More than 100 types of chemical modifications in RNA have been well documented. Recently, several modifications, such as N-methyladenosine (mA), have been detected in mRNA, opening the window into the realm of epitranscriptomics. The mA modification is the most abundant modification in mRNA and non-coding RNA (ncRNA). At the molecular level, mA affects almost all aspects of mRNA metabolism, including splicing, translation, and stability, as well as microRNA (miRNA) maturation, playing essential roles in a range of cellular processes. The mA modification is regulated by three classes of proteins generally referred to as the "writer" (adenosine methyltransferase), "eraser" (mA demethylating enzyme), and "reader" (mA-binding protein). The mA modification is reversibly installed and removed by writers and erasers, respectively. Readers, which are members of the YT521-B homology (YTH) family proteins, selectively bind to RNA and affect its fate in an mA-dependent manner. In this review, we summarize the structures of the functional proteins that modulate the mA modification, and provide our insights into the mA-mediated gene regulation.
Adenosine ; analogs & derivatives ; metabolism ; Animals ; Gene Expression Regulation ; Humans ; Methyltransferases ; chemistry ; metabolism ; RNA, Messenger ; chemistry ; metabolism ; RNA, Untranslated ; chemistry ; metabolism ; RNA-Binding Proteins ; chemistry ; metabolism ; Transcriptome

The CCR4-NOT complex is a highly conserved, multifunctional machinery controlling mRNA metabolism. Its components have been implicated in several aspects of mRNA and protein expression, including transcription initiation, elongation, mRNA degradation, ubiquitination, and protein modification. In this review, we will focus on the role of the CCR4-NOT complex in mRNA degradation. The complex contains two types of deadenylase enzymes, one belonging to the DEDD-type family and one belonging to the EEP-type family, which shorten the poly(A) tails of mRNA. We will review the present state of structure-function analyses into the CCR4-NOT deadenylases and summarize current understanding of their roles in mRNA degradation. We will also review structural and functional work on the Tob/BTG family of proteins, which are known to interact with the CCR4-NOT complex and which have been reported to suppress deadenylase activity in vitro.
Animals ; Humans ; Multiprotein Complexes ; chemistry ; genetics ; metabolism ; Protein Conformation ; RNA, Messenger ; genetics ; metabolism ; Transcription Factors ; chemistry ; genetics ; metabolism

OBJECTIVETo investigate the effects of Astragalus polysaccharides (APS) in inducing the mRNA expression of Agamma- and Ggamma-globin in K562 cells.

METHODSK562 cells were treated with APS at the concentration of 150, 300, and 450 mg/L, with Na-butyrate (NaB)-treated cells serving as the positive control and untreated cells as the blank control. Benzidine staining was used to examine the changes in hemoglobin synthesis in K562 cells after the treatments, and RT-PCR was employed to investigate the mRAN expression of Agamma- and Ggamma-globin.

RESULTSCompared with the untreated cells, APS treatment (300 mg/L) for 48 h resulted in a significant increase of the percentages of benzidine-positive cells from (4.37-/+0.58)% to (15.67-/+1.80)%, and also in significantly increased expression of Agamma-globin and Ggamma-globin mRNAs by 3.59-/+0.16 and 5.02-/+0.81 folds, respectively (P=0.000).

CONCLUSIONAPS potently enhances the mRNA expression of Agamma- and Ggamma-globin in K562 cells and warrants further evaluation as a potential therapeutic agent for beta-thalassemia.

Astragalus membranaceus ; chemistry ; Humans ; K562 Cells ; Polysaccharides ; pharmacology ; RNA, Messenger ; genetics ; metabolism ; gamma-Globins ; genetics ; metabolism

Capillaries ; Cells, Cultured ; Down-Regulation ; Endothelial Cells ; metabolism ; Humans ; Leptin ; genetics ; metabolism ; Ophiopogon ; chemistry ; Polysaccharides ; pharmacology ; RNA, Messenger ; genetics ; metabolism

Alzheimer Disease ; metabolism ; Animals ; Brain ; metabolism ; Calcium-Binding Proteins ; biosynthesis ; chemistry ; genetics ; Gastrointestinal Tract ; metabolism ; Humans ; Islets of Langerhans ; metabolism ; Neoplasms ; metabolism ; RNA, Messenger ; metabolism ; Secretagogins ; Thyroid Gland ; metabolism

OBJECTIVETo investigate if higher hepatocellular glycogen contents can alleviate hepatic ischemia reperfusion injury and its relationship to ICAM-1 gene expression in hepatic sinusoidal cells (HSCs).

METHODSTwenty-one rabbits fed with a standard diet were randomly divided into three groups (n=7 in each). All the animals were subjected to hepatic ischemia reperfusion injury then sacrificed. Before the injury, group A rabbits fasted for 24 hours; group C rabbits had 6 intravenous glucose solution (25%, 20 ml) injections, 4 hours between two injections. Hepatic enzymological changes, hepatic ICAM-1 mRNA expressions and leukocytic counts in the sinusoids were observed.

RESULTSThe liver glycogen contents of the three groups were significantly different. Livers of group A had higher contents of glycogen (9.85+/-0.91 mg/g. wet tissue); in group B they were 38.93+/-5.72; and in group C they were 48.31+/-6.58. Group C animals had the slightest liver function damage. There were no differences in the pre- and post-ischemic ICAM-1 mRNA contents in the three groups. However, livers with a higher content of glycogen showed less expression of ICAM-1 mRNA (group A: 1.398+/-0.365 ng/mg wet tissue; group B: 0.852+/-0.297; group C: 0.366+/-0.183) and lower leukocytic counts. The relationship analysis showed a negative relationship between hepatocellular glycogen and hepatic ICAM-1 mRNA contents (r= -0.965, P less than 0.01).

CONCLUSIONSHepatocellular glycogen is important in protecting liver ischemic reperfusion injury. Also hepatocellular glycogen decreases the expression of ICAM-1 mRNA of HSCs.

Animals ; Female ; Glycogen ; pharmacology ; Hepatocytes ; chemistry ; metabolism ; Intercellular Adhesion Molecule-1 ; genetics ; metabolism ; Liver ; chemistry ; metabolism ; pathology ; Male ; RNA, Messenger ; genetics ; Rabbits ; Reperfusion Injury ; genetics ; metabolism ; pathology

This study was aimed to compare the expression of BMP4 mRNA in the in-vivo tissue engineering bone constructed with Ca/P ceramics against the expression of BMP4 mRNA in the naturally healing bone. 20 porous Ca/P ceramics cylinders with Phi 5 mm x 8 mm were made and implanted into the dorsal muscles of 5 dogs. As control, one molar tooth was pulled out from each dog to create bone defect for the naturally healing bone at the same time. The specimens and the naturally healing bone were harvested at 1, 2, 4, 12 and 24 weeks post-implantation. After RNA extraction and reverse transcription, bone morphogenetic protein 4 (BMP4) and GAPDH mRNA were detected by real-time quantitative polymerase chain reaction (PCR) method. The results showed that the expression level of BMP4 mRNA of the in-vivo tissue engineering bone constructed with Ca/P ceramics was higher than that of the naturally healing bone in the period of experiment. However, the in-vivo tissue engineering bone had the same chronological order of BMP mRNA expression that the naturally healing bone did. As a bone substitute analogous to autologous bone, the in-vivo tissue engineering bone constructed with Ca/P ceramics has the potential for clinical application.
Animals ; Bone Morphogenetic Protein 4 ; genetics ; metabolism ; Bone Substitutes ; chemistry ; Calcium Phosphates ; chemistry ; Ceramics ; chemistry ; Dogs ; Humans ; Implants, Experimental ; RNA, Messenger ; genetics ; metabolism ; Tissue Engineering

OBJECTIVETo establish a HepG2 cell line stably expressing the human cytochrome P450 1A2 and to study its metabolic activity.

METHODSThe human wild-type CYP1A2 cDNA was subcloned into a mammalian expression vector pREP9. A transgenic cell line was established by transfecting the recombinant plasmid of pREP9-CYP1A2 to HepG2 cells. The expression of CYP1A2 mRNA was validated by RT PCR. The metabolic activation of HepG2 CYP1A2 cells on aflatoxin B1 (AFB1) was assayed by cytotoxicity test.

RESULTThe HepG2-CYP1A2 cells expressed CYP1A2 mRNA and could increase the cytotoxicity to AFB1 in comparison with that of wild type HepG2 cells.

CONCLUSIONThe established HepG2-CYP1A2 can express the mRNA and has the metabolic activity to AFB1. The cell line may be useful for testing the toxicity and metabolism of xenobiotics, which might possibly be activated or metabolized by CYP1A2.

Aflatoxin B1 ; metabolism ; Biotransformation ; Cell Line ; Cytochrome P-450 CYP1A2 ; genetics ; metabolism ; DNA, Complementary ; chemistry ; Humans ; RNA, Messenger ; analysis

OBJECTIVE: To study the safety and efficiency of the transfection of antisense oligonucletide into kidney mediated by lipid microbubbles, and to evaluate its potential clinical application. METHODS: The potential and conditions regarding the transfection self-made lipid microbubbles (CY5)-labeled-oligonucleotide (ODN) or CY5-labeled-ODN connective tissue growth factor (CTGF) into the rat kidney were evaluated. Th e safety was evaluated by HE staining, liver and renal function tests. The transfection efficiency was evaluated by fluorescence microscopy. Th e expression of CTGF was detected by RT-PCR and Western blot. RESULTS: Self-made lipid microbubble and/or ultrasound significantly enhanced the efficiency of gene transfer and expression in the kidney. Especially, 85%-90% of total glomerular could be transfected. CY5-labeled-ODN expression could be observed in glomerular, tubular and interstitial area. Th ere was no significant change in blood tests aft er gene transfer. Levels of LDH in 7 days were decreased compared with that at the fi rst day aft er the transfection (P<0.05). CTGF expression was successfully suppressed by transfection of CTGF-antisense-ODN into kidney. CONCLUSION The ultrasound-mediated gene transfer by self-made lipid microbubble could enhance the efficiency of ODN and expression in the rat kidney. Th is self-made lipid microbubbles supplement may be use for transfection of target genes.
Animals ; Connective Tissue Growth Factor ; genetics ; metabolism ; Kidney ; metabolism ; Lipids ; chemistry ; Microbubbles ; Oligonucleotides, Antisense ; genetics ; RNA, Messenger ; Rats ; Transfection ; Ultrasonics

OBJECTIVETo investigate the effects of hydrodynamics-mediated RNAi for Mfn2 gene expression in liver and the levels of blood sugar and fat in mice.

METHODSFifty-six male BALB/c mice were randomly divided into normal control group (NC, n = 8), negative control group (HK, n = 24) and transfection group (Mfn2, n = 24) according to random digits table. 1.5 ml plasmid (negative control or Mfn2 shRNA, 75mug for each mouse) diluted into phosphate buffered solution (PBS) was injected into the HK and Mfn2 groups mice via hydrodynamic intravascular injection. Mfn2 mRNA and protein expression in hepatic tissue was detected by RT-PCR and Western-blot 24 hours, 72 hours and 120 hours respectively after injection. At the same time, the levels of fasted blood sugar (FBS) and triglyceride (TG) were measured.

RESULTSCompared with HK mice, the expressions of Mfn2 mRNA (1.00+/-0.03 vs 1.14+/-0.07, t = 4.027, P = 0.007; 1.01+/-0.053 vs 1.18+/-0.07, t = 4.234, P = 0.006) and protein (7.81+/-0.80 vs 8.01+/-0.08, t = 2.941, P = 0.042; 8.05+/-0.15 vs 8.56+/-0.014, t = 4.883, P = 0.039) decreased markedly in Mfn2 mice in 72 and 120 hours after injection. In the fasting state, in 24 hours after injection, FBS in Mfn2 group was significantly lower than that in HK group [(2.65+/-0.70 vs 5.28+/-0.82) mmol/L, t = 6.879, P value less than 0.01] and TG was also significantly higher than that in HK group [(1.96+/-0.32 vs 1.12+/-0.16) mmol/L, t = -6.711, P value less than 0.01]. No statistical differences found between the NC and HK groups for FBS and TG (F = 1.412, P = 0.26; F = 2.711, P = 0.14). The plasma glucose level in Mfn2 mice was significantly higher than that in HK mice [(7.23+/-0.82 vs 5.18+/-0.69) mmol/L, t = 2.050, P value less than 0.01; (7.00+/-0.67 vs 6.05+/-0.76) mmol/L, t = 3.57, P = 0.023] in 72 and 120 hours after injection. However, no differences found between the two groups for blood TG [(1.53+/-0.27 vs 1.37+/-0.18) mmol/L, t = 0.160, P = 0.23; (1.84+/-0.30 vs 1.52+/-0.37) mmol/L, t = 0.330, P = 0.503].

CONCLUSIONThe data indicate that hydrodynamics- mediated RNAi for Mfn2 gene can effectively inhibit the expression of target gene in mice liver in 72 and 120 hours after shRNA administration, and the inhibition of hepatic Mfn2 can induce glycometabolic and fat metabolic disorder.

Animals ; Blood Glucose ; metabolism ; GTP Phosphohydrolases ; genetics ; metabolism ; Gene Expression ; Hydrodynamics ; Lipids ; blood ; Liver ; chemistry ; metabolism ; Male ; Mice ; Mice, Inbred BALB C ; RNA Interference ; RNA, Messenger ; genetics