Figure 1.

RNASEH1 mutations, transcript, and protein levels. (A) Domains ofhuman RNAse H1 protein (MTS, mitochondrial targeting sequence; HBD, hybrid binding domain; CD, connection domain; catalytic domain). RNase H1 protein sequences from representative species,H. sapiens (Hs, NP_002927) M. musculus (Mm, NP_035405), B. taurus (Bt, NP_001039970), G. gallus (Gg, NP_990329),X. tropicalis (Xt, NP_001096299),D. rerio (Dr, NP_001002659), C. intestinalis (Ci, F6QPH0), D. melanogaster (Dm, NP_995777), C. elegans (Ce, NP_001040786), S. cerevisiae (Sc, Q04740), were extracted from the database and aligned using ClustalW2. Conserved residues found mutated in the patient in exon 4 are boxed in red, while residues in the active site and interacting with DNA or RNA are boxed in yellow and green, respectively. Positions of β strands are marked by blue arrows. (B) Human RNase H1 crystal structure (PDB ID 2QK9) 18 bp DNA(cyan): RNA (magenta) hybrid is shown respectively. Residues in the active site are colored in yellow. Residues Trp¹⁶⁴ (green) and Val¹⁴², or the mutated variant Ile¹⁴² (red), are shown as sticks. (C) RNASEH1 transcript levels in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium assessed by qPCR (probe Hs00268000_m1) and normalized to GAPDH transcript levels. Data are shown as mean ± SD, n = 4, ***p < 0.001. (D) RNASEH1 transcript levels in control (C1) and patient (P) fibroblasts grown in glucose-containing medium assessed by qPCR with probes Hs00268000_m1 (i), Hs01108220_g1 (ii), and Hs01108219_g1 (iii) and normalized to GAPDH transcript levels. Data are shown as mean ± SD, n = 3, ***p < 0.001. (E) Western blot analysis of RNase H1 in control (C1 and C2) and patient (P) fibroblasts grown in either glucose- or galactose-containing medium. GAPDH was used as loading control.