title: Analysis of Posttranscriptional Regulation of CCN Genes.
abstract: Cells generally control the concentration of mRNA by transcriptional and posttranscriptional regulation, so the separate contributions of synthesis and degradation ("decay") cannot be discriminated by the quantification of mRNA. To elucidate the contribution of posttranscriptional regulation, all experimental procedures for the analysis of the total transcript level, transcriptional induction, and degradation of the target mRNA are performed either individually, or in combination. From our experience, measurement of the steady-state levels of the mRNA using quantitative real-time polymerase chain reaction is an essential first step in quantifying ccn2 gene expression level. Subsequently, the effect of transcription rates should be assessed by reporter assays of the ccn2 promoter and nuclear run-on assays. Finally, the stability of ccn2 mRNAs is evaluated in the presence of a metabolic inhibitor actinomycin D, followed by mRNA degradation assays in vitro. Here, we describe the strategic methods used in a series of analyses to elucidate the possible involvement of the posttranscriptional regulatory mechanism of the ccn2 gene and show how this approach can in theory be applied to elucidating the posttranscriptional regulation of other genes belonging to the CCN family.
authors.affiliation: Department of Oral and Maxillofacial Surgery, Faculty of Medicine, Fukuoka University, 7-45-1 Nakakuma, Jonan-ku, Fukuoka, 814-0180, Japan.
authors.forename: Seiji
authors.initials: S
authors.lastname: Kondo
authors.affiliation: Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Dental School/Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Kita-ku, Okayama, 700-8525, Japan.|Department of Membrane Biochemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Kubota
authors.affiliation: Advanced Research Center for Oral and Craniofacial Sciences, Okayama University Dental School/Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, 2-5-1, Shikata-cho, Kita-ku, Okayama, 700-8525, Japan. takigawa@md.okayama-u.ac.jp.
authors.forename: Masaharu
authors.initials: M
authors.lastname: Takigawa
mesh_terms: D020413:3' Untranslated Regions; D055511:CCN Intercellular Signaling Proteins / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D002474:Cell-Free System; D015870:Gene Expression; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D011401:Promoter Regions, Genetic; D012323:RNA Processing, Post-Transcriptional; D020871:RNA Stability; D060888:Real-Time Polymerase Chain Reaction; D017384:Sequence Deletion; D014162:Transfection
chemical_list: D020413:3' Untranslated Regions; D055511:CCN Intercellular Signaling Proteins
keywords: 3′-Untranslated region (3′-UTR); CCN; Gene expression; Posttranscriptional regulation; mRNA degradation