PUB1 is a major nuclear and cytoplasmic polyadenylated RNA-binding protein in Saccharomyces cerevisiae.

Anderson, James T.; Paddy, Michael R.; Swanson, Maurice S.

doi:10.1128/mcb.13.10.6102

Cited by 82 publications

(100 citation statements)

References 50 publications

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“…Denatured antigen was prepared by adding an equal volume of 126 mM Tris⅐HCl (pH 6.8)͞20% glycerol͞4% SDS͞0.005% bromophenol blue to recombinant HuR, heating the sample to 100°C for 3 min, cooling to room temperature, and adding an equal volume of MPL͞TDM adjuvant. Monoclonal antibodies were isolated as described previously (29), except that rapid screening of initial hybridoma supernatants was performed by ELISA, and those scoring positive by ELISA were subsequently examined by immunoblot and cell immunofluorescence analyses. The heavy chain subclass of mAb 3A2 is IgG1.…”

Section: Methodsmentioning

confidence: 99%

HuR binding to cytoplasmic mRNA is perturbed by heat shock

Gallouzi

Brennan

Stenberg

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

294

280

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AU-rich elements (AREs) located in the 3′ untranslated region target the mRNAs encoding many protooncoproteins, cytokines, and lymphokines for rapid degradation. HuR, a ubiquitously expressed member of the embryonic lethal abnormal vision (ELAV) family of RNA-binding proteins, binds ARE sequences and selectively stabilizes ARE-containing reporter mRNAs when overexpressed in transiently transfected cells. HuR appears predominantly nucleoplasmic but has been shown to shuttle between the nucleus and cytoplasm via a novel shuttling sequence HNS. We report generation of a mouse monoclonal antibody 3A2 that both immunoblots and immunoprecipitates HuR protein; it recognizes an epitope located in the first of HuR's three RNA recognition motifs. This antibody was used to probe HuR interactions with mRNA before and after heat shock, a condition that has been reported to stabilize ARE-containing mRNAs. At 37°C, approximately one-third of the cytoplasmic HuR appears polysome associated, and in vivo UV crosslinking reveals that HuR interactions with poly(A) + RNA are predominantly cytoplasmic rather than nuclear. This comprises evidence that HuR directly interacts with mRNA in vivo . After heat shock, 12–15% of HuR accumulates in discrete foci in the cytoplasm, but surprisingly the majority of HuR crosslinks instead to nuclear poly(A) + RNA, whose levels are dramatically increased in the stressed cells. This behavior of HuR differs from that of another ARE-binding protein, hnRNP D, which has been implicated as an effector of mRNA decay rather than mRNA stabilization and of the general pre-RNA-binding protein hnRNP A1. We interpret these differences to mean that the temporal association of HuR with ARE-containing mRNAs is different from that of these other two proteins.

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Section: Methodsmentioning

confidence: 99%

HuR binding to cytoplasmic mRNA is perturbed by heat shock

Gallouzi

Brennan

Stenberg

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

294

280

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“…While the transport of mRNA from the nucleus to the cytoplasm is coupled with a general exchange of hnRNP proteins for cytoplasmic mRNP proteins (46,47), the demonstration of selective hnRNP protein shuttling between the nucleus and cytoplasm (48) suggests that the exchange of nuclear for cytoplasmic RNA-binding proteins continues in the cytoplasm. Analysis of hnRNP-like proteins of lower eukaryotes proves that a continuum of nuclear/ cytoplasmic RNP exchange exists, so specific hnRNP-like proteins either become polyribosomal (49) or co-fractionate with nontranslated mRNP (50). The recent demonstration of a nuclear step governing the cytoplasmic masking of mRNA in Xenopus oocytes suggests that specific nuclear RNP interactions target an mRNA for mRNP3ϩ4 assembly and masking (30,31).…”

Section: Discussionmentioning

confidence: 99%

A Translation Regulatory Particle Containing theXenopus Oocyte Y Box Protein mRNP3+4

Yurkova

Murray

1997

Journal of Biological Chemistry

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In oocytes, nontranslated maternal mRNAs are packaged by protein into messenger ribonucleoprotein particles (mRNPs) that are masked from translation by protein-RNA interactions. Proteins associated with such masked states of mRNAs are particularly abundant in amphibian oocytes. One of these mRNP proteins from Xenopus oocytes, mRNP3؉4 (also called FRG Y2a/b or p54/p56), binds to diverse mRNAs independent of their sequence and is the germ line member of the evolutionarily conserved Y box protein multigene family. Xenopus oocytes contain soluble pools of mRNP3؉4 6 S oligomers, probably dimers, and larger ϳ15 S particles containing mRNP3؉4 and additional proteins. Here we report the purification of this larger form as an ϳ320-kDa particle that contains mRNP3؉4 and nine additional polypeptides, including mRNA-binding polypeptides of 34 and 36 kDa and a doublet of 110/105 kDa that proved to be nucleolin. The particle has a protein kinase activity that phosphorylates its own mRNP3؉4, nucleolin, and a 31-kDa polypeptide component and exhibits translational inhibition in both the wheat germ extract and rabbit reticulocyte lysate systems. The presence of mRNP3؉4 and nucleolin in this large translation regulatory particle suggests that it participates in an early step of mRNP assembly and masking.The packaging of mRNA into stable nontranslated cytoplasmic messenger ribonucleoprotein particles (mRNPs) 1 is an essential mechanism of post-transcriptional regulation of gene expression utilized by both male and female germ cells of vertebrates and invertebrates. Both the nontranslated state and the stability of these translationally masked mRNAs are at least partly mediated by the protein component (1-3), but little is known about the mechanism of translational regulation. Xenopus oocytes provide a rich source of masked mRNPs that can be biochemically characterized, manipulated by microinjection studies, and readily followed during early embryogenesis (4, 5). These mRNA-associated proteins inhibit the translation of ␤-globin mRNA either in vivo following oocyte microinjection or in vitro in a wheat germ extract (WGE) translation system (6), thereby demonstrating their ability to repress translation.Recent studies of the most abundant Xenopus oocyte mRNP polypeptides have begun to address the structure and function of mRNP proteins. These polypeptides were originally named mRNP3ϩ4 (4) and subsequently p54 and p56 (5, 7, 8), pp56 and pp60 (9), or FRG Y2 (mRNP4) (10). Polypeptides mRNP3 and mRNP4 are highly related, probable pseudoalleles (8, 11), and therefore, because their simplest native form appears to be an ϳ6 S dimer (7), they are referred to as a single protein, mRNP3ϩ4. mRNP3ϩ4 particles have also been shown to be the germ line members of an evolutionarily conserved multigene family of nucleic acid-binding proteins, called the Y box proteins (12, 13), that are associated with masked mRNPs during oogenesis and spermatogenesis (8, 14 -16). In various somatic cells, a closely related Y box protein is associated with nontrans...

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“…Aliquots containing 10% of the input cell extracts (IN), 10% of the flowthrough wash (FT), and 100% of the eluate (EL) were resolved by SDS-PAGE and subjected to immunoblot analysis using monoclonal anti-RGS His antibodies (1:500; Qiagen) directed against the tag on His-Gcd10p, and with polyclonal antibodies directed against Gcd10p (1:500), Gcd14p (1:500), or Prt1p(1:1000). (B) Indirect immunofluorescence was used to study the subcellular distribution of HA epitope-tagged forms of Gcd10p, Gcd14p, and Tif34p in strains YJA142 (GCD10-HA; a,b), YJA143 (GCD10; c,d), Hm296 bearing pRC64 (GCD14-HA; g,h), Hm296 bearing pRC62 (GCD14; i,j), and KAY8 (TIF34-HA; k,l), as described previously (Anderson et al 1993). All antibodies were diluted in PBS, 5% non-fat dried milk.…”

Section: Gcd10p Is Required For the 1-methyladenosine Modification Ofmentioning

confidence: 99%

The essential Gcd10p–Gcd14p nuclear complex is required for 1-methyladenosine modification and maturation of initiator methionyl-tRNA

Anderson

Phan²,

Cuesta³

et al. 1998

Genes Dev.

229

327

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Gcd10p andMet maturation. The chromatographic behavior of elongator and initiator tRNA Met on a RPC-5 column indicated that both species are altered structurally in gcd10⌬ cells, and analysis of base modifications revealed that 1-methyladenosine (m 1 A) is undetectable in gcd10⌬ tRNA. Interestingly, gcd10 and gcd14 mutations had no effect on processing or accumulation of elongator tRNA Met , which also contains m 1 A at position 58, suggesting a unique requirement for this base modification in initiator maturation.

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PUB1 is a major nuclear and cytoplasmic polyadenylated RNA-binding protein in Saccharomyces cerevisiae.

Cited by 82 publications

References 50 publications

HuR binding to cytoplasmic mRNA is perturbed by heat shock

HuR binding to cytoplasmic mRNA is perturbed by heat shock

A Translation Regulatory Particle Containing theXenopus Oocyte Y Box Protein mRNP3+4

The essential Gcd10p–Gcd14p nuclear complex is required for 1-methyladenosine modification and maturation of initiator methionyl-tRNA

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