Biomedical subjects

Claire Moore

Publications and source records attributed to Claire Moore.

10 recordsLinked to original sources

PubMed2006

Distinct pathways for snoRNA and mRNA termination.

Transcription termination at mRNA genes is linked to polyadenylation. Cleavage at the poly(A) site generates an entry point for the Rat1/Xrn2 exonuclease, which degrades the downstream transcript to promote termination. Small nucleolar RNAs (snoRNAs) are also transcribed by RNA polymerase II but are not polyadenylated. Chromatin immunoprecipitation experiments show that polyadenylation factors and Rat1 localize to snoRNA genes, but mutations that disrupt poly(A) site cleavage or Rat1 activity do not lead to termination defects at these genes. Conversely, mutations of Nrd1, Sen1, and Ssu72 affect termination at snoRNAs but not at several mRNA genes. The exosome complex was required for 3' trimming, but not termination, of snoRNAs. Both the mRNA and snoRNA pathways require Pcf11 but show differential effects of individual mutant alleles. These results suggest that in yeast the transcribing RNA polymerase II can choose between two distinct termination mechanisms but keeps both options available during elongation.

Blotting, Northern↗

PubMed2006

The C-terminal domains of vertebrate CstF-64 and its yeast orthologue Rna15 form a new structure critical for mRNA 3'-end processing.

Yeast Rna15 and its vertebrate orthologue CstF-64 play critical roles in mRNA 3 '-end processing and in transcription termination downstream of poly(A) sites. These proteins contain N-terminal domains that recognize the poly(A) site, but little is known about their highly conserved C-terminal regions. Here we show by NMR that the C-terminal domains of CstF-64 and Rna15 fold into a three-helix bundle with an uncommon topological arrangement. The structure defines a cluster of evolutionary conserved yet exposed residues we show to be essential for the interaction between Pcf11 and Rna15. Furthermore, we demonstrate that this interaction is critical for the function of Rna15 in 3 '-end processing but dispensable for transcription termination. The C-terminal domain of the Rna15 homologue Pti1 contains critical sequence alterations within this region that are predicted to prevent Pcf11 interaction, providing an explanation for the distinct functions of these two closely related proteins in the 3 '-end formation of RNA polymerase II transcripts. These results define the role of the C-terminal half of Rna15 and provide insight into the network of protein/protein interactions responsible for assembly of the 3 '-end processing apparatus.

Amino Acid Sequence↗

PubMed2006

The role of the Brr5/Ysh1 C-terminal domain and its homolog Syc1 in mRNA 3'-end processing in Saccharomyces cerevisiae.

The cleavage/polyadenylation factor (CPF) of Saccharomyces cerevisiae is thought to provide the catalytic activities of the mRNA 3'-end processing machinery, which include endonucleolytic cleavage at the poly(A) site, followed by synthesis of an adenosine polymer onto the new 3'-end by the CPF subunit Pap1. Because of similarity to other nucleases in the metallo-beta-lactamase family, the Brr5/Ysh1 subunit has been proposed to be the endonuclease. The C-terminal domain of Brr5 lies outside of beta-lactamase homology, and its function has not been elucidated. We show here that this region of Brr5 is necessary for cell viability and mRNA 3'-end processing. It is highly homologous to another CPF subunit, Syc1. Syc1 is not essential, but its removal improves the growth of other processing mutants at restrictive temperatures and restores in vitro processing activity to cleavage/ polyadenylation-defective brr5-1 extract. Our findings suggest that Syc1, by mimicking the essential Brr5 C-terminus, serves as a negative regulator of mRNA 3'-end formation.

Amino Acid Sequence↗

PubMed2005

Regulation of yeast mRNA 3' end processing by phosphorylation.

Recent studies have found that the phosphatase Glc7 associates with the yeast cleavage/polyadenylation factor (CPF), but the role of Glc7 in 3' end processing has not been investigated. Here, we report that depletion of Glc7 causes shortened poly(A) tails in vivo and accumulation of phosphorylated Pta1, a CPF subunit. Removal of Glc7 also gives extract defective for poly(A) addition but normal for cleavage at the poly(A) site. Polyadenylation is rescued by addition of Glc7 or Pta1, but not by phosphorylated Pta1. Moreover, Ypi1, a Glc7-specific inhibitor, or the Cka1 kinase blocks poly(A) addition in wild-type (wt) extract. Pta1 interacts physically and genetically with Glc7, suggesting that Pta1 may also regulate Glc7 or recruit it to CPF. A weakened association of Fip1 with phosphorylated CPF may explain the specific effect on polyadenylation. These results support a model in which poly(A) synthesis is controlled by cycles of phosphorylation and dephosphorylation that require the action of Glc7.

Casein Kinase II↗

PubMed2005

Reviving the exosome.

In the nucleus of eukaryotic cells, RNA decay in the 3'-5' direction is carried out by a complex of exonucleases called the exosome. Surprisingly, the purified exosome shows only weak activity in vitro. Two papers in this issue of Cell, by LaCava et al. (2005) and Wyers et al. (2005), and a third report by Vanacova et al. (2005) help to solve this riddle by characterizing a new RNA decay-activating complex containing a poly(A) polymerase. In addition, they identify new unconventional RNA polymerase II transcription units.

Cell Nucleus↗

PubMed2004

Ssu72 Is an RNA polymerase II CTD phosphatase.

Phosphorylation of serine-2 (S2) and serine-5 (S5) of the C-terminal domain (CTD) of RNA polymerase II (RNAP II) is a dynamic process that regulates the transcription cycle and coordinates recruitment of RNA processing factors. The Fcp1 CTD phosphatase catalyzes dephosphorylation of S2-P. Here, we report that Ssu72, a component of the yeast cleavage/polyadenylation factor (CPF) complex, is a CTD phosphatase with specificity for S5-P. Ssu72 catalyzes CTD S5-P dephosphorylation in association with the Pta1 component of the CPF complex, although its essential role in 3' end processing is independent of catalytic activity. Depletion of Ssu72 impairs transcription in vitro, and this defect can be rescued by recombinant, catalytically active Ssu72. We propose that Ssu72 has a dual role in transcription, one as a CTD S5-P phosphatase that regenerates the initiation-competent, hypophosphorylated form of RNAP II and the other as a factor necessary for cleavage of pre-mRNA and efficient transcription termination.

Carrier Proteins↗

PubMed2004

The essential WD repeat protein Swd2 has dual functions in RNA polymerase II transcription termination and lysine 4 methylation of histone H3.

Swd2, an essential WD repeat protein in Saccharomyces cerevisiae, is a component of two very different complexes: the cleavage and polyadenylation factor CPF and the Set1 methylase, which modifies lysine 4 of histone H3 (H3-K4). It was not known if Swd2 is important for the function of either of these entities. We show here that, in extract from cells depleted of Swd2, cleavage and polyadenylation of the mRNA precursor in vitro are completely normal. However, temperature-sensitive mutations or depletion of Swd2 causes termination defects in some genes transcribed by RNA polymerase II. Overexpression of Ref2, a protein previously implicated in snoRNA 3' end formation and Swd2 recruitment to CPF, can rescue the growth and termination defects, indicating a functional interaction between the two proteins. Some swd2 mutations also significantly decrease global H3-K4 methylation and cause other phenotypes associated with loss of this chromatin modification, such as loss of telomere silencing, hydroxyurea sensitivity, and alterations in repression of INO1 transcription. Even though the two Swd2-containing complexes are both localized to actively transcribed genes, the allele specificities of swd2 defects suggest that the functions of Swd2 in mediating RNA polymerase II termination and H3-K4 methylation are not tightly coupled.

Histone-Lysine N-Methyltransferase↗

PubMed2004

Mutations in the middle domain of yeast poly(A) polymerase affect interactions with RNA but not ATP.

The eukaryotic poly(A) polymerase (PAP) is responsible for the posttranscriptional extension of mRNA 3' ends by the addition of a poly(A) tract. The recently published three-dimensional structures of yeast and bovine PAPs have made a more directed biochemical analysis of this enzyme possible. Based on these structures, the middle domain of PAP was predicted to interact with ATP. However, in this study, we show that mutations of conserved residues in this domain of yeast PAP, Pap1, do not affect interaction with ATP, but instead disrupt the interaction with RNA and affect the enzyme's ability to process substrate lacking 2' hydroxyls at the 3' end. These results are most consistent with a model in which the middle domain of PAP interacts directly with the recently extended RNA and pyrophosphate byproduct.

Adenosine Triphosphate↗

PubMed2002

Coupling of termination, 3' processing, and mRNA export.

In a screen to identify genes required for mRNA export in Saccharomyces cerevisiae, we isolated an allele of poly(A) polymerase (PAP1) and novel alleles encoding several other 3' processing factors. Many newly isolated and some previously described mutants (rna14-48, rna14-49, rna14-64, rna15-58, and pcf11-1 strains) are defective in polymerase II (Pol II) termination but, interestingly, retain the ability to polyadenylate these improperly processed transcripts at the nonpermissive temperature. Deletion of the cis-acting sequences required to couple 3' processing and termination also produces transcripts that fail to exit the nucleus, suggesting that all of these processes (cleavage, termination, and export) are coupled. We also find that several but not all mRNA export mutants produce improperly 3' processed transcripts at the nonpermissive temperature. 3' maturation defects in mRNA export mutants include improper Pol II termination and/or the previously characterized hyperpolyadenylation of transcripts. Importantly, not all mRNA export mutants have defects in 3' processing. The similarity of the phenotypes of some mRNA export mutants and 3' processing mutants indicates that some factors from each process may mechanistically interact to couple mRNA processing and export. Consistent with this assumption, we present evidence that Xpo1p interacts in vivo with several 3' processing factors and that the addition of recombinant Xpo1p to in vitro processing reaction mixtures stimulates 3' maturation. Of the core 3' processing factors tested (Rna14p, Rna15p, Pcf11p, Hrp1p, Fip1p, and Cft1p), only Hrp1p shuttles. Overexpression of Rat8p/Dbp5p suppresses both 3' processing and mRNA export defects found in xpo1-1 cells.

Alleles↗

PubMedDate not supplied

Acute versus chronic vertebral compression fractures treated with kyphoplasty: early results.

BACKGROUND CONTEXT: Kyphoplasty, a minimally invasive technique for fracture reduction and stabilization, has been shown to reduce pain and restore vertebral body height in patients with vertebral compression fractures (VCFs). Analyses comparing treatment outcomes of acute versus chronic VCFs have not yet been reported. PURPOSE: To assess whether kyphoplasty results in better clinical outcome and fracture reduction in patients with either acute or chronic VCFs. STUDY DESIGN: A prospective, consecutive cohort study of patients who underwent kyphoplasty between March 2000 and December 2001 to treat osteoporotic VCFs that were either less than 10 weeks old (acute) or more than 4 months old (chronic). Fifteen subacute fractures (treated 10 to 16 weeks after fracture) were excluded from analyses. PATIENT SAMPLE: Eighty-six VCFs in 47 patients (35 female and 12 male) were treated during 55 kyphoplasty procedures. Mean patient age was 74 years (range, 47 to 91). METHODS: Clinical outcomes were determined by comparison of preoperative and postoperative data from patient-reported indexes (pain assessment, pain medication usage and Oswestry Disability Index for Back Pain). Radiographs were assessed as to percent vertebral collapse, vertebral height restoration and local kyphosis correction. RESULTS: By 2 weeks after surgery, 90% of acute and 87% of chronic fractures were associated with pain relief. Narcotic usage decreased and Oswestry scores improved in almost all patients. Mean vertebral body height significantly improved after kyphoplasty (acute: 58% to 86% of estimated normal vertebral height, p< .001; chronic: 56% to 79% of estimated normal vertebral height, p< .001). Restoration to 89% or greater estimated normal vertebral height was achieved in 60% of acute fractures and 26% of chronic fractures. In addition, more acute fractures were reducible (greater than 80% restoration of height lost) compared with chronic fractures (p= .01). After kyphoplasty, less than 10% correction of height lost occurred in 8% of acute fractures and 20% of chronic fractures. Local kyphosis significantly improved after kyphoplasty (mean local Cobb angle: acute, 15 to 8 degrees, p< .001; chronic, 15 to 10 degrees, p< .001). CONCLUSION: Fracture reduction was best achieved in acute fractures. Symptomatic chronic fractures may also remain candidates for kyphoplasty because pain relief and improvement in patient function are reliable and some kyphosis correction can still be achieved in many of these patients.

Acute Disease↗