scholarly journals Mpe1, a Zinc Knuckle Protein, Is an Essential Component of Yeast Cleavage and Polyadenylation Factor Required for the Cleavage and Polyadenylation of mRNA

2001 ◽  
Vol 21 (24) ◽  
pp. 8346-8356 ◽  
Author(s):  
Le Thuy Anh Vo ◽  
Michèle Minet ◽  
Jean-Marie Schmitter ◽  
François Lacroute ◽  
Françoise Wyers
Keyword(s):  
Rna Binding ◽  
Essential Gene ◽  
Affinity Purification ◽  
Protein A ◽  
Gene Encoding ◽  
Cleavage And Polyadenylation ◽  
The Stability ◽  
Polyadenylation Factor

ABSTRACT In Saccharomyces cerevisiae, in vitro mRNA cleavage and polyadenylation require the poly(A) binding protein, Pab1p, and two multiprotein complexes: CFI (cleavage factor I) and CPF (cleavage and polyadenylation factor). We characterized a novel essential gene,MPE1 (YKL059c), which interacts genetically with the PCF11 gene encoding a subunit of CFI. Mpe1p is an evolutionarily conserved protein, a homolog of which is encoded by the human genome. The protein sequence contains a putative RNA-binding zinc knuckle motif. MPE1 is implicated in the choice ofACT1 mRNA polyadenylation site in vivo. Extracts from a conditional mutant, mpe1-1, or from a wild-type extract immunoneutralized for Mpe1p are defective in 3′-end processing. We used the tandem affinity purification (TAP) method on strains TAP-tagged for Mpe1p or Pfs2p to show that Mpe1p, like Pfs2p, is an integral subunit of CPF. Nevertheless a stable CPF, devoid of Mpe1p, was purified from the mpe1-1 mutant strain, showing that Mpe1p is not directly involved in the stability of this complex. Consistently, Mpe1p is also not necessary for the processive polyadenylation, nonspecific for the genuine pre-mRNA 3′ end, displayed by the CPF alone. However, a reconstituted assay with purified CFI, CPF, and the recombinant Pab1p showed that Mpe1p is strictly required for the specific cleavage and polyadenylation of pre-mRNA. These results show that Mpe1p plays a crucial role in 3′ end formation probably by promoting the specific link between the CFI/CPF complex and pre-mRNA.

scholarly journals Elevated Levels of a U4/U6.U5 snRNP-Associated Protein, Spp381p, Rescue a Mutant Defective in Spliceosome Maturation

1999 ◽  
Vol 19 (1) ◽  
pp. 577-584 ◽  
Author(s):  
Suzanne Lybarger ◽  
Kristopher Beickman ◽  
Vicky Brown ◽  
Neetu Dembla-Rajpal ◽  
Kristin Morey ◽  
...  
Keyword(s):  
Rna Binding ◽  
Protein A ◽  
Temperature Sensitive ◽  
Rna Transcripts ◽  
Binding Domains ◽  
Elevated Expression ◽  
Immune Precipitation ◽  
Ts Mutant

ABSTRACT U4 snRNA release from the spliceosome occurs through an essential but ill-defined Prp38p-dependent step. Here we report the results of a dosage suppressor screen to identify genes that contribute toPRP38 function. Elevated expression of a previously uncharacterized gene, SPP381, efficiently suppresses the growth and splicing defects of a temperature-sensitive (Ts) mutantprp38-1. This suppression is specific in that enhancedSPP381 expression does not alter the abundance of intronless RNA transcripts or suppress the Ts phenotypes of otherprp mutants. Since SPP381 does not suppress aprp38::LEU2 null allele, it is clear that Spp381p assists Prp38p in splicing but does not substitute for it. YeastSPP381 disruptants are severely growth impaired and accumulate unspliced pre-mRNA. Immune precipitation studies show that, like Prp38p, Spp381p is present in the U4/U6.U5 tri-snRNP particle. Two-hybrid analyses support the view that the carboxyl half of Spp381p directly interacts with the Prp38p protein. A putative PEST proteolysis domain within Spp381p is dispensable for the Spp381p–Prp38p interaction and for prp38-1 suppression but contributes to Spp381p function in splicing. Curiously, in vitro, Spp381p may not be needed for the chemistry of pre-mRNA splicing. Based on the in vivo and in vitro results presented here, we propose that two small acidic proteins without obvious RNA binding domains, Spp381p and Prp38p, act in concert to promote U4/U5.U6 tri-snRNP function in the spliceosome cycle.

scholarly journals SYNCRIP, a Member of the Heterogeneous Nuclear Ribonucleoprotein Family, Is Involved in Mouse Hepatitis Virus RNA Synthesis

2004 ◽  
Vol 78 (23) ◽  
pp. 13153-13162 ◽  
Author(s):  
Keum S. Choi ◽  
Akihiro Mizutani ◽  
Michael M. C. Lai
Keyword(s):  
Mammalian Cells ◽  
Rna Synthesis ◽  
Rna Binding ◽  
Hepatitis Virus ◽  
Affinity Purification ◽  
Mouse Hepatitis Virus ◽  
Viral Rna ◽  
Negative Strand

ABSTRACT Several cellular proteins, including several heterogeneous nuclear ribonucleoproteins (hnRNPs), have been shown to function as regulatory factors for mouse hepatitis virus (MHV) RNA synthesis as a result of their binding to the 5′ and 3′ untranslated regions (UTRs) of the viral RNA. Here, we identified another cellular protein, p70, which has been shown by UV cross-linking to bind both the positive- and negative-strand UTRs of MHV RNA specifically. We purified p70 with a a one-step RNA affinity purification procedure with the biotin-labeled 5′-UTR. Matrix-assisted laser desorption ionization (MALDI)-mass spectrometry identified it as synaptotagmin-binding cytoplasmic RNA-interacting protein (SYNCRIP). SYNCRIP is a member of the hnRNP family and localizes largely in the cytoplasm. The p70 was cross-linked to the MHV positive- or negative-strand UTR in vitro and in vivo. The bacterially expressed SYNCRIP was also able to bind to the 5′-UTR of both strands. The SYNCRIP-binding site was mapped to the leader sequence of the 5′-UTR, requiring the UCUAA repeat sequence. To investigate the functional significance of SYNCRIP in MHV replication, we expressed a full-length or a C-terminally truncated form of SYNCRIP in mammalian cells expressing the MHV receptor. The overexpression of either form of SYNCRIP inhibited syncytium formation induced by MHV infection. Furthermore, downregulation of the endogenous SYNCRIP with a specific short interfering RNA delayed MHV RNA synthesis; in contrast, overexpression or downregulation of SYNCRIP did not affect MHV translation. These results suggest that SYNCRIP may be directly involved in MHV RNA replication as a positive regulator. This study identified an additional cellular hnRNP as an MHV RNA-binding protein potentially involved in viral RNA synthesis.

scholarly journals Yeast Pab1 interacts with Rna15 and participates in the control of the poly(A) tail length in vitro.

1997 ◽  
Vol 17 (7) ◽  
pp. 3694-3701 ◽  
Author(s):  
N Amrani ◽  
M Minet ◽  
M Le Gouar ◽  
F Lacroute ◽  
F Wyers
Keyword(s):  
Wild Type Strain ◽  
Tail Length ◽  
Anion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Wild Type ◽  
Cleavage And Polyadenylation ◽  
Two Hybrid ◽  
Polyadenylation Factor

In Saccharomyces cerevisiae, the single poly(A) binding protein, Pab1, is the major ribonucleoprotein associated with the poly(A) tails of mRNAs in both the nucleus and the cytoplasm. We found that Pab1 interacts with Rna15 in two-hybrid assays and in coimmunoprecipitation experiments. Overexpression of PAB1 partially but specifically suppressed the rna15-2 mutation in vivo. RNA15 codes for a component of the cleavage and polyadenylation factor CF I, one of the four factors needed for pre-mRNA 3'-end processing. We show that Pab1 and CF I copurify in anion-exchange chromatography. These data suggest that Pab1 is physically associated with CF I. Extracts from a thermosensitive pab1 mutant and from a wild-type strain immunoneutralized for Pab1 showed normal cleavage activity but a large increase in poly(A) tail length. A normal tail length was restored by adding recombinant Pab1 to the mutant extract. The longer poly(A) tails were not due to an inhibition of exonuclease activities. Pab1 has previously been implicated in the regulation of translation initiation and in cytoplasmic mRNA stability. Our data indicate that Pab1 is also a part of the 3'-end RNA-processing complex and thus participates in the control of the poly(A) tail lengths during the polyadenylation reaction.

scholarly journals Structural and Functional Analysis of an mRNP Complex That Mediates the High Stability of Human β-Globin mRNA

2001 ◽  
Vol 21 (17) ◽  
pp. 5879-5888 ◽  
Author(s):  
Jia Yu ◽  
J. Eric Russell
Keyword(s):  
Mrna Stability ◽  
Rna Binding ◽  
Assembly Sequence ◽  
Regulatory Pathway ◽  
Globin Mrna ◽  
Mrnp Complex ◽  
The Stability ◽  
High Level

ABSTRACT Human globins are encoded by mRNAs exhibiting high stabilities in transcriptionally silenced erythrocyte progenitors. Unlike α-globin mRNA, whose stability is enhanced by assembly of a specific messenger RNP (mRNP) α complex on its 3′ untranslated region (UTR), neither the structure(s) nor the mechanism(s) that effects the high-level stability of human β-globin mRNA has been identified. The present work describes an mRNP complex assembling on the 3′ UTR of the β-globin mRNA that exhibits many of the properties of the stability-enhancing α complex. The β-globin mRNP complex is shown to contain one or more factors homologous to αCP, a 39-kDa RNA-binding protein that is integral to α-complex assembly. Sequence analysis implicates a specific 14-nucleotide pyrimidine-rich track within its 3′ UTR as the site of β-globin mRNP assembly. The importance of this track to mRNA stability is subsequently verified in vivo using mice expressing human β-globin transgenes that contain informative mutations in this region. In combination, the in vitro and in vivo analyses indicate that the high stabilities of the α- and β-globin mRNAs are maintained through related mRNP complexes that may share a common regulatory pathway.

scholarly journals p53 Stabilization and Accumulation Induced by Human Vaccinia-Related Kinase 1

2004 ◽  
Vol 24 (23) ◽  
pp. 10366-10380 ◽  
Author(s):  
Francisco M. Vega ◽  
Ana Sevilla ◽  
Pedro A. Lazo
Keyword(s):  
Small Interfering Rna ◽  
Protein A ◽  
Cellular Functions ◽  
Active Suppression ◽  
Tumor Susceptibility ◽  
Human Kinome ◽  
The Stability ◽  
Interfering Rna

ABSTRACT Variations in intracellular levels of p53 regulate many cellular functions and determine tumor susceptibility. Major mechanisms modulating p53 levels include phosphorylation and interaction of p53 with specific ubiquitin ligases that promote its degradation. N-terminal phosphorylation regulates the interaction of p53 with several regulatory molecules. Vaccinia-related kinase 1 (VRK1) is the prototype of a new Ser-Thr kinase family in the human kinome. VRK1 is located in the nucleus outside the nucleolus. Overexpression of VRK1 increases the stability of p53 by a posttranslational mechanism leading to its accumulation by a mechanism independent of the Chk2 kinase. Catalytically inactive VRK1 protein (a K179E mutant) does not induce p53 accumulation. VRK1 phosphorylates human p53 in Thr18 and disrupts p53-Mdm2 interaction in vitro, although a significant decrease in p53 ubiquitination by Mdm2 in vivo was not detected. VRK1 kinase does not phosphorylate Mdm2. VRK1-mediated p53 stabilization was also detected in Mdm2−/− cells. VRK1 also has an additive effect with MdmX or p300 to stabilize p53, and p300 coactivation and acetylation of p53 is enhanced by VRK1. The p53 stabilized by VRK1 is transcriptionally active. Suppression of VRK1 expression by specific small interfering RNA provokes several defects in proliferation, situating the protein in the regulation of this process. VRK1 might function as a switch controlling the proteins that interact with p53 and thus modifying its stability and activity. We propose VRK1 as the first step in a new pathway regulating p53 activity during cell proliferation.

scholarly journals HuR counteracts miR-330 to promote STAT3 translation during inflammation-induced muscle wasting

2019 ◽  
Vol 116 (35) ◽  
pp. 17261-17270 ◽  
Author(s):  
Souad Mubaid ◽  
Jennifer F. Ma ◽  
Amr Omer ◽  
Kholoud Ashour ◽  
Xian J. Lian ◽  
...  
Keyword(s):  
Muscle Wasting ◽  
Rna Binding ◽  
Fiber Formation ◽  
Muscle Loss ◽  
Translation Inhibition ◽  
The Stability ◽  
Transcription 3 ◽  
Cellular Movement

Debilitating cancer-induced muscle wasting, a syndrome known as cachexia, is lethal. Here we report a posttranscriptional pathway involving the RNA-binding protein HuR as a key player in the onset of this syndrome. Under these conditions, HuR switches its function from a promoter of muscle fiber formation to become an inducer of muscle loss. HuR binds to the STAT3 (signal transducer and activator of transcription 3) mRNA, which encodes one of the main effectors of this condition, promoting its expression both in vitro and in vivo. While HuR does not affect the stability and the cellular movement of this transcript, HuR promotes the translation of the STAT3 mRNA by preventing miR-330 (microRNA 330)–mediated translation inhibition. To achieve this effect, HuR directly binds to a U-rich element in the STAT3 mRNA-3′untranslated region (UTR) located within the vicinity of the miR-330 seed element. Even though the binding sites of HuR and miR-330 do not overlap, the recruitment of either one of them to the STAT3-3′UTR negatively impacts the binding and the function of the other factor. Therefore, together, our data establish the competitive interplay between HuR and miR-330 as a mechanism via which muscle fibers modulate, in part, STAT3 expression to determine their fate in response to promoters of muscle wasting.

scholarly journals Constitutive centromere-associated network contacts confer differential stability on CENP-A nucleosomes in vitro and in the cell

2018 ◽  
Vol 29 (6) ◽  
pp. 751-762 ◽  
Author(s):  
Shengya Cao ◽  
Keda Zhou ◽  
Zhening Zhang ◽  
Karolin Luger ◽  
Aaron F. Straight
Keyword(s):  
Protein A ◽  
Histone H3 ◽  
Intrinsic Property ◽  
Cell Cycles ◽  
Centromere Protein A ◽  
Histone H3 Variant ◽  
Differential Stability ◽  
The Stability

Eukaryotic centromeres are defined by the presence of nucleosomes containing the histone H3 variant, centromere protein A (CENP-A). Once incorporated at centromeres, CENP-A nucleosomes are remarkably stable, exhibiting no detectable loss or exchange over many cell cycles. It is currently unclear whether this stability is an intrinsic property of CENP-A containing chromatin or whether it arises from proteins that specifically associate with CENP-A chromatin. Two proteins, CENP-C and CENP-N, are known to bind CENP-A human nucleosomes directly. Here we test the hypothesis that CENP-C or CENP-N stabilize CENP-A nucleosomes in vitro and in living cells. We show that CENP-N stabilizes CENP-A nucleosomes alone and additively with CENP-C in vitro. However, removal of CENP-C and CENP-N from cells, or mutating CENP-A so that it no longer interacts with CENP-C or CENP-N, had no effect on centromeric CENP-A stability in vivo. Thus, the stability of CENP-A nucleosomes in chromatin does not arise solely from its interactions with CENP-C or CENP-N.

scholarly journals Mammalian Peptidylglycine α-Amidating Monooxygenase mRNA Expression Can Be Modulated by the La Autoantigen

2005 ◽  
Vol 25 (17) ◽  
pp. 7505-7521 ◽  
Author(s):  
Fabienne Brenet ◽  
Nadège Dussault ◽  
Jonas Borch ◽  
Géraldine Ferracci ◽  
Christine Delfino ◽  
...  
Keyword(s):  
Rna Binding ◽  
Stop Codon ◽  
Affinity Purification ◽  
Nuclear Retention ◽  
Clear Cut ◽  
La Protein ◽  
Mass Spectrometry Identification ◽  
Reporter Assays

ABSTRACT Peptidylglycine α-amidating monooxygenase (PAM; EC 1.14.17.3) catalyzes the COOH-terminal α-amidation of peptidylglycine substrates, yielding amidated products. We have previously reported a putative regulatory RNA binding protein (PAM mRNA-BP) that binds specifically to the 3′ untranslated region (UTR) of PAM-mRNA. Here, the PAM mRNA-BP was isolated and revealed to be La protein using affinity purification onto a 3′ UTR PAM RNA, followed by tandem mass spectrometry identification. We determined that the core binding sequence is approximately 15-nucleotides (nt) long and is located 471 nt downstream of the stop codon. Moreover, we identified the La autoantigen as a protein that specifically binds the 3′ UTR of PAM mRNA in vivo and in vitro. Furthermore, La protein overexpression caused a nuclear retention of PAM mRNAs and resulted in the down-regulation of endogenous PAM activity. Most interestingly, the nuclear retention of PAM mRNA is lost upon expressing the La proteins that lack a conserved nuclear retention element, suggesting a direct association between PAM mRNA and La protein in vivo. Reporter assays using a chimeric mRNA that combined luciferase and the 3′ UTR of PAM mRNA demonstrated a decrease of the reporter activity due to an increase in the nuclear localization of reporter mRNAs, while the deletion of the 15-nt La binding site led to their clear-cut cytoplasmic relocalization. The results suggest an important role for the La protein in the modulation of PAM expression, possibly by mechanisms that involve a nuclear retention and perhaps a processing of pre-PAM mRNA molecules.

scholarly journals τ91, an Essential Subunit of Yeast Transcription Factor IIIC, Cooperates with τ138 in DNA Binding

1998 ◽  
Vol 18 (1) ◽  
pp. 1-9 ◽  
Author(s):  
Rosalía Arrebola ◽  
Nathalie Manaud ◽  
Sophie Rozenfeld ◽  
Marie-Claude Marsolier ◽  
Olivier Lefebvre ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Essential Gene ◽  
Open Reading Frames ◽  
Class Iii ◽  
Gene Encoding ◽  
Promoter Recognition

ABSTRACT Transcription factor IIIC (TFIIIC) (or τ) is a large multisubunit and multifunctional factor required for transcription of all class III genes in Saccharomyces cerevisiae. It is responsible for promoter recognition and TFIIIB assembly. We report here the cloning and characterization of TFC6, an essential gene encoding the 91-kDa polypeptide, τ91, present in affinity-purified TFIIIC. τ91 has a predicted molecular mass of 74 kDa. It harbors a central cluster of His and Cys residues and has basic and acidic amino acid regions, but it shows no specific similarity to known proteins or predicted open reading frames. The TFIIIC subunit status of τ91 was established by the following biochemical and genetic evidence. Antibodies to τ91 bound TFIIIC-DNA complexes in gel shift assays; in vivo, a B block-deficient U6 RNA gene (SNR6) harboring GAL4 binding sites was reactivated by fusing the GAL4 DNA binding domain to τ91; and a point mutation in TFC6 (τ91-E330K) was found to suppress the thermosensitive phenotype of a tfc3-G349Emutant affected in the B block binding subunit (τ138). The suppressor mutation alleviated the DNA binding and transcription defects of mutant TFIIIC in vitro. These results indicated that τ91 cooperates with τ138 for DNA binding. Recombinant τ91 by itself did not interact with a tRNA gene, although it showed a strong affinity for single-stranded DNA.

scholarly journals Trypanosoma brucei Translation Initiation Factor Homolog EIF4E6 Forms a Tripartite Cytosolic Complex with EIF4G5 and a Capping Enzyme Homolog

2014 ◽  
Vol 13 (7) ◽  
pp. 896-908 ◽  
Author(s):  
Eden R. Freire ◽  
Amaranta M. Malvezzi ◽  
Ajay A. Vashisht ◽  
Joanna Zuberek ◽  
Edwin A. Saada ◽  
...  
Keyword(s):  
Transcriptional Control ◽  
Rna Binding ◽  
Regulation Of Gene Expression ◽  
Protein A ◽  
Translation Initiation Factor ◽  
Initiation Factor ◽  
Nucleoside Triphosphate ◽  
Rna Interaction

ABSTRACT Trypanosomes lack the transcriptional control characteristic of the majority of eukaryotes that is mediated by gene-specific promoters in a one-gene–one-promoter arrangement. Rather, their genomes are transcribed in large polycistrons with no obvious functional linkage. Posttranscriptional regulation of gene expression must thus play a larger role in these organisms. The eIF4E homolog TbEIF4E6 binds mRNA cap analogs in vitro and is part of a complex in vivo that may fulfill such a role. Knockdown of TbEIF4E6 tagged with protein A-tobacco etch virus protease cleavage site-protein C to approximately 15% of the normal expression level resulted in viable cells that displayed a set of phenotypes linked to detachment of the flagellum from the length of the cell body, if not outright flagellum loss. While these cells appeared and behaved as normal under stationary liquid culture conditions, standard centrifugation resulted in a marked increase in flagellar detachment. Furthermore, the ability of TbEIF4E6-depleted cells to engage in social motility was reduced. The TbEIF4E6 protein forms a cytosolic complex containing a triad of proteins, including the eIF4G homolog TbEIF4G5 and a hypothetical protein of 70.3 kDa, referred to as TbG5-IP. The TbG5-IP analysis revealed two domains with predicted secondary structures conserved in mRNA capping enzymes: nucleoside triphosphate hydrolase and guanylyltransferase. These complex members have the potential for RNA interaction, either via the 5′ cap structure for TbEIF4E6 and TbG5-IP or through RNA-binding domains in TbEIF4G5. The associated proteins provide a signpost for future studies to determine how this complex affects capped RNA molecules.

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