Apr 26, 2023

B., Rossire J. snRNPs, but not C/D box snoRNP components, into nucleoplasmic foci, and also for merging these factors into canonical CBs. Altogether, our data suggest that CBs have a modular structure with distinct domains for spliceosomal U snRNPs and snoRNPs. INTRODUCTION The interphase nucleus contains many morphologically distinct substructures, called nuclear bodies, which are nonCmembrane-bound, stable cellular compartments involved in the fidelity and efficiency of gene expression. One of the most extensively studied nuclear bodies, the Cajal body (CB), was originally identified more than 100 years ago (Cajal, 1903 ). CBs are present in the nucleus of vertebrates, invertebrates and plants in varying number and size depending on the cell type (Gall, 2000 ; Cioce and Lamond, 2005 ). They are molecularly defined by the presence of the protein coilin, which was discovered through the analysis of patient autoimmune sera (Andrade egg extract, no visible foci of spliceosomal components can be observed in pronuclei formed in vitro (Bauer and Gall, 1997 ). Maleimidoacetic Acid Similarly, mouse embryonic fibroblast (MEF) cells lacking functional coilin fail to concentrate spliceosomal U snRNPs in the nucleus (Tucker (2004) and harvested after 72 h or 48 h, respectively. Real-Time RT-PCR hTGS1, SMN, and coilin knockdowns were analyzed by the relative quantification based on the relative amount of target mRNA in knockdown cells versus target mRNA in control cells with a one-step real-time RT-PCR using SYBR Green (QuantiTec, Beaverton, OR; SYBR Green RT-PCR Kit). For normalization of the target genes the intronless glutamate dehydrogenase 2 (GLUD2) was used as an endogenous standard. Total RNA was isolated from HeLa SS6 cells treated with GL2 control siRNA and with siRNAs targeting hTGS1, SMN, or coilin (RNeasy Mini Kit, Qiagen) and on-column was treated with DNase1. Total RNA, 12.5 ng from each sample, was used Maleimidoacetic Acid for analysis. The following PCR primers, which are exon/exon-spanning for all but GLUD2, were used: hTGS1-F 5-GGCTATTACATCAGAGACAGTGG-3; hTGS1-R: 5-GAATCAAGTTCACTTTCATCCAGGC-3; SMN-F: 5-TGCATTTACCCAGCTACCATTG-3; SMN-R: 5-GATCGGACAGATTTTGCTCCTC-3; Coilin-F: 5-CTTGAGAGAACCTGGGAAATTTG-3; Coilin-R: 5-GTCTTGGGTCAATCAACTCTTTCC-3; GLUD2-F: 5-TCGTGGAGGACAAGTTGGTG-3; and GLUD2-R: 5-TTGCAGGGCTTGATGATCCG-3. All real-time RT-PCRs were performed Rabbit polyclonal to ACAD9 with the DNA Engine Opticon 1 System (Bio-Rad, Richmond, CA). The specificity of real-time RT-PCR products was verified by high-resolution gel electrophoresis, which showed a single product with the desired length in all cases. In addition, an Opticon melting-curve analysis was performed, which resulted in single-product-specific melting curves. The transcripts investigated showed real-time PCR efficiencies ranging from 1.8 to 2.1 (Pfaffl, 2001 ) in the range from 0.2 to 25 ng total RNA input with high linearity (Pearson 0.98). Relative quantification of knockdown versus control mRNA levels was accomplished according to the Pfaffl method (Pfaffl, Maleimidoacetic Acid 2001 ). Additionally, controls without RT mix were performed for each RNA to verify that there was no residual DNA contamination. S100 Extract Preparation and In Vitro Hypermethylation Assay Cells were harvested with a rubber policeman, washed in phosphate-buffered saline (PBS; pH 7.4), resuspended in one packed-cell volume of Roeder buffer A (Dignam and 4C. Subsequently, the supernatant was dialyzed against reconstitution buffer (50 mM HEPES, pH 7.4; 50 mM KCl; 5 mM MgCl2; 5% glycerol; 0.5 mM DTE; 0.5 mM phenylmethylsulfonyl fluoride). The concentration of the S100 extract was adjusted to 2 mg/ml, and the extract was then stored at ?80C until use. The in vitro m3G-cap hypermethylation assay was performed according to Plessel (1994) . In brief, 100 l of S100 extract (2 mg/ml) was incubated, in the presence of 0.1 mM strain, and recombinant proteins were purified using glutathione-Sepharose beads (Amersham) essentially as described by Smith and Johnson (1988) . Immunoblotting, Indirect Immunofluorescence, Fluorescence In Situ Hybridization, and Fluorescence Microscopy For Western blot analysis, 50 g of total cell extract was separated on an 13% SDS-polyacrylamide gel, transferred to a nitrocellulose membrane (Schleicher and Schll, Keene, NH), and immunostained with antibodies by using the ECL detection kit as described by the manufacturer (Amersham). Indirect immunofluorescence experiments with siRNA-transfected cells were performed as previously described (Ingelfinger (1992) . The sequence of the U2 oligonucleotide used for FISH was as follows: 5-GAACAGATACTACACTTGATCTTAGCCAAAAGGCCGAGAAGC-3. The oligonucleotide was labeled at its 5 and 3 ends with Alexa-488 (Molecular Probes). Fluorescence samples were visualized under identical conditions for control and knockdown.

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