Osteoblasts orchestrate bone formation through the secretion of type I collagen and other constituents of the matrix on which hydroxyapatite crystals mineralize. Here, we show that TENT5A, whose mutations were found in congenital bone disease osteogenesis imperfecta patients, is a cytoplasmic poly(A) polymerase playing a crucial role in regulating bone mineralization. Direct RNA sequencing revealed that TENT5A is induced during osteoblast differentiation and polyadenylates mRNAs encoding Col1α1, Col1α2, and other secreted proteins involved in osteogenesis, increasing their expression. We postulate that TENT5A, possibly together with its paralog TENT5C, is responsible for the wave of cytoplasmic polyadenylation of mRNAs encoding secreted proteins occurring during bone mineralization. Importantly, the Tent5a knockout (KO) mouse line displays bone fragility and skeletal hypomineralization phenotype resulting from quantitative and qualitative collagen defects. Thus, we report a biologically relevant posttranscriptional regulator of collagen production and, more generally, bone formation.
- MeSH
- buněčná diferenciace MeSH
- fyziologická kalcifikace genetika MeSH
- kolagen typu I, řetězec alfa 1 genetika metabolismus MeSH
- kolagen typu I genetika metabolismus MeSH
- lidé MeSH
- messenger RNA genetika metabolismus MeSH
- modely nemocí na zvířatech MeSH
- myši knockoutované MeSH
- myši MeSH
- neurotrofní faktory genetika metabolismus MeSH
- nukleotidyltransferasy genetika metabolismus MeSH
- oční proteiny genetika metabolismus MeSH
- osteoblasty metabolismus patologie MeSH
- osteogenesis imperfecta genetika metabolismus patologie MeSH
- osteogeneze genetika MeSH
- osteonektin genetika metabolismus MeSH
- poly(A)-polymerasa genetika metabolismus MeSH
- polyadenylace MeSH
- protein - isoformy nedostatek genetika MeSH
- sekvenční analýza RNA MeSH
- serpiny genetika metabolismus MeSH
- signální transdukce MeSH
- vývojová regulace genové exprese MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The title method is applied to one of the analyzed amplicons. The new approach is used for identification of C282Y (C), H63D (H), and S65C (S) mutations in the HFE gene. In a group of DNA samples from 40 patients with suspected hereditary hemochromatosis we amplified specific parts of HFE exon 2 (208 bp) and exon 4 (189 bp) by multiplex PCR. Purified amplicons were subsequently used in a multiplex cycle sequencing reaction containing a hyperadenylated reverse primer for exon 2. Adenylation with a 105-nucleotide polyadenylated tail attached to the 5' end of the primer significantly prolonged the migration time of the exon 2 amplicons in the electrophoretic capillary. This enabled us to obtain separated sequencing data for both the studied amplicons. The numbers of identified zygotes and allelic frequencies in the patients are given. We found six subjects with the risk of hereditary hemochromatosis (15 %). The new method is a faster and cheap alternative to dideoxynucleotide sequencing using standard sequencing primers. Hyperadenylation makes it possible to obtain complete information about the presence or absence of C282Y, H63D and S65C mutations in the HFE gene from one-capillary electrophoretic analysis.
- MeSH
- 3' nepřekládaná oblast MeSH
- 5' nepřekládaná oblast * MeSH
- DNA vazebné proteiny * MeSH
- DNA-polymerasa I MeSH
- elektroforéza v agarovém gelu * MeSH
- genetické nemoci vrozené diagnóza MeSH
- hemochromatóza * diagnóza epidemiologie prevence a kontrola MeSH
- lidé MeSH
- mikro RNA MeSH
- mutační analýza DNA * MeSH
- poly(A)-polymerasa MeSH
- polyadenylace MeSH
- polymerázová řetězová reakce * MeSH
- polynukleotidfosforylasa MeSH
- proteiny vázající RNA MeSH
- sekvenční analýza DNA * MeSH
- techniky amplifikace nukleových kyselin * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
