Epitope Tagging of Proteins at the Native Chromosomal Loci of Genes in Mice and in Cultured Vertebrate Cells

Chen, Yen-I G.; Maika, Shanna D.; Stevens, Scott W.

doi:10.1016/j.jmb.2006.06.052

Cited by 9 publications

(10 citation statements)

References 21 publications

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“…Using our recently developed CLEP tagging procedure (22), we tagged the SmD3 polypeptide in chicken DT40 cells by introducing a TAP tag (24) at the native genomic locus. For each experiment, 6 l of SmD3-TAP-DT40 cells were harvested and processed as described for purification of supraspliceosomes from HeLa cells.…”

Section: Resultsmentioning

confidence: 99%

“…To date, such factors have generally been elucidated via genetic or molecular strategies focused on individual pre-mRNAs. However, with the introduction of the CLEP tagging technology to more cell types (22), it may be possible to rapidly enumerate factors that function in regulated or alternative splicing.…”

Section: Resultsmentioning

confidence: 99%

“…Six liters of SmD3-TAP DT40 cells (22) were grown in Dulbecco's modified Eagle media supplemented with 5% chicken serum and 2.5% Fetalplex (Gemini Bio-Products) to a density of 7.5 × 10 5 cells/ml for TAP purification. Cells were harvested by centrifugation (1000 x g for 5 min), washed twice with ice-cold PBS, allowed to swell in 10 ml of TM buffer (10 mM Tris–Cl pH 7.5, 3 mM MgCl 2 ) with 0.2 mM PMSF, 1 µg/ml leupeptin, and 1 µg/ml pepstatin for 10 min ice, and lysed with 25 strokes of a Dounce homogenizer at 4°C.…”

Section: Methodsmentioning

confidence: 99%

“…We have chosen the chicken DT40 system to compare with the HeLa system for a number of reasons. First, we have shown that working with this rapidly growing cell type, which possesses high rates of homologous recombination, allows for downstream experimental flexibility in epitope-tagging of other genes (22). The evolutionary distance between human and chicken will also allow us to assess the evolutionary conservation of the machinery as well as validating novel co-purifying factors.…”

Section: Introductionmentioning

confidence: 99%

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Proteomic analysis of in vivo-assembled pre-mRNA splicing complexes expands the catalog of participating factors

Chen

Moore

et al. 2007

Nucleic Acids Research

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104

117

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Previous compositional studies of pre-mRNA processing complexes have been performed in vitro on synthetic pre-mRNAs containing a single intron. To provide a more comprehensive list of polypeptides associated with the pre-mRNA splicing apparatus, we have determined the composition of the bulk pre-mRNA processing machinery in living cells. We purified endogenous nuclear pre-mRNA processing complexes from human and chicken cells comprising the massive (>200S) supraspliceosomes (a.k.a. polyspliceosomes). As expected, RNA components include a heterogeneous mixture of pre-mRNAs and the five spliceosomal snRNAs. In addition to known pre-mRNA splicing factors, 5′ end binding factors, 3′ end processing factors, mRNA export factors, hnRNPs and other RNA binding proteins, the protein components identified by mass spectrometry include RNA adenosine deaminases and several novel factors. Intriguingly, our purified supraspliceosomes also contain a number of structural proteins, nucleoporins, chromatin remodeling factors and several novel proteins that were absent from splicing complexes assembled in vitro. These in vivo analyses bring the total number of factors associated with pre-mRNA to well over 300, and represent the most comprehensive analysis of the pre-mRNA processing machinery to date.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Proteomic analysis of in vivo-assembled pre-mRNA splicing complexes expands the catalog of participating factors

Chen

Moore

et al. 2007

Nucleic Acids Research

Self Cite

104

117

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“…The ease of generating temperature-sensitive mutant alleles has allowed a detailed functional characterization of individual splicing factors. In contrast, the function of the mammalian spliceosome has mostly been studied by proteomic analysis of spliceosome intermediates (15)(16)(17)(18)(19)(20)(21). The isolation and comparison of different spliceosome intermediates has given insights into the dynamics of the spliceosome and revealed that excision of an intron from a pre-mRNA occurs largely by the same sequential path in yeast and higher eukaryotes (15,16,19,20).…”

mentioning

confidence: 99%

Drosophila MFAP1 Is Required for Pre-mRNA Processing and G2/M Progression

Andersen

Tapon

2008

Journal of Biological Chemistry

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The mammalian spliceosome has mainly been studied using proteomics. The isolation and comparison of different splicing intermediates has revealed the dynamic association of more than 200 splicing factors with the spliceosome, relatively few of which have been studied in detail. Here, we report the characterization of the Drosophila homologue of microfibril-associated protein 1 (dMFAP1), a previously uncharacterized protein found in some human spliceosomal fractions (Jurica, M. S., and Moore, M. J. (2003) Mol. Cell 12, 5-14). We show that dMFAP1 binds directly to the Drosophila homologue of Prp38p (dPrp38), a tri-small nuclear ribonucleoprotein component (Xie, J., Beickman, K., Otte, E., and Rymond, B. C. (1998) EMBO J. 17, 2938 -2946), and is required for pre-mRNA processing. dMFAP1, like dPrp38, is essential for viability, and our in vivo data show that cells with reduced levels of dMFAP1 or dPrp38 proliferate more slowly than normal cells and undergo apoptosis. Consistent with this, double-stranded RNA-mediated depletion of dPrp38 or dMFAP1 causes cells to arrest in G 2 /M, and this is paralleled by a reduction in mRNA levels of the mitotic phosphatase string/ cdc25. Interestingly double-stranded RNA-mediated depletion of a wide range of core splicing factors elicits a similar phenotype, suggesting that the observed G 2 /M arrest might be a general consequence of interfering with spliceosome function.Splicing of pre-mRNAs is a catalytic reaction that involves two successive trans-esterification steps. This process is carried out by a highly conserved ribonucleoprotein complex, called the spliceosome. The spliceosome consists of five snRNAs (U1, U2, U4, U5, and U6) and more than 200 proteins, making it one of the largest and most complex molecular machines studied (1). Spliceosome assembly is a sequential process that is initiated by the recruitment of the U1 small nuclear ribonucleoprotein (snRNP) 2 to the 5Ј-splice donor site to form a "commitment complex" ("E complex" in mammals) (3, 4). Subsequently, recruitment of the U2 snRNP to the branch site by the U2 auxiliary factor of 65 kDa (U2AF65) generates the "pre-spliceosome" ("A complex" in mammals) (5-8). A preformed U4/U6.U5 tri-snRNP unit then joins the U1-U2-pre-mRNA complex to form the "complete spliceosome" ("B complex" in mammals). To activate the complete spliceosome several conformational rearrangements must take place (reviewed in Ref.9). These include unwinding of base pairings between U1 and the 5Ј-splice site and between U6 and U4, as well as the formation of a new base pair interaction between U5 and U6 snRNPs and the 5Ј-splice site and U6 and U2. As a result, U1 and U4 snRNPs are released and an active spliceosome is formed (10, 11). The yeast RNA helicases Prp28p and Brr2p are both implicated in the structural rearrangements that take place during the activation step of the spliceosome (11). Prp28p is thought to be important for the unwinding of base pairings between U1 and the 5Ј-splice site (12) and Brr2p is implicated in unwinding the U4/U6...

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Is the disease course of rheumatoid arthritis becoming milder?: Time trends since 1985 in an inception cohort of early rheumatoid arthritis

Welsing¹,

Fransen²,

Riel³

2005

Arthritis & Rheumatism

111

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Objective. Based on comparisons of short-term cohort studies or cross-sectional samples of patients from different calendar times, it has been suggested that present patients with rheumatoid arthritis (RA) have a milder disease course compared with that of patients in past decades. This study was undertaken to investigate whether the course of disease activity and functional disability in patients with RA has become milder over the past several years.Methods. We used the Nijmegen inception cohort of early RA, which included all patients with newly diagnosed RA who had attended the department of rheumatology at Radboud University Nijmegen Medical Centre since 1985. Patients were assessed for disease activity by the Disease Activity Score in 28 joints (DAS28) every 3 months and for functional disability by the Health Assessment Questionnaire (HAQ) disability index (DI) every 6 months. Within the total cohort, 4 subcohorts were defined, based on the date of inclusion of the patients (1985-1990, 1990-1995, 1995-2000, 2000-2005). To investigate whether the course of disease activity and functional disability (over time) was different between the subcohorts, longitudinal regression analysis (linear mixed models) was used, with the DAS28 and HAQ DI over time as outcome variables, respectively, and subcohort as the independent variable, correcting for baseline demographic and clinical characteristics. The treatment strategy was compared between the subcohorts.Results. The DAS28 at baseline and over the first 5 years of disease was lower in the more recent subcohorts. The HAQ DI did not show improvement but instead a trend toward worsening functional disability. Using longitudinal regression it was shown that disease activity improved early in the disease course and stabilized thereafter, and that this improvement was greater in patients in the more recent subcohorts and in patients with a higher baseline DAS28. Initially, the HAQ DI also improved but stabilized thereafter, and this initial improvement was less pronounced in patients in the more recent subcohorts and was greater for patients with a higher baseline HAQ DI. The treatment strategy was more aggressive in the more recent subcohorts, as shown by a shorter duration from diagnosis to the start of treatment with prednisone or disease-modifying antirheumatic drugs (DMARDs), and a greater prevalence of DMARD therapy.

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Epitope Tagging of Proteins at the Native Chromosomal Loci of Genes in Mice and in Cultured Vertebrate Cells

Cited by 9 publications

References 21 publications

Proteomic analysis of in vivo-assembled pre-mRNA splicing complexes expands the catalog of participating factors

Proteomic analysis of in vivo-assembled pre-mRNA splicing complexes expands the catalog of participating factors

Drosophila MFAP1 Is Required for Pre-mRNA Processing and G2/M Progression

Is the disease course of rheumatoid arthritis becoming milder?: Time trends since 1985 in an inception cohort of early rheumatoid arthritis

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