scholarly journals Polyadenylation of maternal mRNA during oocyte maturation: poly(A) addition in vitro requires a regulated RNA binding activity and a poly(A) polymerase.

1992 ◽  
Vol 11 (13) ◽  
pp. 5021-5032 ◽  
Author(s):  
C.A. Fox ◽  
M.D. Sheets ◽  
E. Wahle ◽  
M. Wickens
Keyword(s):  
Oocyte Maturation ◽  
Rna Binding ◽  
Binding Activity ◽  
Maternal Mrna

DNA Binding Activity of Marine Shrimp LvProfilin

2021 ◽  
Author(s):  
Yanisa Laoong-u-thai ◽  
Warapond Wanna ◽  
Autaipohn Kaikaew
Keyword(s):  
Dna Binding ◽  
Rna Binding ◽  
3D Structure ◽  
Binding Activity ◽  
Shrimp Farming ◽  
Binding Prediction ◽  
3D Protein Structure ◽  
Dna Binding Activity ◽  
Marine Shrimp

Shrimp farming is an important business in Thailand and worldwide. The study of molecular biology and biochemical pathway of the key molecules controlling muscle growth is an essential to improve shrimp livestock. Profilin is a pivotal protein in muscle formation, especially actin protein. Its nuclear function has been reported in many species for gene regulation. Here in this work, we characterized the function of LvProfilin, a marine shrimp profilin from Litopenaeus vannamei, both in silico and in vitro. The phylogenetic tree of LvProfilin among organisms and its 3D protein structure showed that LvProfilin was highly conserved among shrimp and arthropods. The homology modeling of its 3D structure revealed 3 alpha-helices and 6 beta-strands similar to most eukaryotic profilins. To interpret its possible function, the gene expression of LvProfilin in various tissues was performed. We found that this gene was expressed in various tissues. This result may imply that LvProfilin could share a common function in all tissues. Nuclear activity has been a promising function of LvProfilin. We performed a DNA/RNA binding prediction analysis using DRNApred. The result indicated that Lysine-90 and Threonine-91 were the putative DNA-binding sites with the probability of 63.12% and 54.16%, respectively. Its binding activity was confirmed in vitro which bound stronger to single strand DNA than double strand DNA. To our best knowledge, this is the first report of DNA binding activity of profilin in invertebrates.

scholarly journals The multiple RNA-binding domains of the mRNA poly(A)-binding protein have different RNA-binding activities.

1991 ◽  
Vol 11 (7) ◽  
pp. 3419-3424 ◽  
Author(s):  
C G Burd ◽  
E L Matunis ◽  
G Dreyfuss
Keyword(s):  
Rna Binding ◽  
Binding Protein ◽  
Consensus Sequence ◽  
Binding Activity ◽  
Yeast Cells ◽  
Yeast Saccharomyces Cerevisiae ◽  
Amino Terminal ◽  
Binding Domains ◽  
Rna Binding Domains

The poly(A)-binding protein (PABP) is the major mRNA-binding protein in eukaryotes, and it is essential for viability of the yeast Saccharomyces cerevisiae. The amino acid sequence of the protein indicates that it consists of four ribonucleoprotein consensus sequence-containing RNA-binding domains (RBDs I, II, III, and IV) and a proline-rich auxiliary domain at the carboxyl terminus. We produced different parts of the S. cerevisiae PABP and studied their binding to poly(A) and other ribohomopolymers in vitro. We found that none of the individual RBDs of the protein bind poly(A) specifically or efficiently. Contiguous two-domain combinations were required for efficient RNA binding, and each pairwise combination (I/II, II/III, and III/IV) had a distinct RNA-binding activity. Specific poly(A)-binding activity was found only in the two amino-terminal RBDs (I/II) which, interestingly, are dispensable for viability of yeast cells, whereas the activity that is sufficient to rescue lethality of a PABP-deleted strain is in the carboxyl-terminal RBDs (III/IV). We conclude that the PABP is a multifunctional RNA-binding protein that has at least two distinct and separable activities: RBDs I/II, which most likely function in binding the PABP to mRNA through the poly(A) tail, and RBDs III/IV, which may function through binding either to a different part of the same mRNA molecule or to other RNA(s).

scholarly journals Phosphorylation of Bluetongue Virus Nonstructural Protein 2 Is Essential for Formation of Viral Inclusion Bodies

2005 ◽  
Vol 79 (15) ◽  
pp. 10023-10031 ◽  
Author(s):  
Jens Modrof ◽  
Kostas Lymperopoulos ◽  
Polly Roy
Keyword(s):  
Bluetongue Virus ◽  
Inclusion Bodies ◽  
Rna Binding ◽  
Nonstructural Protein ◽  
Binding Activity ◽  
Nonstructural Protein 2 ◽  
The Matrix ◽  
Infected Cells ◽  
Viral Inclusion

ABSTRACT In bluetongue virus (BTV)-infected cells, large cytoplasmic aggregates are formed, termed viral inclusion bodies (VIBs), which are believed to be the sites of viral replication and morphogenesis. The BTV nonstructural protein NS2 is the major component of VIBs. NS2 undergoes intracellular phosphorylation and possesses a strong single-stranded RNA binding activity. By changing phosphorylated amino acids to alanines and aspartates, we have mapped the phosphorylated sites of NS2 to two serine residues at positions 249 and 259. Since both of these serines are within the context of protein kinase CK2 recognition signals, we have further examined if CK2 is involved in NS2 phosphorylation by both intracellular colocalization and an in vitro phosphorylation assay. In addition, we have utilized the NS2 mutants to determine the role of phosphorylation on NS2 activities. The data obtained demonstrate that NS2 phosphorylation is not necessary either for its RNA binding properties or for its ability to interact with the viral polymerase VP1. However, phosphorylated NS2 exhibited VIB formation while unmodified NS2 failed to assemble as VIBs although smaller oligomeric forms of NS2 were readily formed. Our data reveal that NS2 phosphorylation controls VIBs formation consistent with a model in which NS2 provides the matrix for viral assembly.

scholarly journals Multifunctional Roles for the N-Terminal Basic Motif of Alfalfa mosaic virus Coat Protein: Nucleolar/Cytoplasmic Shuttling, Modulation of RNA-Binding Activity, and Virion Formation

2012 ◽  
Vol 25 (8) ◽  
pp. 1093-1103 ◽  
Author(s):  
Mari Carmen Herranz ◽  
Vicente Pallas ◽  
Frederic Aparicio
Keyword(s):  
Coat Protein ◽  
Mosaic Virus ◽  
Nuclear Export ◽  
Rna Binding ◽  
Nuclear Export Signal ◽  
Alfalfa Mosaic Virus ◽  
Binding Activity ◽  
Systemic Movement ◽  
Virion Formation

In addition to virion formation, the coat protein (CP) of Alfalfa mosaic virus (AMV) is involved in the regulation of replication and translation of viral RNAs, and in cell-to-cell and systemic movement of the virus. An intriguing feature of the AMV CP is its nuclear and nucleolar accumulation. Here, we identify an N-terminal lysine-rich nucleolar localization signal (NoLS) in the AMV CP required to both enter the nucleus and accumulate in the nucleolus of infected cells, and a C-terminal leucine-rich domain which might function as a nuclear export signal. Moreover, we demonstrate that AMV CP interacts with importin-α, a component of the classical nuclear import pathway. A mutant AMV RNA 3 unable to target the nucleolus exhibited reduced plus-strand RNA synthesis and cell-to-cell spread. Moreover, virion formation and systemic movement were completely abolished in plants infected with this mutant. In vitro analysis demonstrated that specific lysine residues within the NoLS are also involved in modulating CP-RNA binding and CP dimerization, suggesting that the NoLS represents a multifunctional domain within the AMV CP. The observation that nuclear and nucleolar import signals mask RNA-binding properties of AMV CP, essential for viral replication and translation, supports a model in which viral expression is carefully modulated by a cytoplasmic/nuclear balance of CP accumulation.

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.

Messenger RNAs in metaphase II oocytes correlate with successful embryo development to the blastocyst stage

Zygote ◽  
2012 ◽  
Vol 22 (1) ◽  
pp. 69-79 ◽  
Author(s):  
Fernando Henrique Biase ◽  
Robin Edward Everts ◽  
Rosane Oliveira ◽  
Weruska Karyna Freitas Santos-Biase ◽  
Giovana Krempel Fonseca Merighe ◽  
...  
Keyword(s):  
Embryo Development ◽  
Rna Binding ◽  
Blastocyst Stage ◽  
Messenger Rnas ◽  
Cell Stage ◽  
Maternal Mrna ◽  
Expression Of Genes ◽  
Oocyte Cytoplasm ◽  
Metaphase Ii Oocytes

SummaryThe mRNAs accumulated in oocytes provide support for embryo development until embryo genomic activation. We hypothesized that the maternal mRNA stock present in bovine oocytes is associated with embryo development until the blastocyst stage. To test our hypothesis, we analyzed the transcriptome of the oocyte and correlated the results with the embryo development. Our goal was to identify genes expressed in the oocyte that correlate with its ability to develop to the blastocyst stage. A fraction of oocyte cytoplasm was biopsied using micro-aspiration and stored for further expression analysis. Oocytes were activated chemically, cultured individually and classified according to their capacity to develop in vitro to the blastocyst stage. Microarray analysis was performed on mRNA extracted from the oocyte cytoplasm fractions and correlated with its ability to develop to the blastocyst stage (good quality oocyte) or arrest at the 8–16-cell stage (bad quality oocyte). The expression of 4320 annotated genes was detected in the fractions of cytoplasm that had been collected from oocytes matured in vitro. Gene ontology classification revealed that enriched gene expression of genes was associated with certain biological processes: ‘RNA processing’, ‘translation’ and ‘mRNA metabolic process’. Genes that are important to the molecular functions of ‘RNA binding’ and ‘translation factor activity, RNA binding’ were also enriched in oocytes. We identified 29 genes with differential expression between the two groups of oocytes compared (good versus bad quality). The content of mRNAs expressed in metaphase II oocytes influences the activation of the embryonic genome and enables further develop to the blastocyst stage.

scholarly journals Bacillus subtilis Aconitase Is Required for Efficient Late-Sporulation Gene Expression

2006 ◽  
Vol 188 (17) ◽  
pp. 6396-6405 ◽  
Author(s):  
Alisa W. Serio ◽  
Kieran B. Pechter ◽  
Abraham L. Sonenshein
Keyword(s):  
Bacillus Subtilis ◽  
Transcriptional Activation ◽  
Rna Binding ◽  
Regulatory Protein ◽  
Binding Activity ◽  
High Catalytic Activity ◽  
Spore Coat ◽  
Mobility Shift ◽  
Gel Mobility Shift

ABSTRACT Bacillus subtilis aconitase, encoded by the citB gene, is homologous to the bifunctional eukaryotic protein IRP-1 (iron regulatory protein 1). Like IRP-1, B. subtilis aconitase is both an enzyme and an RNA binding protein. In an attempt to separate the two activities of aconitase, the C-terminal region of the B. subtilis citB gene product was mutagenized. The resulting strain had high catalytic activity but was defective in sporulation. The defect was at a late stage of sporulation, specifically affecting expression of σK-dependent genes, many of which are important for spore coat assembly and require transcriptional activation by GerE. Accumulation of gerE mRNA and GerE protein was delayed in the aconitase mutant strain. Pure B. subtilis aconitase bound to the 3′ untranslated region of gerE mRNA in in vitro gel mobility shift assays, strongly suggesting that aconitase RNA binding activity may stabilize gerE mRNA in order to allow efficient GerE synthesis and proper timing of spore coat assembly.

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