真核生物mRNA上的修饰核苷及在发育调控中的作用

信使RNA (Messenger RNA,mRNA)上的表观修饰对于转录本的稳定性和翻译活性有重要影响。在不同生物体的不同发育时期和不同组织器官中,特异转录本不同位点存在的核苷修饰影响mRNA的前体剪切、成熟mRNA的稳定性以及其翻译为蛋白质的效率。目前已知的170多种修饰核苷中在mRNA上发现的只占极少数,由于mRNA的丰度低、组织和发育特异性强等特点,研究mRNA特异位点的核苷修饰有很大的技术难度。近些年随着meRIP等技术的进步,mRNA核苷修饰功能的研究得到了长足的发展,特别是针对m6A、m5C等甲基化修饰的研究已经相当深入。文中简要回顾近年来mRNA核苷修饰领域的研究进展,对不同位点和不同类型的修饰核苷在不同物种生长发育中的调控作一总结,并对未来的研究热点和技术瓶颈展开讨论。     

Genetically acquired diabetes: adipocyte guanine nucleotide regulatory protein expression and adenylate cyclase regulation

Adipocyte membranes from diabetic (db/db) animals showed marked elevations in the levels of α-subunits for G_i-1 which were almost twice those in membranes from their normal, lean littermates. In contrast, no apparent differences were noted for levels of the α-subunits of G_i-2 and G_i-3, and 42 and 45 kDa forms of G_s and for G-protein β-subunits. Adenylate cyclase specific activity was similar in membranes from both normal and diabetic animals under basal conditions and also when stimulated by optimal concentrations of either NaF or forsckolin. In contrast, the ability of isoprenaline, glucagon and secretin to stimulate adenylate cyclase activity was greater in membranes from normal animals compared with membranes from diabetic animals. Receptor-mediated inhibition of adenylate cyclase, as assessed using PGE₁ and nicotinate, was similar using membranes from both sources, but PIA (phenylisopropyladenosine) was a slightly more effective inhibitor in membranes from diabetic animals. A doubling in the expression of G₁-1 thus appears to have little discernible effect upon the inhibitory regulation of adenylate cyclase.     

Altered expression of inhibitory guanine nucleotide regulatory proteins (Gi-proteins) in experimental hepatocellular carcinoma

Guanine nucleotide regulatory proteins (G-proteins) play an important role in the onset and progression of malignancy. We hypothesized that alterations in inhibitory G-protein (Gi) expression and/or function may contribute to cellular invasion and formation of hepatocellular carcinoma (HCC). H4IIE hepatoma cells were inoculated directly into the liver parenchyma of ACI strain rats, and membranes were prepared from HCC livers and adjacent nonneoplastic livers 12 days following the initial inoculation. Expression of inhibitory Giα proteins was determined by Western blot analysis and changes in the functional activity of these proteins confirmed by pertussis toxin catalyzed ADP ribosylation and adenylyl cyclase activity. Inhibitory Giα1, Giα1/2, and Giα3 protein expression was significantly elevated in HCC when compared to adjacent nonneoplastic liver and sham-operated hepatic tissue. Pertussis toxin catalyzed ADP ribosylation of Giα substrates was significantly enhanced in HCC concomitant with increased basal and stimulated adenylyl cyclase activity following uncoupling of Gi-proteins with manganese ions. The role of Gi-proteins in cellular proliferation was confirmed using cultured H4IIE cells and normal hepatocytes. In quiescent H4IIE cells, mastoparan (Giα activator) increased [³H] thymidine incorporation and cell growth in a dose-dependent manner, whereas both pertussis toxin (a Gi-protein inhibitor) and 8-bromo-cAMP inhibited mitogenesis. In contrast, in isolated cultured hepatocytes, mastoparan inhibited [³H] thymidine incorporation, while pertussis toxin and 8-bromo-cAMP were mitogenic. We conclude that HCC is associated with marked changes in Giα-protein expression in vivo and in vitro, direct activation of which leads to increased mitogenesis in H4IIE cells in vitro. J. Cell. Physiol. 175:295–304, 1998. © 1998 Wiley-Liss, Inc.     

Genetically acquired diabetes: adipocyte guanine nucleotide regulatory protein expression and adenylate cyclase regulation.

Adipocyte membranes from diabetic (db/db) animals showed marked elevations in the levels of alpha-subunits for Gi-1 which were almost twice those found in membranes from their normal, lean littermates. In contrast, no apparent differences were noted for levels of the alpha-subunits of Gi-2 and Gi-3, the 42 and 45 kDa forms of Gs and for G-protein beta-subunits. Adenylate cyclase specific activity was similar in membranes from both normal and diabetic animals under basal conditions and also when stimulated by optimal concentrations of either NaF or forskolin. In contrast, the ability of isoprenaline, glucagon and secretin to stimulate adenylate cyclase activity was greater in membranes from normal animals compared with membranes from diabetic animals. Receptor-mediated inhibition of adenylate cyclase, as assessed using PGE1 and nicotinate, was similar using membranes from both sources, but PIA (phenylisopropyladenosine) was a slightly more effective inhibitor in membranes from diabetic animals. A doubling in the expression of Gi-1 thus appears to have little discernible effect upon the inhibitory regulation of adenylate cyclase.     

Ethanol and guanine nucleotide binding proteins: a selective interaction

Guanine nucleotide binding proteins (G proteins) play key roles in signal transduction, including the coupling of hormone and neurotransmitter receptors to adenylate cyclase, ion channels, and polyphosphoinositide metabolism. One member of this family of proteins, Gs, appears to represent a specific site of action of ethanol in the central nervous system. Ethanol is often perceived as a nonspecific drug, and its anesthetic effects may in fact arise from relatively nonspecific interactions with cell membrane lipids. However, recent investigations point to a selective effect of low concentrations of ethanol to promote the activation of Gs, and thus to enhance adenylate cyclase activity. Ethanol seems to have little or no effect on the function of other identified G proteins. After chronic ingestion of ethanol by animals, or chronic exposure of cells in culture to ethanol, the sensitivity of adenylate cyclase to stimulation by guanine nucleotides and agonists that act via Gs is decreased. The mechanism of this change may involve qualitative and/or quantitative alterations in Gs, and seems to vary in different cell types. Studies of human platelets and lymphocytes also reveal differences in adenylate cyclase activity between alcoholics and control subjects. The differences are consistent with involvement of Gs, and do not appear to reverse upon cessation of alcohol exposure. The results suggest that the platelet and/or lymphocyte adenylate cyclase system may provide a biochemical marker of genetic predisposition to alcoholism.     

Tissue specific expression and developmental regulation of two genes coding for rat fatty acid binding proteins

We have examined the tissue distribution and developmental regulation of two low molecular weight cytosolic fatty acid binding proteins. Based on their initial site of isolation, they have been referred to as liver and intestinal fatty acid binding proteins (FABP). Cloned cDNAs were used to probe blots of RNAs extracted from a wide variety of adult rat tissues as well as small intestine and liver RNA obtained from fetal, suckling, and weaning animals. The highest concentrations of "liver" FABP mRNA were found in small intestine and liver. "Intestinal" FABP mRNA is most abundant in small bowel RNA while only trace amounts were encountered in liver. Both mRNAs were detectable in stomach, colon, pancreas, spleen, lung, heart, testes, adrenal, and brain RNA at 1-8% the concentrations observed in small intestine. Accumulation of both mRNAs in the small intestinal epithelium increases during development. The mRNAs are first detectable between the 19th and 21st day of gestation. They undergo a coordinated 3-4-fold increase in concentration within the first 24 h after birth. Thereafter, gut levels of intestinal FABP mRNA remain constant during the suckling period while liver FABP mRNA increases an additional 2-fold. Liver FABP mRNA levels are also induced in hepatocytes during the first postnatal day but subsequently do not change during the suckling and weaning phase, despite marked alterations in hepatic fatty acid metabolism. These observations support the concept that the major role of these proteins is to facilitate the entry of lipids into cells and/or their subsequent intracellular transport and compartmentalization. The data also raise questions about the identity of extragastrointestinal FABPs.     

Differential regulation of three sodium channel messenger RNAs in the rat central nervous system during development.

The levels of the mRNAs encoding sodium channels I, II and III in various regions of the developing rat central nervous system (from embryonal day 10 to postnatal day 90) have been examined by blot hybridization analysis with specific probes. The three sodium channel mRNAs exhibit different temporal and regional expression patterns. The expression of sodium channel I mRNA rises after a lag phase to adult levels during the second and third postnatal weeks with stronger increases in caudal regions of the brain and in spinal cord. Sodium channel II mRNA increases steadily until the first postnatal week, keeping high adult levels in rostral regions of the brain or reaching low adult levels after the second postnatal week in most caudal regions of the brain and in spinal cord; cerebellum shows low levels during the first two postnatal weeks but high adult levels. In all regions, sodium channel III mRNA attains maximum levels around birth and decreases during the first and second postnatal weeks to reach variable low adult levels. These results suggest that sodium channel III is expressed predominantly at fetal and early postnatal stages and sodium channel I predominantly at late postnatal stages, whereas sodium channel II is expressed throughout the developmental stages studied with greater regional variability.     

Differential effects of fluoride on adenylate cyclase activity and guanine nucleotide regulation of agonist high-affinity receptor binding.

Fluoride ion, presumably an Al3+-F- complex, has been proposed to activate the guanine nucleotide regulatory protein (G-protein) of the visual system, transducin, by associating with GDP at the nucleotide-binding site and thus mimicking the effects of non-hydrolysable GTP analogues [Bigay, Deterre, Pfister & Chabre (1985) FEBS Lett. 191, 181-85]. We have examined this proposed model by using the adenylate cyclase complexes of frog erythrocytes, S49 lymphoma cells and human platelets. Preincubation of plasma membranes from frog erythrocytes and S49 cells with 20 mM-fluoride for 20 min at 30 degrees C strongly stimulated adenylate cyclase activity. In contrast, the preactivated membranes were still able to bind beta-adrenergic agonist with high affinity, as determined by radioligand-binding techniques. Moreover, high-affinity agonist binding in fluoride-treated membranes was fully sensitive to guanine nucleotide, which decreased beta-adrenergic-receptor affinity for agonist. Very similar results were obtained for [3H]prostaglandin E1 binding to S49 membranes pretreated with fluoride. Incubation of human platelet membranes with increasing concentrations of fluoride (1-50 mM) resulted in biphasic regulation of adenylate cyclase activity, with inhibition observed at concentrations greater than 10 mM. Preincubation of platelet membranes with 20 mM-fluoride did not affect agonist high-affinity binding to alpha 2-adrenergic receptors, nor receptor regulation by guanine nucleotide. These results suggest that the model developed from the study of transducin may not be generally applicable to the G-proteins of the adenylate cyclase system.     

Differential coupling of smooth muscle and liver vasopressin (V1) receptors to guanine nucleotide binding proteins

We report the reconstitution of the smooth muscle vasopressin V1 receptor functionally coupled to a pertussis toxin-insensitive guanine nucleotide-binding protein. This V1 receptor was spontaneously coupled to this guanine nucleotide-binding protein upon solubilization in the absence of agonist, in contrast to our earlier report on the liver V1 receptor, which required agonist for coupling. The smooth muscle V1 receptor was reconstituted as a high affinity receptor (Kd = 5 nM), with a slow rate of agonist dissociation. Upon the addition of guanosine 5'-thiotriphosphate, there was a decrease in receptor affinity (Kd = 30 nM) concomitant with an increase in the rate of ligand dissociation. The ability of the smooth muscle V1 receptor to spontaneously couple to a guanine nucleotide-binding protein(s) suggests that in the absence of agonist it exists as a high affinity receptor. The smooth muscle V1 receptor may, therefore, be more sensitive to plasma concentrations of vasopressin than its liver homologue.     

Immunochemical detection of guanine nucleotide binding proteins mono-ADP-ribosylated by bacterial toxins

Rabbits immunized with ADP-ribose chemically conjugated to carrier proteins developed antibodies reactive against guanine nucleotide binding proteins (G proteins) that had been mono-ADP-ribosylated by bacterial toxins. Antibody reactivity on immunoblots was strictly dependent on incubation of substrate proteins with both toxin and NAD and was quantitatively related to the extent of ADP-ribosylation. Gi, Go, and transducin (ADP-ribosylated by pertussis toxin) and elongation factor II (EF-II) (ADP-ribosylated by pseudomonas exotoxin) all reacted with ADP-ribose antibodies. ADP-ribose antibodies detected the ADP-ribosylation of an approximately 40-kilodalton (kDa) membrane protein related to Gi in intact human neutrophils incubated with pertussis toxin and the ADP-ribosylation of an approximately 90-kDa cytosolic protein, presumably EF-II, in intact HUT-102 cells incubated with pseudomonas exotoxin. ADP-ribose antibodies represent a novel tool for the identification and study of G proteins and other substrates for bacterial toxin ADP-ribosylation.     

Distinct guanine nucleotide binding and release properties of the three Gi proteins

The native pertussis toxin sensitive GTP-binding proteins (Gi proteins) were individually resolved, and their guanine nucleotide binding and release properties were studied. Gi2 and Gi3, the two major GTP-binding proteins of human erythrocytes, were purified to apparent homogeneity by fast protein liquid chromatography. Gi1 was purified from bovine brain. The three proteins bound 0.6-0.85 mol of guanosine 5'-O-(thio-triphosphate (GTP gamma S)/mol of protein with similar affinities (KD(app) = 50-100 nM). The rate of [35S]GTP gamma S binding to Gi2 was 5-8-fold faster than to Gi1 or Gi3 at 2 mm Mg2+. There were no observable differences in the binding characteristics between bovine brain Gi1 and human erythrocyte Gi3. At 50 mM Mg2+, all three Gi proteins exhibited fast binding, although Gi1 and Gi3 were marginally slower than Gi2. All three Gi proteins exhibited different rates of [32P]GDP release at 2 mM Mg2+. GDP release from Gi2 was severalfold faster than that from Gi1 or Gi3. GDP release rates from Gi1 and Gi3 were similar, although Gi3 was somewhat (60-80%) faster than Gi1. These data indicate that rates of GDP release and GTP binding may be independently regulated for these three proteins and that the relative proportions of Gi2/Gi1 or Gi2/Gi3 will be a crucial factor in determining the kinetics of signal transduction through Gi-coupled effectors.     

Pertussis vaccine reduces agonist binding to the rat heart muscarinic receptor and its guanine nucleotide modulation

The injection ofBordetella pertussis, inactivated by merthiolate, causes a 2-fold increase in the IC50 of carbamylcholine (carbachol) in displacing [³H];-L(–) quinuclidinyl benzilate binding ([³H]QNB) to the receptor. In control animals, 50 M Gpp(NH)p causes a 6-fold decrease in the affinity of carbachol binding, whereas after vaccination the reduction is only 1.6-fold. After pertussis treatment there is no alteration in the affinity and number of [³H]QNB binding sites of to the muscarinic receptor.     

Differential regulation of keratin 8 and 18 messenger RNAs in differentiating F9 cells

F9 embryonal carcinoma cells (F9EC) can be induced to differentiate in vitro into epithelial cells expressing keratin 8 (K8) and keratin 18 (K18). cDNAs corresponding to K8 and K18 mRNAs were cloned and used to study the change in the abundance of these mRNAs during differentiation of F9 cells into parietal endoderm-like cells by treatment with retinoic acid (RA) or with RA and dibutyryl cAMP (Bt₂cAMP). Using an RNase protection assay, it was determined that K8 mRNA was induced slightly before K18 mRNA and that it accumulated to a greater extent than K18 mRNA. Furthermore, differentiation in presence of Bt₂cAMP plus RA resulted in an earlier induction of the two mRNAs and a higher level of expression of K8 mRNA. These results indicate that K8 and K18 mRNAs are regulated differently in F9 cells.     

Physiological correlation between nucleoside-diphosphate kinase and the enzyme-associated guanine nucleotide binding proteins

The physiological correlation between NDP-kinase and the enzyme-associated guanine nucleotide binding proteins (G1 and G2) has been studied in vitro. It was found that incubation of the phosphoenzyme (enzyme-bound high-energy phosphate intermediate) of NDP-kinases with one of the nucleoside 5'-diphosphates (NDPs) in the presence of divalent cations (Mg2+ and Ca2+) results in the formation of nucleoside 5'-triphosphates (NTPs) within 40 sec even at low temperatures (below 4 degrees C) without strict base-specificity; and high-energy phosphates on the phosphoenzyme can transfer preferentially to GDP on the guanine nucleotide binding proteins (G1, G2 and r-p21 protein) in the presence of 0.25 mM Ca2+ or 1 mM Mg2+ even if any other NDPs are present in the reaction mixtures. These observations suggest that NDP-kinase may be responsible for the phosphate-transfer between GDP on the guanine nucleotide binding proteins and its phosphoenzyme.     

Differential regulation of keratin 8 and 18 messenger RNAs in differentiating F9 cells.

F9 embryonal carcinoma cells (F9EC) can be induced to differentiate in vitro into epithelial cells expressing keratin 8 (K8) and keratin 18 (K18). cDNAs corresponding to K8 and K18 mRNAs were cloned and used to study the change in the abundance of these mRNAs during differentiation of F9 cells into parietal endoderm-like cells by treatment with retinoic acid (RA) or with RA and dibutyryl cAMP (Bt2cAMP). Using an RNase protection assay, it was determined that K8 mRNA was induced slightly before K18 mRNA and that it accumulated to a greater extent than K18 mRNA. Furthermore, differentiation in presence of Bt2cAMP plus RA resulted in an earlier induction of the two mRNAs and a higher level of expression of K8 mRNA. These results indicate that K8 and K18 mRNAs are regulated differently in F9 cells.