The Role of the Schizosaccharomyces pombe gar2 Protein in Nucleolar Structure and Function Depends on the Concerted Action of its Highly Charged N Terminus and its RNA-binding Domains

Nonribosomal nucleolar protein gar2 is required for 18S rRNA and 40S ribosomal subunit production in Schizosaccharomyces pombe. We have investigated the consequences of the absence of each structural domain of gar2 on cell growth, 18S rRNA production, and nucleolar structure. Deletion of gar2 RNA-binding domains (RBDs) causes stronger inhibition of growth and 18S rRNA accumulation than the absence of the whole protein, suggesting that other factors may be titrated by its remaining N-terminal basic/acidic serine-rich domain. These drastic functional defects correlate with striking nucleolar hypertrophy. Point mutations in the conserved RNP1 motifs of gar2 RBDs supposed to inhibit RNA–protein interactions are sufficient to induce severe nucleolar modifications but only in the presence of the N-terminal domain of the protein. Gar2 and its mutants also distribute differently in glycerol gradients: gar2 lacking its RBDs is found either free or assembled into significantly larger complexes than the wild-type protein. We propose that gar2 helps the assembly on rRNA of factors necessary for 40S subunit synthesis by providing a physical link between them. These factors may be recruited by the N-terminal domain of gar2 and may not be released if interaction of gar2 with rRNA is impaired.

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Ribosomal protein L7a binds RNA through two distinct RNA-binding domains

Biochemical Journal ◽

10.1042/bj20040371 ◽

2004 ◽

Vol 385 (1) ◽

pp. 289-299 ◽

Cited By ~ 17

Author(s):

Giulia RUSSO ◽

Monica CUCCURESE ◽

Gianluca MONTI ◽

Annapina RUSSO ◽

Angela AMORESANO ◽

...

Keyword(s):

Ribosomal Protein ◽

Rna Binding ◽

Limited Proteolysis ◽

Ribosomal Subunit ◽

The Other ◽

Protein Domain ◽

Binding Motif ◽

Nucleic Acid Binding ◽

Binding Domains ◽

Rna Binding Domains

The human ribosomal protein L7a is a component of the major ribosomal subunit. We previously identified three nuclear-localization-competent domains within L7a, and demonstrated that the domain defined by aa (amino acids) 52–100 is necessary, although not sufficient, to target the L7a protein to the nucleoli. We now demonstrate that L7a interacts in vitro with a presumably G-rich RNA structure, which has yet to be defined. We also demonstrate that the L7a protein contains two RNA-binding domains: one encompassing aa 52–100 (RNAB1) and the other encompassing aa 101–161 (RNAB2). RNAB1 does not contain any known nucleic-acid-binding motif, and may thus represent a new class of such motifs. On the other hand, a specific region of RNAB2 is highly conserved in several other protein components of the ribonucleoprotein complex. We have investigated the topology of the L7a–RNA complex using a recombinant form of the protein domain that encompasses residues 101–161 and a 30mer poly(G) oligonucleotide. Limited proteolysis and cross-linking experiments, and mass spectral analyses of the recombinant protein domain and its complex with poly(G) revealed the RNA-binding region.

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Characterization of the RNA-Binding Domains in the Replicase Proteins of Tomato Bushy Stunt Virus

Journal of Virology ◽

10.1128/jvi.77.17.9244-9258.2003 ◽

2003 ◽

Vol 77 (17) ◽

pp. 9244-9258 ◽

Cited By ~ 96

Author(s):

K. S. Rajendran ◽

Peter D. Nagy

Keyword(s):

Rna Binding ◽

Tomato Bushy Stunt Virus ◽

Binding Motif ◽

Mobility Shift ◽

Terminal Domain ◽

Binding Domains ◽

Replicase Proteins ◽

Rna Binding Domains ◽

Gel Mobility Shift

ABSTRACT Tomato bushy stunt virus (TBSV), a tombusvirus with a nonsegmented, plus-stranded RNA genome, codes for two essential replicase proteins. The sequence of one of the replicase proteins, namely p33, overlaps with the N-terminal domain of p92, which contains the signature motifs of RNA-dependent RNA polymerases (RdRps) in its nonoverlapping C-terminal portion. In this work, we demonstrate that both replicase proteins bind to RNA in vitro based on gel mobility shift and surface plasmon resonance measurements. We also show evidence that the binding of p33 to single-stranded RNA (ssRNA) is stronger than binding to double-stranded RNA (dsRNA), ssDNA, or dsDNA in vitro. Competition experiments with ssRNA revealed that p33 binds to a TBSV-derived sequence with higher affinity than to other nonviral ssRNA sequences. Additional studies revealed that p33 could bind to RNA in a cooperative manner. Using deletion derivatives of the Escherichia coli-expressed recombinant proteins in gel mobility shift and Northwestern assays, we demonstrate that p33 and the overlapping domain of p92, based on its sequence identity with p33, contain an arginine- and proline-rich RNA-binding motif (termed RPR, which has the sequence RPRRRP). This motif is highly conserved among tombusviruses and related carmoviruses, and it is similar to the arginine-rich motif present in the Tat trans-activator protein of human immunodeficiency virus type 1. We also find that the nonoverlapping C-terminal domain of p92 contains additional RNA-binding regions. Interestingly, the location of one of the RNA-binding domains in p92 is similar to the RNA-binding domain of the NS5B RdRp protein of hepatitis C virus.

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Molecular analysis of a novel Schizosaccharomyces pombe gene containing two RNP consensus-sequence RNA-binding domains

Current Genetics ◽

10.1007/s002940050051 ◽

1996 ◽

Vol 29 (4) ◽

pp. 307-315 ◽

Cited By ~ 1

Author(s):

R. W. VanHoy ◽

J. A. Wise

Keyword(s):

Schizosaccharomyces Pombe ◽

Molecular Analysis ◽

Rna Binding ◽

Consensus Sequence ◽

Binding Domains ◽

Rna Binding Domains

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Faculty Opinions recommendation of Comprehensive Identification of RNA-Binding Domains in Human Cells.

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

10.3410/f.726579306.793531390 ◽

2017 ◽

Author(s):

Julie Forman-Kay ◽

Andrew Chong

Keyword(s):

Rna Binding ◽

Human Cells ◽

Binding Domains ◽

Rna Binding Domains

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In Caenorhabditis elegans, the RNA-Binding Domains of the Cytoplasmic Polyadenylation Element Binding Protein FOG-1 Are Needed to Regulate Germ Cell Fates

Genetics ◽

10.1093/genetics/159.4.1617 ◽

2001 ◽

Vol 159 (4) ◽

pp. 1617-1630

Author(s):

Suk-Won Jin ◽

Nancy Arno ◽

Adam Cohen ◽

Amy Shah ◽

Qijin Xu ◽

...

Keyword(s):

Caenorhabditis Elegans ◽

Germ Cell ◽

Rna Binding ◽

Dominant Negative ◽

Missense Mutations ◽

Biochemical Tests ◽

Cell Fates ◽

Cytoplasmic Polyadenylation ◽

Binding Domains ◽

Rna Binding Domains

Abstract FOG-1 controls germ cell fates in the nematode Caenorhabditis elegans. Sequence analyses revealed that FOG-1 is a cytoplasmic polyadenylation element binding (CPEB) protein; similar proteins from other species have been shown to bind messenger RNAs and regulate their translation. Our analyses of fog-1 mutations indicate that each of the three RNA-binding domains of FOG-1 is essential for activity. In addition, biochemical tests show that FOG-1 is capable of binding RNA sequences in the 3′-untranslated region of its own message. Finally, genetic assays reveal that fog-1 functions zygotically, that the small fog-1 transcript has no detectable function, and that missense mutations in fog-1 cause a dominant negative phenotype. This last observation suggests that FOG-1 acts in a complex, or as a multimer, to regulate translation. On the basis of these data, we propose that FOG-1 binds RNA to regulate germ cell fates and that it does so by controlling the translation of its targets. One of these targets might be the fog-1 transcript itself.

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The N-terminal domain of Sxl protein disrupts Sxl autoregulation in females and promotes female-specific splicing of tra in males

Development ◽

10.1242/dev.126.13.2841 ◽

1999 ◽

Vol 126 (13) ◽

pp. 2841-2853 ◽

Cited By ~ 1

Author(s):

G. Deshpande ◽

G. Calhoun ◽

P.D. Schedl

Keyword(s):

Fusion Protein ◽

Rna Binding ◽

Chimeric Protein ◽

Dominant Negative ◽

Terminal Domain ◽

Binding Domains ◽

Transcriptional Regulatory ◽

Dominant Negative Activity ◽

Female Specific

Sex determination in Drosophila depends upon the post-transcriptional regulatory activities of the Sex-lethal (Sxl) gene. Sxl maintains the female determined state and activates female differentiation pathways by directing the female-specific splicing of Sxl and tra pre-mRNAs. While there is compelling evidence that Sxl proteins regulate splicing by directly binding to target RNAs, previous studies indicate that the two Sxl RNA-binding domains are not in themselves sufficient for biological activity and that an intact N-terminal domain is also critical for splicing function. To further investigate the functions of the Sxl N terminus, we ectopically expressed a chimeric protein consisting of the N-terminal 99 amino acids fused to ss-galactosidase. The Nss-gal fusion protein behaves like a dominant negative, interfering with the Sxl autoregulatory feedback loop and killing females. This dominant negative activity can be attributed to the recruitment of the fusion protein into the large Sxl:Snf splicing complexes that are found in vivo and the consequent disruption of these complexes. In addition to the dominant negative activity, the Nss-gal fusion protein has a novel gain-of-function activity in males: it promotes the female-specific processing of tra pre-mRNAs. This novel activity is discussed in light of the blockage model for the tra splicing regulation.

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