The mitotic phosphorylation of p54nrb modulates its RNA binding activity

2011 ◽  
Vol 89 (4) ◽  
pp. 423-433 ◽  
Author(s):  
Céline Bruelle ◽  
Mikaël Bédard ◽  
Stéphanie Blier ◽  
Martin Gauthier ◽  
Abdulmaged M. Traish ◽  
...  
Keyword(s):  
Rna Binding ◽  
Binding Activity ◽  
Site Directed Mutagenesis ◽  
Binding Assays ◽  
Non Coding Rna ◽  
Protein Partners ◽  
Biochemical Assays ◽  
Mitotic Phosphorylation

The RNA-binding protein p54nrb is involved in many nuclear processes including transcription, RNA processing, and retention of hyperedited RNAs. In interphase cells, p54nrb localizes to the nucleoplasm and concentrates with protein partners in the paraspeckles via an interaction with the non-coding RNA Neat1. During mitosis, p54nrb becomes multiphosphorylated and the effects of this modification are not known. In the present study, we show that p54nrb phosphorylation does not affect the interactions with its protein partners but rather diminishes its general RNA-binding ability. Biochemical assays indicate that in vitro phosphorylation of a GST-p54nrb construct by CDK1 abolishes the interaction with 5′ splice site RNA sequence. Site-directed mutagenesis shows that the threonine 15 residue, located N-terminal to the RRM tandem domains of p54nrb, is involved in this inhibition. In vivo analysis reveals that Neat1 ncRNA co-immunoprecipitates with p54nrb in either interphase or mitotic cells, suggesting that p54nrb–Neat1 interaction is not modulated by phosphorylation. Accordingly, in vitro phosphorylated GST-p54nrb still interacts with PIR-1 RNA, a G-rich Neat1 sequence known to interact with p54nrb. In vitro RNA binding assays show that CDK1-phosphorylation of a GST-p54nrb construct abolishes its interaction with homoribopolymers poly(A), poly(C), and poly(U) but not with poly(G). These data suggest that p54nrb interaction with RNA could be selectively modulated by phosphorylation during mitosis.

scholarly journals The 5' leader of a chloroplast mRNA mediates the translational requirements for two nucleus-encoded functions in Chlamydomonas reinhardtii.

1994 ◽  
Vol 14 (8) ◽  
pp. 5268-5277 ◽  
Author(s):  
W Zerges ◽  
J D Rochaix
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Rna Binding ◽  
Mrna Translation ◽  
Binding Activity ◽  
Leader Sequence ◽  
Sequence Motif ◽  
Wild Type ◽  
Chloroplast Mrna

In the green alga Chlamydomonas reinhardtii, the nuclear mutations F34 and F64 have been previously shown to abolish the synthesis of the photosystem II core polypeptide subunit P6, which is encoded by the chloroplast psbC gene. In this report the functions encoded by F34 and F64 are shown to be required for translation of the psbC mRNA, on the basis of the finding that the expression of a heterologous reporter gene fused to the psbC 5' nontranslated leader sequence requires wild-type F34 and F64 alleles in vivo. Moreover, a point mutation in the psbC 5' nontranslated leader sequence suppresses this requirement for wild-type F34 function. In vitro RNA-protein cross-linking studies reveal that chloroplast protein extracts from strains carrying the F64 mutation contain an approximately 46-kDa RNA-binding protein. The absence of the RNA-binding activity of this protein in chloroplast extracts of wild-type strains suggests that it is related to the role of the F64-encoded function for psbC mRNA translation. The binding specificity of this protein appears to be for an AU-rich RNA sequence motif.

ARCD-1, an apobec-1-related cytidine deaminase, exerts a dominant negative effect on C to U RNA editing

2001 ◽  
Vol 281 (6) ◽  
pp. C1904-C1916 ◽  
Author(s):  
Shrikant Anant ◽  
Debnath Mukhopadhyay ◽  
Vakadappu Sankaranand ◽  
Susan Kennedy ◽  
Jeffrey O. Henderson ◽  
...  
Keyword(s):  
Rna Editing ◽  
Rna Binding ◽  
Cytidine Deaminase ◽  
Binding Activity ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Mrna Editing ◽  
Apob Mrna

Mammalian apolipoprotein B (apoB) C to U RNA editing is catalyzed by a multicomponent holoenzyme containing a single catalytic subunit, apobec-1. We have characterized an apobec-1 homologue, ARCD-1, located on chromosome 6p21.1, and determined its role in apoB mRNA editing. ARCD-1 mRNA is ubiquitously expressed; phylogenetic analysis reveals it to be a distant member of the RNA editing family. Recombinant ARCD-1 demonstrates cytidine deaminase and apoB RNA binding activity but does not catalyze C to U RNA editing, either in vitro or in vivo. Although not competent itself to mediate deamination of apoB mRNA, ARCD-1 inhibits apobec-1-mediated C to U RNA editing. ARCD-1 interacts and heterodimerizes with both apobec-1 and apobec-1 complementation factor (ACF) and localizes to both the nucleus and cytoplasm of transfected cells. Together, the data suggest that ARCD-1 is a novel cytidine deaminase that interacts with apobec-1 and ACF to inhibit apoB mRNA editing, possibly through interaction with other protein components of the apoB RNA editing holoenzyme.

scholarly journals The RNA Binding Activity of a Ribosome Biogenesis Factor, Nucleophosmin/B23, Is Modulated by Phosphorylation with a Cell Cycle-dependent Kinase and by Association with Its Subtype

2002 ◽  
Vol 13 (6) ◽  
pp. 2016-2030 ◽  
Author(s):  
Mitsuru Okuwaki ◽  
Masafumi Tsujimoto ◽  
Kyosuke Nagata
Keyword(s):  
Cell Cycle ◽  
Ribosome Biogenesis ◽  
Cellular Localization ◽  
Rna Binding ◽  
Intracellular Localization ◽  
Binding Activity ◽  
Cyclin B ◽  
Cycle Dependent

Nucleophosmin/B23 is a nucleolar phosphoprotein. It has been shown that B23 binds to nucleic acids, digests RNA, and is localized in nucleolar granular components from which preribosomal particles are transported to cytoplasm. The intracellular localization of B23 is significantly changed during the cell cycle. Here, we have examined the cellular localization of B23 proteins and the effect of mitotic phosphorylation of B23.1 on its RNA binding activity. Two splicing variants of B23 proteins, termed B23.1 and B23.2, were complexed both in vivo and in vitro. The RNA binding activity of B23.1 was impaired by hetero-oligomer formation with B23.2. Both subtypes of B23 proteins were phosphorylated during mitosis by cyclin B/cdc2. The RNA binding activity of B23.1 was repressed through cyclin B/cdc2-mediated phosphorylation at specific sites in B23. Thus, the RNA binding activity of B23.1 is stringently modulated by its phosphorylation and subtype association. Interphase B23.1 was mainly localized in nucleoli, whereas B23.2 and mitotic B23.1, those of which were incapable of binding to RNA, were dispersed throughout the nucleoplasm and cytoplasm, respectively. These results suggest that nucleolar localization of B23.1 is mediated by its ability to associate with RNA.

scholarly journals Gle1 Regulates RNA Binding of the DEAD-Box Helicase Ded1 in Its Complex Role in Translation Initiation

2017 ◽  
Vol 37 (21) ◽  
Author(s):  
Peyman P. Aryanpur ◽  
Chelsea A. Regan ◽  
John M. Collins ◽  
Telsa M. Mittelmeier ◽  
David M. Renner ◽  
...  
Keyword(s):  
Gene Expression ◽  
Rna Binding ◽  
Mrna Export ◽  
Binding Assays ◽  
Cellular Mechanisms ◽  
Ribonucleoprotein Complexes ◽  
Dead Box

ABSTRACT DEAD-box proteins (DBPs) are required in gene expression to facilitate changes to ribonucleoprotein complexes, but the cellular mechanisms and regulation of DBPs are not fully defined. Gle1 is a multifunctional regulator of DBPs with roles in mRNA export and translation. In translation, Gle1 modulates Ded1, a DBP required for initiation. However, DED1 overexpression causes defects, suggesting that Ded1 can promote or repress translation in different contexts. Here we show that GLE1 expression suppresses the repressive effects of DED1 in vivo and Gle1 counteracts Ded1 in translation assays in vitro. Furthermore, both Ded1 and Gle1 affect the assembly of preinitiation complexes. Through mutation analysis and binding assays, we show that Gle1 inhibits Ded1 by reducing its affinity for RNA. Our results are consistent with a model wherein active Ded1 promotes translation but inactive or excess Ded1 leads to translation repression. Gle1 can inhibit either role of Ded1, positioning it as a gatekeeper to optimize Ded1 activity to the appropriate level for translation. This study suggests a paradigm for finely controlling the activity of DEAD-box proteins to optimize their function in RNA-based processes. It also positions the versatile regulator Gle1 as a potential node for the coordination of different steps of gene expression.

scholarly journals The Gly/Arg-rich (GAR) domain of Xenopus nucleolin facilitates in vitro nucleic acid binding and in vivo nucleolar localization.

1993 ◽  
Vol 4 (11) ◽  
pp. 1189-1204 ◽  
Author(s):  
M A Heine ◽  
M L Rankin ◽  
P J DiMario
Keyword(s):  
Nucleic Acid ◽  
Binding Activity ◽  
Full Length ◽  
Nucleic Acid Binding ◽  
Binding Assays ◽  
Nucleic Acid Probes ◽  
E Coli ◽  
Indirect Immunofluorescence Staining

Epitope-tagged Xenopus nucleolin was expressed in Escherichia coli cells and in Xenopus oocytes either as a full-length wild-type protein or as a truncation that lacked the distinctive carboxy glycine/arginine-rich (GAR) domain. Both full-length and truncated versions of nucleolin were tagged at their amino termini with five tandem human c-myc epitopes. Whether produced in E. coli or in Xenopus, epitope-tagged full-length nucleolin bound nucleic acid probes in in vitro filter binding assays. Conversely, the E. coli-expressed GAR truncation failed to bind the nucleic acid probes, whereas the Xenopus-expressed truncation maintained slight binding activity. Indirect immunofluorescence staining showed that myc-tagged full-length nucleolin properly localized to the dense fibrillar regions within the multiple nucleoli of Xenopus oocyte nuclei. The epitope-tagged GAR truncation also translocated to the oocyte nuclei, but it failed to efficiently localize to the nucleoli. Our results show that the carboxy GAR domain must be present for nucleolin to efficiently bind nucleic acids in vitro and to associate with nucleoli in vivo.

scholarly journals MSY2 and MSY4 Bind a Conserved Sequence in the 3′ Untranslated Region of Protamine 1 mRNA In Vitro and In Vivo

2001 ◽  
Vol 21 (20) ◽  
pp. 7010-7019 ◽  
Author(s):  
Flaviano Giorgini ◽  
Holly G. Davies ◽  
Robert E. Braun
Keyword(s):  
Untranslated Region ◽  
Rna Binding ◽  
Mutational Analysis ◽  
Binding Activity ◽  
Mobility Shift ◽  
Conserved Sequence ◽  
Mobility Shift Assay ◽  
Protamine 1

ABSTRACT Y-box proteins are major constituents of ribonucleoprotein particles (RNPs) which contain translationally silent mRNAs in gametic cells. We have recently shown that a sequence-specific RNA binding activity present in spermatogenic cells contains the two Y-box proteins MSY2 and MSY4. We show here that MSY2 and MSY4 bind a sequence, 5′-UCCAUCA-3′, present in the 3′ untranslated region of the translationally repressed protamine 1 (Prm1) mRNA. Using pre- and post-RNase T1-digested substrate RNAs, it was determined that MSY2 and MSY4 can bind an RNA of eight nucleotides containing the MSY2 and MSY4 binding site. Single nucleotide mutations in the sequence eliminated the binding of MSY2 and MSY4 in an electrophoretic mobility shift assay, and the resulting mutants failed to compete for binding in a competition assay. A consensus site of UACCACAUCCACU(subscripts indicate nucleotides which do not disrupt YRS binding by MSY2 and MSY4), denoted the Y-box recognition site (YRS), was defined from this mutational analysis. These mutations in the YRS were further characterized in vivo using a novel application of the yeast three-hybrid system. Experiments with transgenic mice show that disruption of the YRS in vivo relieves Prm1-like repression of a reporter gene. The conservation of the RNA binding motifs among Y-box protein family members raises the possibility that other Y-box proteins may have previously unrecognized sequence-specific RNA binding activities.

scholarly journals The cAMP pathway regulates mRNA decay through phosphorylation of the RNA-binding protein TIS11b/BRF1

2016 ◽  
Vol 27 (24) ◽  
pp. 3841-3854 ◽  
Author(s):  
Felicitas Rataj ◽  
Séverine Planel ◽  
Agnès Desroches-Castan ◽  
Juliette Le Douce ◽  
Khadija Lamribet ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Interactions ◽  
Mrna Decay ◽  
Rna Binding ◽  
Phosphorylation Site ◽  
P38 Map Kinase ◽  
Site Directed Mutagenesis ◽  
Protein Protein Interactions

TPA-inducible sequence 11b/butyrate response factor 1 (TIS11b/BRF1) belongs to the tristetraprolin (TTP) family of zinc-finger proteins, which bind to mRNAs containing AU-rich elements in their 3′-untranslated region and target them for degradation. Regulation of TTP family function through phosphorylation by p38 MAP kinase and Akt/protein kinase B signaling pathways has been extensively studied. In contrast, the role of cAMP-dependent protein kinase (PKA) in the control of TTP family activity in mRNA decay remains largely unknown. Here we show that PKA activation induces TIS11b gene expression and protein phosphorylation. Site-directed mutagenesis combined with kinase assays and specific phosphosite immunodetection identified Ser-54 (S54) and Ser-334 (S334) as PKA target amino acids in vitro and in vivo. Phosphomimetic mutation of the C-terminal S334 markedly increased TIS11b half-life and, unexpectedly, enhanced TIS11b activity on mRNA decay. Examination of protein–protein interactions between TIS11b and components of the mRNA decay machinery revealed that mimicking phosphorylation at S334 enhances TIS11b interaction with the decapping coactivator Dcp1a, while preventing phosphorylation at S334 potentiates its interaction with the Ccr4-Not deadenylase complex subunit Cnot1. Collectively our findings establish for the first time that cAMP-elicited phosphorylation of TIS11b plays a key regulatory role in its mRNA decay-promoting function.

scholarly journals Arginine-Rich Regions Mediate the RNA Binding and Regulatory Activities of the Protein Encoded by the Drosophila melanogaster suppressor of sable Gene

2000 ◽  
Vol 20 (21) ◽  
pp. 8198-8208 ◽  
Author(s):  
Michael A. Turnage ◽  
Paul Brewer-Jensen ◽  
Wen-Li Bai ◽  
Lillie L. Searles
Keyword(s):  
Drosophila Melanogaster ◽  
Rna Binding ◽  
Consensus Sequence ◽  
Polytene Chromosomes ◽  
Ectopic Expression ◽  
Binding Activity ◽  
Suppressor Of Sable ◽  
Rna Accumulation

ABSTRACT The Drosophila melanogaster suppressor of sable gene,su(s), encodes a novel, 150-kDa nuclear RNA binding protein, SU(S), that negatively regulates RNA accumulation from mutant alleles of other genes that have transposon insertions in the 5′ transcribed region. In this study, we delineated the RNA binding domain of SU(S) and evaluated its relevance to SU(S) function in vivo. As a result, we have defined two arginine-rich motifs (ARM1 and ARM2) that mediate the RNA binding activity of SU(S). ARM1 is required for in vitro high-affinity binding of SU(S) to small RNAs that were previously isolated by SELEX (binding site selection assay) and that contain a common consensus sequence. ARM1 is also required for the association of SU(S) with larval polytene chromosomes in vivo. ARM2 promotes binding of SU(S) to SELEX RNAs that lack the consensus sequence and apparently is neither necessary nor sufficient for the stable polytene chromosome association of SU(S). Use of the GAL4/UAS system to drive ectopic expression of su(s) cDNA transgenes revealed two previously unknown properties of SU(S). First, overexpression of SU(S) is lethal. Second, SU(S) negatively regulates expression of su(s)intronless cDNA transgenes, and the ARMs are required for this effect. Considering these and previous results, we propose that SU(S) binds to the 5′ region of nascent transcripts and inhibits RNA production in a manner that can be overcome by splicing complex assembly.

scholarly journals CUS2, a Yeast Homolog of Human Tat-SF1, Rescues Function of Misfolded U2 through an Unusual RNA Recognition Motif

1998 ◽  
Vol 18 (9) ◽  
pp. 5000-5009 ◽  
Author(s):  
Dong Yan ◽  
Rhonda Perriman ◽  
Haller Igel ◽  
Kenneth J. Howe ◽  
Megan Neville ◽  
...  
Keyword(s):  
Rna Binding ◽  
Rna Binding Proteins ◽  
Normal Activity ◽  
Binding Activity ◽  
Rna Recognition Motif ◽  
Rna Recognition ◽  
Recognition Motif ◽  
Yeast Homolog

ABSTRACT A screen for suppressors of a U2 snRNA mutation identified CUS2, an atypical member of the RNA recognition motif (RRM) family of RNA binding proteins. CUS2 protein is associated with U2 RNA in splicing extracts and interacts with PRP11, a subunit of the conserved splicing factor SF3a. Absence of CUS2 renders certain U2 RNA folding mutants lethal, arguing that a normal activity of CUS2 is to help refold U2 into a structure favorable for its binding to SF3b and SF3a prior to spliceosome assembly. Both CUS2 function in vivo and the in vitro RNA binding activity of CUS2 are disrupted by mutation of the first RRM, suggesting that rescue of misfolded U2 involves the direct binding of CUS2. Human Tat-SF1, reported to stimulate Tat-specific, transactivating region-dependent human immunodeficiency virus transcription in vitro, is structurally similar to CUS2. Anti-Tat-SF1 antibodies coimmunoprecipitate SF3a66 (SAP62), the human homolog of PRP11, suggesting that Tat-SF1 has a parallel function in splicing in human cells.

scholarly journals Structure of AadA fromSalmonella enterica: a monomeric aminoglycoside (3′′)(9) adenyltransferase

2015 ◽  
Vol 71 (11) ◽  
pp. 2267-2277 ◽  
Author(s):  
Yang Chen ◽  
Joakim Näsvall ◽  
Shiying Wu ◽  
Dan I. Andersson ◽  
Maria Selmer
Keyword(s):  
Adenosine Monophosphate ◽  
Site Directed Mutagenesis ◽  
Titration Calorimetry ◽  
Atp Binding ◽  
Binding Assays ◽  
Aminoglycoside Resistance ◽  
X Ray Scattering ◽  
Sad Phasing

Aminoglycoside resistance is commonly conferred by enzymatic modification of drugs by aminoglycoside-modifying enzymes such as aminoglycoside nucleotidyltransferases (ANTs). Here, the first crystal structure of an ANT(3′′)(9) adenyltransferase, AadA fromSalmonella enterica, is presented. AadA catalyses the magnesium-dependent transfer of adenosine monophosphate from ATP to the two chemically dissimilar drugs streptomycin and spectinomycin. The structure was solved using selenium SAD phasing and refined to 2.5 Å resolution. AadA consists of a nucleotidyltransferase domain and an α-helical bundle domain. AadA crystallizes as a monomer and is a monomer in solution as confirmed by small-angle X-ray scattering, in contrast to structurally similar homodimeric adenylating enzymes such as kanamycin nucleotidyltransferase. Isothermal titration calorimetry experiments show that ATP binding has to occur before binding of the aminoglycoside substrate, and structure analysis suggests that ATP binding repositions the two domains for aminoglycoside binding in the interdomain cleft. Candidate residues for ligand binding and catalysis were subjected to site-directed mutagenesis.In vivoresistance andin vitrobinding assays support the role of Glu87 as the catalytic base in adenylation, while Arg192 and Lys205 are shown to be critical for ATP binding.

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