated with induction of ETR-3 protein expression during differentiation of C2C12 myoblasts. During heart development, the switch in cTNT splicing correlated with a transition in ETR-3 protein isoforms. We propose that ETR-3 is a major regulator of cTNT alternative splicing and that the CELF family plays an important regulatory role in cell-specific alternative splicing during normal development and disease.

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A Plasmodium yoelii Mei2-Like RNA Binding Protein Is Essential for Completion of Liver Stage Schizogony

Infection and Immunity ◽

10.1128/iai.01417-15 ◽

2016 ◽

Vol 84 (5) ◽

pp. 1336-1345 ◽

Cited By ~ 8

Author(s):

Dorender A. Dankwa ◽

Marshall J. Davis ◽

Stefan H. I. Kappe ◽

Ashley M. Vaughan

Keyword(s):

Rna Binding ◽

Rna Binding Proteins ◽

Plasmodium Yoelii ◽

Parasite Development ◽

Mammalian Host ◽

Mosquito Vector ◽

Wild Type ◽

Liver Stage ◽

Content Type ◽

Stage Development

Plasmodiumparasites employ posttranscriptional regulatory mechanisms as their life cycle transitions between host cell invasion and replication within both the mosquito vector and mammalian host. RNA binding proteins (RBPs) provide one mechanism for modulation of RNA function. To explore the role ofPlasmodiumRBPs during parasite replication, we searched for RBPs that might play a role during liver stage development, the parasite stage that exhibits the most extensive growth and replication. We identified a parasite ortholog of theMei2(Meiosisinhibited 2) RBP that is conserved amongPlasmodiumspecies (PlasMei2) and exclusively transcribed in liver stage parasites. Epitope-taggedPlasmodium yoeliiPlasMei2 was expressed only during liver stage schizogony and showed an apparent granular cytoplasmic location. Knockout ofPlasMei2(plasmei2−) inP. yoeliionly affected late liver stage development. TheP. yoeliiplasmei2−liver stage size increased progressively until late in development, similar to wild-type parasite development. However,P. yoeliiplasmei2−liver stage schizonts exhibited an abnormal DNA segregation phenotype and failed to form exoerythrocytic merozoites. Consequently the cellular integrity ofP. yoeliiplasmei2−liver stages became increasingly compromised late in development and the majority ofP. yoeliiplasmei2−underwent cell death by the time wild-type liver stages mature and release merozoites. This resulted in a complete block ofP. yoeliiplasmei2−transition from liver stage to blood stage infection in mice. Our results show for the first time the importance of aPlasmodiumRBP in the coordinated progression of late liver stage schizogony and maturation of new invasive forms.

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Target selection by natural and redesigned PUF proteins

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1508501112 ◽

2015 ◽

Vol 112 (52) ◽

pp. 15868-15873 ◽

Cited By ~ 22

Author(s):

Douglas F. Porter ◽

Yvonne Y. Koh ◽

Brett VanVeller ◽

Ronald T. Raines ◽

Marvin Wickens

Keyword(s):

Rna Binding ◽

Target Selection ◽

Mutant Protein ◽

Sequence Specificity ◽

Wild Type ◽

Rna Recognition Motifs ◽

Puf Proteins ◽

Binding Domains ◽

Recognition Motifs

Pumilio/fem-3 mRNA binding factor (PUF) proteins bind RNA with sequence specificity and modularity, and have become exemplary scaffolds in the reengineering of new RNA specificities. Here, we report the in vivo RNA binding sites of wild-type (WT) and reengineered forms of the PUF protein Saccharomyces cerevisiae Puf2p across the transcriptome. Puf2p defines an ancient protein family present throughout fungi, with divergent and distinctive PUF RNA binding domains, RNA-recognition motifs (RRMs), and prion regions. We identify sites in RNA bound to Puf2p in vivo by using two forms of UV cross-linking followed by immunopurification. The protein specifically binds more than 1,000 mRNAs, which contain multiple iterations of UAAU-binding elements. Regions outside the PUF domain, including the RRM, enhance discrimination among targets. Compensatory mutants reveal that one Puf2p molecule binds one UAAU sequence, and align the protein with the RNA site. Based on this architecture, we redesign Puf2p to bind UAAG and identify the targets of this reengineered PUF in vivo. The mutant protein finds its target site in 1,800 RNAs and yields a novel RNA network with a dramatic redistribution of binding elements. The mutant protein exhibits even greater RNA specificity than wild type. The redesigned protein decreases the abundance of RNAs in its redesigned network. These results suggest that reengineering using the PUF scaffold redirects and can even enhance specificity in vivo.

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