Characterization of the 5' and 3' untranslated regions in murine mdm2 mRNAs

The murine double minute 2 (mdm2) gene is essential for embryogenesis in mice that express the p53 tumor suppressor protein. Mdm2 levels must be regulated tightly because overexpression of mdm2 contributes to tumorigenesis. We investigated whether the 5' and 3' untranslated regions (UTRs) of murine mdm2 affect the expression of MDM2 proteins. Induction of mdm2 expression by p53 results in synthesis of an mdm2 mRNA with a short 5' UTR. The long 5' UTR increases internal initiation of translation of a minor MDM2 protein, p76(MDM2), without affecting the efficiency of translation of the full-length p90(MDM2). We discovered two alternative 3' untranslated regions in murine mdm2 mRNA expressed in the testis. The longer 3' UTR contains a consensus instability element, but mdm2 mRNAs containing the long and short 3' UTRs have comparable half-lives. The 3' UTRs do not affect either initiation codon use or translation efficiency. Thus, the murine 5' UTR, but not the 3'UTR, influences the ratio of the two MDM2 proteins but neither UTR affects MDM2 abundance significantly.     

Evidence for low GluR2 AMPA receptor subunit expression at synapses in the rat basolateral amygdala

Fast excitatory synaptic responses in basolateral amygdala (BLA) neurons are mainly mediated by ionotropic glutamate receptors of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) subtype. AMPA receptors containing an edited GluR2 subunit are calcium impermeable, whereas those that lack this subunit are calcium permeable and also inwardly rectifying. Here, we sought to determine the extent to which synapses in the rat BLA have AMPA receptors with GluR2 subunits. We assessed GluR2 protein expression in the BLA by immunocytochemistry with a GluR2 subunit-specific antiserum at the light and electron microscopic level; for comparison, a parallel examination was carried out in the hippocampus. We also recorded from amygdala brain slices to examine the voltage-dependent properties of AMPA receptor- mediated evoked synaptic currents in BLA principal neurons. At the light microscopic level, GluR2 immunoreactivity was localized to the perikarya and proximal dendrites of BLA neurons; dense labeling was also present over the pyramidal cell layer of hippocampal subfields CA1 and CA3. In electron micrographs from the BLA, most of the synapses were asymmetrical with pronounced postsynaptic densities (PSD). They contained clear, spherical vesicles apposed to the PSD and were predominantly onto spines (86%), indicating that they are mainly with BLA principal neurons. Only 11% of morphological synapses in the BLA were onto postsynaptic elements that showed GluR2 immunoreactivity, in contrast to hippocampal subfields CA1 and CA3 in which 76% and 71% of postsynaptic elements were labeled (p < 0.001). Synaptic staining in the BLA and hippocampus, when it occurred, was exclusively postsynaptic, and particularly heavy over the PSD. In whole-cell voltage clamp recordings, 72% of BLA principal neurons exhibited AMPA receptor-mediated synaptic currents evoked by external capsule stimulation that were inwardly rectifying. Although BLA principal neurons express perikaryal and proximal dendritic GluR2 immunoreactivity, few synapses onto these neurons express GluR2, and a preponderance of principal neurons have inwardly rectifying AMPA-mediated synaptic currents, suggesting that targeting of GluR2 to synapses is restricted. Many BLA synaptic AMPA receptors are likely to be calcium permeable and could play roles in synaptic plasticity, epileptogenesis and excitoxicity.     

Data mining of imperfect double-stranded RNA in 3' untranslated regions of eukaryotic mRNAs

Recent developments in the study of RNA silencing indicate that double-stranded RNA (dsRNA) can be used in eukaryotes to block expression of a corresponding cellular gene. There is also a large class of small non-coding RNAs having potential to form a distinct, stable stem-loop in numbers of eukaryotic genomes. We had reported that a large imperfect dsRNA structure with hundreds of base-pairs (bp) in the 3' untranslated region (3' UTR) of cytotoxic ribonuclease was correlated with the translation suppression. In this study, we search for such dsRNAs in a 3' UTR database. The occurrence rate of large dsRNA in 3' UTRs ranges from 0.01% in plant to 0.30% in vertebrate mRNAs. However, small imperfect dsRNAs of ~ 30 bp are much more prevalent than large ones. The small dsRNAs are statistically very significant and uniquely well-ordered. Most of them have the conserved structural features of pre-miRNAs. Our data mining of the dsRNAs in the 3' UTR database can be used to explore RNA-based regulation of gene expression.     

Surface Expression of the AMPA Receptor Subunits GluR1, GluR2, and GluR4 in Stably Transfected Baby Hamster Kidney Cells

Abstract: The surface expression of the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptor (GluR) subunits GluR1, GluR2, and GluR4 was studied in cultures of stably transfected baby hamster kidney (BHK)-570 cells. Two methods were used to quantify surface expression: cross-linking with the membrane-impermeant reagent bis(sulfosuccinimidyl)suberate (BS³) and labeling of surface receptors with the membrane-impermeant biotinylating reagent sulfosuccinimidyl 2-(biotinamido)ethyl-1,3-dithiopropionate (NHS-ss-biotin) followed by precipitation with neutravidin beads. Western blot analyses of control versus treated cultures revealed that, for all three GluR subunits examined, 25–40% of the total GluR population is located in the plasma membrane of the BHK-570 cells. This finding was corroborated by analyses of the surface expression of [³H]AMPA binding sites in the GluR-expressing BHK-570 cells performed via the biotinylation/precipitation method; these studies revealed that 30–40% of the total binding site population is found in the plasma membrane. Analyses of combinations of the subunits, both GluR1 + GluR2 and GluR2 + GluR4, revealed that heteromeric combinations of the subunits are not trafficked to the surface more efficiently than homomeric receptors. For each of the three subunits, western blots revealed two distinct bands; removal of surface receptors reduced immunoreactivity for the upper band of each subunit by >90%, whereas immunoreactivity for the lower band was reduced by only 10–20%. Treatment of extracts from the various cell lines with glycopeptidase F resulted in the collapse of the two bands into a single band of lower molecular weight, suggesting that the two original bands represent differentially glycosylated forms of the same polypeptides. These data indicate that the majority of the stably expressed GluR subunits in these cell lines are incompletely glycosylated and that complete glycosylation is associated with trafficking of the GluR subunits to the cell surface.     

Translational control of maskin mRNA by its 3' untranslated region

BACKGROUND INFORMATION: Maskin is a member of the TACC (transforming acidic coiled-coil) domain proteins found in Xenopus laevis oocytes and embryos. It has been implicated in the co-ordination of the spindle and has been reported to mediate translational repression of cyclin B1 mRNA. RESULTS: In the present study, we report that maskin mRNA is translationally repressed at the level of initiation in stage 4 oocytes and becomes activated in stage 6 oocytes. The translational repression of maskin mRNA correlates with the presence of a short poly(A) tail on this mRNA in stage 4 oocytes. The 3'-UTR (untranslated region) of maskin can confer the translational regulation to a reporter mRNA, and so can the 3'-UTR of human TACC3. A conserved GUCU repeat element was found to repress translation in both stage 4 and stage 6 oocytes, but deletion of this element did not abrogate repression in stage 4 oocytes. UV cross-linking experiments indicated that overlapping sets of proteins bind efficiently to both the maskin and the cyclin B1 3'-UTRs. As reported previously, CPEB [CPE (cytoplasmic polyadenylation element)-binding protein] binds to the cyclin B1 3'-UTR, but its binding to the maskin 3'-UTR is minimal. By RNA affinity chromatography and MS, we identified the EDEN-BP [EDEN (embryonic deadenylation element)-binding protein] as one of the proteins binding to both the maskin and the cyclin B1 3'-UTRs. CONCLUSIONS: Maskin mRNA is translationally regulated by at least two repressor elements and an activation element. One of the repessor elements is the evolutionarily conserved GUCU repeat. EDEN-BP binds to both the maskin and cyclin B1 3'-UTRs, indicating it may be involved in the deadenylation of these mRNAs.     

晚时相长时程增强翻转前后大鼠海马CA1区AMPAR/GluR2的表达

晚时相长时程增强(late-phase long-term potentiation,L-LTP)对于海马长期记忆的维持具有非常重要的作用,然而L-LTP可被诱导之后的神经元活动所翻转。本实验旨在研究海马CA1区L-LTP的翻转是否有突触前机制的参与以及L-LTP翻转前后AMPARs的表达有无变化。实验采用海马脑薄片细胞外场电位记录技术,使用强直刺激(high-frequency stimulation,HFS)诱导出CA1区L-LTP,2h后用两组间隔10min的高强度的双脉冲低频刺激(high-intensity paired-pulse low frequency stimulation,HI-PP-LFS)诱导L-LTP翻转。在LTP诱导前、诱导2h后、翻转后均给予一个双脉冲刺激,观察双脉冲比值(paired-pulse ratio,PPR)的变化;另一方面,实验通过免疫荧光组织化学方法观察AMPAR/GluR2在L-LTP翻转前后海马CA1区表达的变化。结果显示,L-LTP诱导后2h,HI-PP-LFS可诱导L-LTP的部分翻转(翻转率为61.79%±14.51%)。LTP诱导前、诱导2h后、翻转后PPR均大于1,表现为双脉冲易化(paired-pulse facilitation,PPF),且三者大小顺序为:LTP诱导后LTP翻转后LTP诱导前;在海马CA1区AMPAR/GluR2亚单位的表达方面,对照组、LTP组及LTP翻转组之间没有显著差异。上述结果提示,海马CA1区L-LTP维持与翻转均有突触前机制的参与,但L-LTP诱导与翻转前后AMPAR/GluR2表达没有发生变化。     

Characterization and evolution of 5' and 3' untranslated regions in eukaryotes

Untranslated regions (UTRs) in eukaryotes play a significant role in the regulation of translation and mRNA half-life, as well as interacting with specific RNA-binding proteins. However, UTRs receive less attention than more crucial elements such as genes, and the basic structural and evolutionary characteristics of UTRs of different species, and the relationship between these UTRs and the genome size and species gene number is not well understood. To address these questions, we performed a comparative analysis of 5' and 3' untranslated regions of different species by analyzing the basic characteristics of 244,976 UTRs from three eukaryote kingdoms (Plantae, Fungi, and Protista). The results showed that the UTR lengths and SSR frequencies in UTRs increased significantly with increasing species gene number while the length and G+C content in 5' UTRs and different types of repetitive sequences in 3' UTRs increased with the increase of genome size. We also found that the sequence length of 5' UTRs was significantly positively correlated with the presence of transposons and SSRs while the sequence length of 3' UTRs was significantly positively correlated with the presence of tandem repeat sequences. These results suggested that evolution of species complexity from lower organisms to higher organisms is accompanied by an increase in the regulatory complexity of UTRs, mediated by increasing UTR length, increasing G+C content of 5' UTRs, and insertion and expansion of repetitive sequences.     

JKTBP1 is involved in stabilization and IRES-dependent translation of NRF mRNAs by binding to 5' and 3' untranslated regions

Heterogeneous nuclear ribonucleoprotein D-like protein (JKTBP) 1 was implicated in cap-independent translation by binding to the internal ribosome entry site in the 5′ untranslated region (UTR) of NF-κB-repressing factor (NRF). Two different NRF mRNAs have been identified so far, both sharing the common 5′ internal ribosome entry site but having different length of 3′ UTRs. Here, we used a series of DNA and RNA luciferase reporter constructs comprising 5′, 3′ or both NRF UTRs to study the effect of JKTBP1 on translation of NRF mRNA variants. The results indicate that JKTBP1 regulates the level of NRF protein expression by binding to both NRF 5′ and 3′ UTRs. Using successive deletion and point mutations as well as RNA binding studies, we define two distinct JKTBP1 binding elements in NRF 5′ and 3′ UTRs. Furthermore, JKTBP1 requires two distinct RNA binding domains to interact with NRF UTRs and a short C-terminal region for its effect on NRF expression. Together, our study shows that JKTBP1 contributes to NRF protein expression via two disparate mechanisms: mRNA stabilization and cap-independent translation. By binding to 5′ UTR, JKTBP1 increases the internal translation initiation in both NRF mRNA variants, whereas its binding to 3′ UTR elevated primarily the stability of the major NRF mRNA. Thus, JKTBP1 is a key regulatory factor linking two pivotal control mechanisms of NRF gene expression: the cap-independent translation initiation and mRNA stabilization.     

Changes in phosphorylation of the NMDA receptor in the rat hippocampus induced by status epilepticus

Systemic administration of pilocarpine preceded by lithium induces status epilepticus (SE) that results in neurodegeneration and may lead to the development of spontaneous recurrent seizures. We investigated the effect of Li/pilocarpine-induced SE on phosphorylation of the NMDA receptor in rat hippocampus. Phosphorylation of NR1 by PKC on Ser890 was decreased to 45% of control values immediately following 1 h of SE. During the first 3 h following the termination of SE, phosphorylation of Ser890 increased 4-fold before declining to control values by 24 h. Phosphorylation of NR1 by PKA was also depressed relative to controls immediately following SE and transiently increased above control values upon the termination of SE. SE was accompanied by a general increase in tyrosine phosphorylation of hippocampal proteins that lasted for several hours following the termination of seizures. Tyrosine phosphorylation of the NR2A and NR2B subunits of the NMDAR increased 3-4-fold over control values during SE, continued to increase during the first hour following SE and then declined to control levels by 24 h. SE resulted in the activation of Src and Pyk2 associated with the postsynaptic apparatus, suggesting a role for these enzymes in the SE-induced increase in tyrosine phosphorylation. Changes in phosphorylation of the NMDA receptor may play a role in the pathophysiological consequences of SE.     

Wistar大鼠海马CA1区、CA3区和齿状回区的解剖分割

目的:在体视显微镜下分割Wistar大鼠海马CA1区、CA3区和齿状回(DG)区。方法:24只健康Wistar大鼠,分组如下:①6只大鼠取脑后硫堇染色,观察海马各区细胞形态;②6只大鼠分离出海马,体视显微镜下观察海马形态并分割CA1区、CA3区和DG区,各区分别切片后硫堇染色;③12只大鼠检测海马各区HSP 70的表达。结果:①大脑冠状切片硫堇染色清晰显示出海马CA1区、CA3区和DG区;②体视显微镜下,在海马腹侧面,沿着CA1区和DG区之间的海马沟可分割开CA1区和DG区,沿着CA3区和DG区之间的裂隙可分割开CA3区和DG区;分割后的海马各区细胞形态结构与整体大脑冠状切片上相对应区域的细胞形态结构一致;③Western blot结果显示:与对照组相比,脑缺血组HSP 70的表达在海马CA3+DG区明显上调、而在CA1无明显变化,这一结果与免疫组织化学结果一致。结论:上述方法可比较明确地分割Wistar大鼠海马CA1区、CA3区和DG区,分割得到的各区组织可用于蛋白质表达的检测。     

A genome‐scale analysis of mRNAs targeting to plant mitochondria: upstream AUGs in 5' untranslated regions reduce mitochondrial association

Intracellular sorting of mRNAs is an essential process for regulating gene expression and protein localization. Most mitochondrial proteins are nuclear‐encoded and imported into the mitochondria through post‐translational or co‐translational processes. In the latter case, mRNAs are found to be enriched in the vicinity of mitochondria. A genome‐scale analysis of mRNAs associated with mitochondria has been performed to determine plant cytosolic mRNAs targeted to the mitochondrial surface. Many messengers encoding mitochondrial proteins were found associated with mitochondria. These mRNAs correspond to particular functions and complexes, such as respiration or mitoribosomes, which indicates a coordinated control of mRNA localization within metabolic pathways. In addition, upstream AUGs in 5' untranslated regions (UTRs), which modulate the translation efficiency of downstream sequences, were found to negatively affect the association of mRNAs with mitochondria. A mutational approach coupled with in vivo mRNA visualization confirmed this observation. Moreover, this technique allowed the identification of 3'‐UTRs as another essential element for mRNA localization at the mitochondrial surface. Therefore, this work offers new insights into the mechanism, function and regulation of the association of cytosolic mRNAs with plant mitochondria.     

Monocistronic mRNAs containing defective hepatitis C virus-like picornavirus internal ribosome entry site elements in their 5' untranslated regions are efficiently translated in cells by a cap-dependent mechanism

The initiation of protein synthesis on mRNAs within eukaryotic cells is achieved either by a 5' cap-dependent mechanism or through internal initiation directed by an internal ribosome entry site (IRES). Picornavirus IRES elements, located in the 5' untranslated region (5'UTR), contain extensive secondary structure and multiple upstream AUG codons. These features can be expected to inhibit cap-dependent initiation of translation. However, we have now shown that certain mutant hepatitis C virus-like picornavirus IRES elements (from porcine teschovirus-1 and avian encephalomyelitis virus), which are unable to direct internal initiation, are not significant barriers to efficient translation of capped monocistronic mRNAs that contain these defective elements within their 5'UTRs. Moreover, the translation of these mRNAs is highly sensitive to the expression of an enterovirus 2A protease (which induces cleavage of eIF4G) and is also inhibited by hippuristanol, a specific inhibitor of eIF4A function, in contrast to their parental wild-type IRES elements. These results provide a possible basis for the evolution of viral IRES elements within the context of functional mRNAs that are translated by a cap-dependent mechanism.     

Involvement of P2X7 receptors in the regulation of neurotransmitter release in the rat hippocampus

Although originally cloned from rat brain, the P2X7 receptor has only recently been localized in neurones, and functional responses mediated by these neuronal P2X7 receptors (P2X7 R) are largely unknown. Here we studied the effect of P2X7 R activation on the release of neurotransmitters from superfused rat hippocampal slices. ATP (1-30 mm) and other ATP analogues elicited concentration-dependent [3 H]GABA outflow, with the following rank order of potency: benzoylbenzoylATP (BzATP) > ATP > ADP. PPADS, the non-selective P2-receptor antagonist (3-30 microm), Brilliant blue G (1-100 nm) the P2X7 -selective antagonist and Zn2+ (0.1-30 microm) inhibited, whereas lack of Mg2+ potentiated the response by ATP. In situ hybridization revealed that P2X7 R mRNA is expressed in the neurones of the cell body layers in the hippocampus. P2X7 R immunoreactivity was found in excitatory synaptic terminals in CA1 and CA3 region targeting the dendrites of pyramidal cells and parvalbumin labelled structures. ATP (3-30 microm) and BzATP (0.6-6 microm) elicited concentration-dependent [14 C]glutamate efflux, and blockade of the kainate receptor-mediated transmission by CNQX (10-100 microm) and gadolinium (100 microm), decreased ATP evoked [3 H]GABA efflux. The Na+ channel blocker TTX (1 microm), low temperature (12 degrees C), and the GABA uptake blocker nipecotic acid (1 mm) prevented ATP-induced [3 H]GABA efflux. Brilliant blue G and PPADS also reduced electrical field stimulation-induced [3 H]GABA efflux. In conclusion, P2X7 Rs are localized to the excitatory terminals in the hippocampus, and their activation regulates the release of glutamate and GABA from themselves and from their target cells.