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The bacterial ribonuclease P holoenzyme requires specific, conserved residues for efficient catalysis and substrate positioning.
Reiter NJ, Osterman AK, Mondragón A
(2012) Nucleic Acids Res 40: 10384-93
MeSH Terms: Bacterial Proteins, Biocatalysis, Catalytic Domain, Models, Molecular, Mutagenesis, Site-Directed, RNA Precursors, RNA, Transfer, Ribonuclease P, Thermotoga maritima
Show Abstract · Added January 28, 2014
RNase P is an RNA-based enzyme primarily responsible for 5'-end pre-tRNA processing. A structure of the bacterial RNase P holoenzyme in complex with tRNAPhe revealed the structural basis for substrate recognition, identified the active site location, and showed how the protein component increases functionality. The active site includes at least two metal ions, a universal uridine (U52), and P RNA backbone moieties, but it is unclear whether an adjacent, bacterially conserved protein loop (residues 52-57) participates in catalysis. Here, mutagenesis combined with single-turnover reaction kinetics demonstrate that point mutations in this loop have either no or modest effects on catalytic efficiency. Similarly, amino acid changes in the 'RNR' region, which represent the most conserved region of bacterial RNase P proteins, exhibit negligible changes in catalytic efficiency. However, U52 and two bacterially conserved protein residues (F17 and R89) are essential for efficient Thermotoga maritima RNase P activity. The U52 nucleotide binds a metal ion at the active site, whereas F17 and R89 are positioned >20 Å from the cleavage site, probably making contacts with N(-4) and N(-5) nucleotides of the pre-tRNA 5'-leader. This suggests a synergistic coupling between transition state formation and substrate positioning via interactions with the leader.
0 Communities
1 Members
0 Resources
9 MeSH Terms
Tailoring of membrane proteins by alternative splicing of pre-mRNA.
Mittendorf KF, Deatherage CL, Ohi MD, Sanders CR
(2012) Biochemistry 51: 5541-56
MeSH Terms: Alternative Splicing, Alzheimer Disease, Animals, Humans, Introns, Membrane Proteins, Organ Specificity, Protein Folding, Protein Sorting Signals, Protein Transport, RNA Precursors, RNA, Messenger
Show Abstract · Added March 7, 2014
Alternative splicing (AS) of RNA is a key mechanism for diversification of the eukaryotic proteome. In this process, different mRNA transcripts can be produced through altered excision and/or inclusion of exons during processing of the pre-mRNA molecule. Since its discovery, AS has been shown to play roles in protein structure, function, and localization. Dysregulation of this process can result in disease phenotypes. Moreover, AS pathways are promising therapeutic targets for a number of diseases. Integral membrane proteins (MPs) represent a class of proteins that may be particularly amenable to regulation by alternative splicing because of the distinctive topological restraints associated with their folding, structure, trafficking, and function. Here, we review the impact of AS on MP form and function and the roles of AS in MP-related disorders such as Alzheimer's disease.
0 Communities
2 Members
0 Resources
12 MeSH Terms
Systematic two-hybrid and comparative proteomic analyses reveal novel yeast pre-mRNA splicing factors connected to Prp19.
Ren L, McLean JR, Hazbun TR, Fields S, Vander Kooi C, Ohi MD, Gould KL
(2011) PLoS One 6: e16719
MeSH Terms: Models, Biological, Organisms, Genetically Modified, Protein Binding, Protein Structure, Tertiary, Proteomics, RNA Precursors, RNA Splicing, RNA Splicing Factors, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Schizosaccharomyces, Spliceosomes, Transcription Factors, Two-Hybrid System Techniques, Yeasts
Show Abstract · Added March 5, 2014
Prp19 is the founding member of the NineTeen Complex, or NTC, which is a spliceosomal subcomplex essential for spliceosome activation. To define Prp19 connectivity and dynamic protein interactions within the spliceosome, we systematically queried the Saccharomyces cerevisiae proteome for Prp19 WD40 domain interaction partners by two-hybrid analysis. We report that in addition to S. cerevisiae Cwc2, the splicing factor Prp17 binds directly to the Prp19 WD40 domain in a 1:1 ratio. Prp17 binds simultaneously with Cwc2 indicating that it is part of the core NTC complex. We also find that the previously uncharacterized protein Urn1 (Dre4 in Schizosaccharomyces pombe) directly interacts with Prp19, and that Dre4 is conditionally required for pre-mRNA splicing in S. pombe. S. pombe Dre4 and S. cerevisiae Urn1 co-purify U2, U5, and U6 snRNAs and multiple splicing factors, and dre4Δ and urn1Δ strains display numerous negative genetic interactions with known splicing mutants. The S. pombe Prp19-containing Dre4 complex co-purifies three previously uncharacterized proteins that participate in pre-mRNA splicing, likely before spliceosome activation. Our multi-faceted approach has revealed new low abundance splicing factors connected to NTC function, provides evidence for distinct Prp19 containing complexes, and underscores the role of the Prp19 WD40 domain as a splicing scaffold.
0 Communities
3 Members
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15 MeSH Terms
The solution structure of the ADAR2 dsRBM-RNA complex reveals a sequence-specific readout of the minor groove.
Stefl R, Oberstrass FC, Hood JL, Jourdan M, Zimmermann M, Skrisovska L, Maris C, Peng L, Hofr C, Emeson RB, Allain FH
(2010) Cell 143: 225-37
MeSH Terms: Adenosine Deaminase, Amino Acid Sequence, Animals, Cell Line, Humans, Mice, Models, Molecular, Molecular Sequence Data, Mutation, Nuclear Magnetic Resonance, Biomolecular, RNA Precursors, RNA, Double-Stranded, RNA-Binding Proteins, Rats, Receptors, AMPA, Sequence Alignment
Show Abstract · Added May 28, 2014
Sequence-dependent recognition of dsDNA-binding proteins is well understood, yet sequence-specific recognition of dsRNA by proteins remains largely unknown, despite their importance in RNA maturation pathways. Adenosine deaminases that act on RNA (ADARs) recode genomic information by the site-selective deamination of adenosine. Here, we report the solution structure of the ADAR2 double-stranded RNA-binding motifs (dsRBMs) bound to a stem-loop pre-mRNA encoding the R/G editing site of GluR-2. The structure provides a molecular basis for how dsRBMs recognize the shape, and also more surprisingly, the sequence of the dsRNA. The unexpected direct readout of the RNA primary sequence by dsRBMs is achieved via the minor groove of the dsRNA and this recognition is critical for both editing and binding affinity at the R/G site of GluR-2. More generally, our findings suggest a solution to the sequence-specific paradox faced by many dsRBM-containing proteins that are involved in post-transcriptional regulation of gene expression.
Copyright © 2010 Elsevier Inc. All rights reserved.
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1 Members
0 Resources
16 MeSH Terms
Circulating RNA transcripts identify therapeutic response in cystic fibrosis lung disease.
Saavedra MT, Hughes GJ, Sanders LA, Carr M, Rodman DM, Coldren CD, Geraci MW, Sagel SD, Accurso FJ, West J, Nick JA
(2008) Am J Respir Crit Care Med 178: 929-38
MeSH Terms: Acute Disease, Adult, Anti-Bacterial Agents, Biomarkers, Cohort Studies, Cystic Fibrosis, Cytokines, Female, Forced Expiratory Volume, Gene Expression Profiling, Humans, Leukocytes, Male, Oligonucleotide Array Sequence Analysis, Predictive Value of Tests, Prospective Studies, RNA Precursors, ROC Curve, Reproducibility of Results, Reverse Transcriptase Polymerase Chain Reaction, Spirometry, Young Adult
Show Abstract · Added November 17, 2011
RATIONALE - Circulating leukocyte RNA transcripts are systemic markers of inflammation, which have not been studied in cystic fibrosis (CF) lung disease. Although the standard assessment of pulmonary treatment response is FEV(1), a measure of airflow limitation, the lack of systemic markers to reflect changes in lung inflammation critically limits the testing of proposed therapeutics.
OBJECTIVES - We sought to prospectively identify and validate peripheral blood leukocyte genes that could mark resolution of pulmonary infection and inflammation using a model by which RNA transcripts could increase the predictive value of spirometry.
METHODS - Peripheral blood mononuclear cells were isolated from 10 patients with CF and acute pulmonary exacerbations before and after therapy. RNA expression profiling revealed that 10 genes significantly changed with treatment when compared with matched non-CF and control subjects with stable CF to establish baseline transcript abundance. Peripheral blood mononuclear cell RNA transcripts were prospectively validated, using real-time polymerase chain reaction amplification, in an independent cohort of acutely ill patients with CF (n = 14). Patients who responded to therapy were analyzed using general estimating equations and multiple logistic regression, such that changes in FEV(1)% predicted were regressed with transcript changes.
MEASUREMENTS AND MAIN RESULTS - Three genes, CD64, ADAM9, and CD36, were significant and independent predictors of a therapeutic response beyond that of FEV(1) alone (P < 0.05). In both cohorts, receiver operating characteristic analysis revealed greater accuracy when genes were combined with FEV(1).
CONCLUSIONS - Circulating mononuclear cell transcripts characterize a response to the treatment of pulmonary exacerbations. Even in small patient cohorts, changes in gene expression in conjunction with FEV(1) may enhance current outcomes measures for treatment response.
0 Communities
1 Members
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22 MeSH Terms
Structure and sequence determinants required for the RNA editing of ADAR2 substrates.
Dawson TR, Sansam CL, Emeson RB
(2004) J Biol Chem 279: 4941-51
MeSH Terms: Adenosine Deaminase, Alternative Splicing, Animals, Base Pairing, Base Sequence, Binding, Competitive, Cell Line, Exons, Humans, In Vitro Techniques, Introns, Kinetics, Models, Molecular, Molecular Sequence Data, Mutation, Nucleic Acid Conformation, RNA Editing, RNA Precursors, RNA-Binding Proteins, Rats, Recombinant Proteins, Substrate Specificity, Transfection
Show Abstract · Added July 12, 2010
ADAR2 is a double-stranded RNA-specific adenosine deaminase involved in the editing of mammalian RNAs by the site-specific conversion of adenosine to inosine. We have demonstrated previously that ADAR2 can modify its own pre-mRNA, leading to the creation of a proximal 3'-splice junction containing a non-canonical adenosine-inosine (A-I) dinucleotide. Alternative splicing to this proximal acceptor shifts the reading frame of the mature mRNA transcript, resulting in the loss of functional ADAR2 expression. Both evolutionary sequence conservation and mutational analysis support the existence of an extended RNA duplex within the ADAR2 pre-mRNA formed by base-pairing interactions between regions approximately 1.3-kilobases apart in intron 4 and exon 5. Characterization of ADAR2 pre-mRNA transcripts isolated from adult rat brain identified 16 editing sites within this duplex region, and sites preferentially modified by ADAR1 and ADAR2 have been defined using both tissue culture and in vitro editing systems. Statistical analysis of nucleotide sequences surrounding edited and non-edited adenosine residues have identified a nucleotide sequence bias correlating with ADAR2 site preference and editing efficiency. Among a mixed population of ADAR substrates, ADAR2 preferentially favors its own transcript, yet mutation of a poor substrate to conform to the defined nucleotide bias increases the ability of that substrate to be modified by ADAR2. These data suggest that both sequence and structural elements are required to define adenosine moieties targeted for specific ADAR2-mediated deamination.
1 Communities
1 Members
0 Resources
23 MeSH Terms
Modulation of RNA editing by functional nucleolar sequestration of ADAR2.
Sansam CL, Wells KS, Emeson RB
(2003) Proc Natl Acad Sci U S A 100: 14018-23
MeSH Terms: Adenosine Deaminase, Amino Acid Sequence, Animals, Base Sequence, Cell Line, Cell Nucleolus, In Vitro Techniques, Mice, Molecular Sequence Data, NIH 3T3 Cells, RNA Editing, RNA Precursors, RNA, Double-Stranded, RNA, Ribosomal, RNA-Binding Proteins, Rats, Recombinant Fusion Proteins, Substrate Specificity
Show Abstract · Added January 17, 2014
The adenosine deaminases that act on RNA (ADARs) catalyze the site-specific conversion of adenosine to inosine (A to I) in primary mRNA transcripts, thereby affecting the splicing pattern or coding potential of mature mRNAs. Although the subnuclear localization of A-to-I editing has not been precisely defined, ADARs have been shown to act before splicing, suggesting that they function near nucleoplasmic sites of transcription. Here we demonstrate that ADAR2, a member of the vertebrate ADAR family, is concentrated in the nucleolus, a subnuclear domain disparate from the sites of mRNA transcription. Selective inhibition of ribosomal RNA synthesis or the introduction of mutations in the double-stranded RNA-binding domains within ADAR2 results in translocation of the protein to the nucleoplasm, suggesting that nucleolar association of ADAR2 depends on its ability to bind to ribosomal RNA. Fluorescence recovery after photobleaching reveals that ADAR2 can shuttle rapidly between subnuclear compartments. Enhanced translocation of endogenous ADAR2 from the nucleolus to the nucleoplasm results in increased editing of endogenous ADAR2 substrates. These observations indicate that the nucleolar localization of ADAR2 represents an important mechanism by which RNA editing can be modulated by the sequestration of enzymatic activity from potential RNA substrates in the nucleoplasm.
1 Communities
2 Members
0 Resources
18 MeSH Terms
Proteomics analysis reveals stable multiprotein complexes in both fission and budding yeasts containing Myb-related Cdc5p/Cef1p, novel pre-mRNA splicing factors, and snRNAs.
Ohi MD, Link AJ, Ren L, Jennings JL, McDonald WH, Gould KL
(2002) Mol Cell Biol 22: 2011-24
MeSH Terms: Amino Acid Sequence, Animals, Cell Cycle Proteins, Humans, Macromolecular Substances, Mass Spectrometry, Microscopy, Electron, Molecular Sequence Data, Molecular Weight, Multiprotein Complexes, Mutation, Proteome, Proto-Oncogene Proteins c-myb, RNA Precursors, RNA Splicing, RNA, Messenger, RNA, Small Nuclear, RNA-Binding Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Schizosaccharomyces, Schizosaccharomyces pombe Proteins, Sequence Homology, Amino Acid, Thermodynamics
Show Abstract · Added April 18, 2013
Schizosaccharomyces pombe Cdc5p and its Saccharomyces cerevisiae ortholog, Cef1p, are essential Myb-related proteins implicated in pre-mRNA splicing and contained within large multiprotein complexes. Here we describe the tandem affinity purification (TAP) of Cdc5p- and Cef1p-associated complexes. Using transmission electron microscopy, we show that the purified Cdc5p complex is a discrete structure. The components of the S. pombe Cdc5p/S. cerevisiae Cef1p complexes (termed Cwfs or Cwcs, respectively) were identified using direct analysis of large protein complex (DALPC) mass spectrometry (A. J. Link et al., Nat. Biotechnol. 17:676-682, 1999). At least 26 proteins were detected in the Cdc5p/Cef1p complexes. Comparison of the polypeptides identified by S. pombe Cdc5p purification with those identified by S. cerevisiae Cef1p purification indicates that these two yeast complexes are nearly identical in composition. The majority of S. pombe Cwf proteins and S. cerevisiae Cwc proteins are known pre-mRNA splicing factors including core Sm and U2 and U5 snRNP components. In addition, the complex contains the U2, U5, and U6 snRNAs. Previously uncharacterized proteins were also identified, and we provide evidence that several of these novel factors are involved in pre-mRNA splicing. Our data represent the first comprehensive analysis of CDC5-associated proteins in yeasts, describe a discrete highly conserved complex containing novel pre-mRNA splicing factors, and demonstrate the power of DALPC for identification of components in multiprotein complexes.
0 Communities
5 Members
0 Resources
24 MeSH Terms
Removal of a single alpha-tubulin gene intron suppresses cell cycle arrest phenotypes of splicing factor mutations in Saccharomyces cerevisiae.
Burns CG, Ohi R, Mehta S, O'Toole ET, Winey M, Clark TA, Sugnet CW, Ares M, Gould KL
(2002) Mol Cell Biol 22: 801-15
MeSH Terms: Base Sequence, Cell Cycle, Cell Cycle Proteins, DEAD-box RNA Helicases, DNA, Fungal, DNA-Binding Proteins, Fungal Proteins, Genes, Fungal, Introns, Microtubules, Mitosis, Mutation, Phenotype, Protein Kinases, Protein-Serine-Threonine Kinases, RNA Helicases, RNA Precursors, RNA Splicing, RNA Splicing Factors, RNA, Fungal, RNA, Messenger, RNA-Binding Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Schizosaccharomyces pombe Proteins, Temperature, Tubulin
Show Abstract · Added March 5, 2014
Genetic and biochemical studies of Schizosaccharomyces pombe and Saccharomyces cerevisiae have identified gene products that play essential functions in both pre-mRNA splicing and cell cycle control. Among these are the conserved, Myb-related CDC5 (also known as Cef1p in S. cerevisiae) proteins. The mechanism by which loss of CDC5/Cef1p function causes both splicing and cell cycle defects has been unclear. Here we provide evidence that cell cycle arrest in a new temperature-sensitive CEF1 mutant, cef1-13, is an indirect consequence of defects in pre-mRNA splicing. Although cef1-13 cells harbor global defects in pre-mRNA splicing discovered through intron microarray analysis, inefficient splicing of the alpha-tubulin-encoding TUB1 mRNA was considered as a potential cause of the cef1-13 cell cycle arrest because cef1-13 cells arrest uniformly at G(2)/M with many hallmarks of a defective microtubule cytoskeleton. Consistent with this possibility, cef1-13 cells possess reduced levels of total TUB1 mRNA and alpha-tubulin protein. Removing the intron from TUB1 in cef1-13 cells boosts TUB1 mRNA and alpha-tubulin expression to near wild-type levels and restores microtubule stability in the cef1-13 mutant. As a result, cef1-13 tub1Deltai cells progress through mitosis and their cell cycle arrest phenotype is alleviated. Removing the TUB1 intron from two other splicing mutants that arrest at G(2)/M, prp17Delta and prp22-1 strains, permits nuclear division, but suppression of the cell cycle block is less efficient. Our data raise the possibility that although cell cycle arrest phenotypes in prp mutants can be explained by defects in pre-mRNA splicing, the transcript(s) whose inefficient splicing contributes to cell cycle arrest is likely to be prp mutant dependent.
0 Communities
2 Members
0 Resources
27 MeSH Terms
Use of high specific activity StarFire oligonucleotide probes to visualize low-abundance pre-mRNA splicing intermediates in S. pombe.
Behlke MA, Dames SA, McDonald WH, Gould KL, Devor EJ, Walder JA
(2000) Biotechniques 29: 892-7
MeSH Terms: Oligonucleotide Probes, RNA Precursors, RNA Splicing, RNA, Fungal, Schizosaccharomyces
Show Abstract · Added March 5, 2014
An oligonucleotide labeling system was developed that can produce radiolabeled hybridization probes with tenfold or more higher specific activity than is obtained by traditional 5'-end-labeling with polynucleotide kinase. Yet the system is as rapid and simple as kinase labeling. The reaction uses the Klenow fragment of E. coli DNA polymerase to add alpha-32P-dA residues to the 3'-end of an oligonucleotide in a primer-extension reaction. Unlike other methods of radioactive tailing (e.g., terminal transferase), a single species is produced of both known length and known specific activity. The reaction is efficient, and over 90% of probe molecules are routinely labeled. Using this method of labeling, an oligonucleotide was shown to be tenfold more sensitive in detecting target DNA sequences in a dot blot hybridization assay, compared to the same oligonucleotide labeled using polynucleotide kinase. Northern blots of Schizosaccharomyces pombe RNA were probed with an oligonucleotide specific for intron 1 of the tf2d gene, a TATA-box binding transcription factor. Kinase-labeled tf2d probe detected only unspliced RNA, while the same oligonucleotide labeled using the new method detected both unspliced tf2d RNA and rare pre-mRNA splicing intermediates.
0 Communities
2 Members
0 Resources
5 MeSH Terms