Tissue-Specific Expression of the Gene Encoding a Mouse RNA Binding Protein Homologous to Human HuD Antigen

We have cloned and sequenced cDNAs encoding a mouse RNA-bindingprotein that is homologous to human HuD antigen. The amino acidsequence deduced from the nucleotide sequence has revealed thatthe mouse HuD protein is identicalto the human counterpart exceptfor two amino-acid substitutions outside the three RNA recognitionmotifs (RRMs) and a difference in the N-terminus. The mouseHuD gene produces two major brain-specific mRNAs (3.7 kb and4.4 kb) and a minor testis-specific mRNA (1.3 kb), which isindicative of alternative RNA processing. These results suggestthat the mouse HuD homolog is a member of the tissue-specificRNA-binding protein family, possibly involved in RNA metabolismin the nervous system.     

冷诱导RNA结合蛋白在急性脑外伤后的表达变化及其促炎作用研究

目的:观察小鼠急性脑外伤后,冷诱导RNA结合蛋白(CIRP)的表达变化情况及其对炎症因子的影响,旨在探讨CIRP在神经系统损伤性疾病中的作用,为该疾病的临床治疗提供新的思路。方法:成年小鼠随机分为正常组(n=10)和手术组(n=36),用液压打击法建立小鼠急性脑外伤模型,利用HE染色法观察模型建立后小鼠大脑的损伤情况;损伤后24、48 h时间点,采用实时PCR检测大脑损伤部位CIRP和炎症因子肿瘤坏死因子α(TNFα)m RNA的表达,免疫组织化学法检测CIRP蛋白水平的表达;分析CIRP表达变化与炎症因子TNFα相关性。结果:急性脑外伤后小鼠脑损伤部位的CIRP m RNA及蛋白表达水平呈上升趋势,CIRP表达与炎症因子TNFα表达正相关。结论:CIRP可能参与了小鼠脑外伤的炎性反应过程。     

Antibodies Against the Bovine Brain Glutamate Binding Protein

Abstract: Antibodies against the purified bovine brain glutamate binding protein (GBP) were raised in rabbits. Both nonderivatized and dinitrobenzene-derivatized GBP produced strong immunological responses in rabbits. Using the enzyme-linked immunosorbent assay (ELISA), we have quantified the antibody production and determined the specificity of the antibodies. Bovine brain GBP and the analogous protein from rat brain interacted most strongly with the antibodies. A bacterial glutamate-aspartate binding protein, as well as the enzymes glutamate dehydrogenase (EC 1.4.1.3), glutamine synthetase (EC 6.3.1.2), and γ-glutamyl transpeptidase (EC 2.3.2.2), showed little or no cross-reactivity with the anti-GBP antibodies. A crude bacterial glutamate decarboxylase (EC 4.1.1.15) preparation gave a small to moderate cross-reaction with the anti-GBP antibodies. The sensitivity of the ELISA assay and the specificity of the antibodies were such that GBP levels as low as 3–10 ng could be detected.     

Organization of the Human Protein 4.1 Genomic Locus: New Insights into the Tissue-Specific Alternative Splicing of the Pre-mRNA

Protein 4.1 is a globular 80-kDa component of the erythrocyte membrane skeleton that enhances spectrin–actin interaction via its internal 10-kDa domain. Previous studies have shown that protein 4.1 mRNA is expressed as multiple alternatively spliced isoforms, resulting from the inclusion or exclusion of small cassette sequences called motifs. By tissue screening for protein 4.1 isoforms, we have observed new features of an already complex pattern of alternative splicing within the spectrin/actin binding domain. In particular, we found a new 51-nt exon that is present almost exclusively in muscle tissue. In addition, we have isolated multiple genomic clones spanning over 200 kb, containing the entire erythroid and nonerythroid coding sequence of the human locus. The exon/intron structure has now been characterized; with the exception of a 17-nt motif, all of the alternatively spliced motifs correspond to individual exons. The 3′-untranslated region (UTR) has also been completely sequenced using various PCR and genomic-sequencing methods. The 3′ UTR, over 3 kb, accounts for one-half of the mature mRNA.     

The p23 Protein of Citrus Tristeza Virus Controls Asymmetrical RNA Accumulation

Citrus tristeza virus (CTV), a member of the Closteroviridae, has a 19.3-kb positive-stranded RNA genome that is organized into 12 open reading frames (ORFs) with the 10 3' genes expressed via a nested set of nine or ten 3'-coterminal subgenomic mRNAs (sgRNAs). Relatively large amounts of negative-stranded RNAs complementary to both genomic and sgRNAs accumulate in infected cells. As is characteristic of RNA viruses, wild-type CTV produced more positive than negative strands, with the plus-to-minus ratios of genomic and sgRNAs estimated at 10 to 20:1 and 40 to 50:1, respectively. However, a mutant with all of the 3' genes deleted replicated efficiently, but produced plus to minus strands at a markedly decreased ratio of 1 to 2:1. Deletion analysis of 3'-end genes revealed that the p23 ORF was involved in asymmetric RNA accumulation. A mutation which caused a frameshift after the fifth codon resulted in nearly symmetrical RNA accumulation, suggesting that the p23 protein, not a cis-acting element within the p23 ORF, controls asymmetric accumulation of CTV RNAs. Further in-frame deletion mutations in the p23 ORF suggested that amino acid residues 46 to 180, which contained RNA-binding and zinc finger domains, were indispensable for asymmetrical RNA accumulation, while the N-terminal 5 to 45 and C-terminal 181 to 209 amino acid residues were not absolutely required. Mutation of conserved cysteine residues to alanines in the zinc finger domain resulted in loss of activity of the p23 protein, suggesting involvement of the zinc finger in asymmetric RNA accumulation. The absence of p23 gene function was manifested by substantial increases in accumulation of negative-stranded RNAs and only modest decreases in positive-stranded RNAs. Moreover, the substantial decrease in the accumulation of negative-stranded coat protein (CP) sgRNA in the presence of the functional p23 gene resulted in a 12- to 15-fold increase in the expression of the CP gene. Apparently the excess negative-stranded sgRNA reduces the availability of the corresponding positive-stranded sgRNA as a messenger. Thus, the p23 protein controls asymmetric accumulation of CTV RNAs by downregulating negative-stranded RNA accumulation and indirectly increases expression of 3' genes.     

冷诱导RNA结合蛋白研究进展

冷诱导RNA结合蛋白(CIRP)广泛表达于各种组织细胞中,但低温(32℃)诱导下表达明显增加。随着研究的不断深入,科学家们发现CIRP还可以被其他应激条件如紫外线、缺氧、渗透压等诱导高表达。CIRP是一种重要的生理活性物质,其表达量的改变,直接影响体内心率、体温、内分泌等有节律性变化的生理过程。现已证实它广泛参与缺氧神经元保护、癌症发生、神经及胚胎发育、生物钟调节等一系列生化过程。我们对冷诱导RNA结合蛋白的最新研究进展加以综述,旨在初步阐明该蛋白的功能,为利用该蛋白治疗临床相关疾病奠定理论基础。     

Sensory Regulation of Neuroligins and Neurexin I in the Honeybee Brain

Background

Neurexins and neuroligins, which have recently been associated with neurological disorders such as autism in humans, are highly conserved adhesive proteins found on synaptic membranes of neurons. These binding partners produce a trans-synaptic bridge that facilitates maturation and specification of synapses. It is believed that there exists an optimal spatio-temporal code of neurexin and neuroligin interactions that guide synapse formation in the postnatal developing brain. Therefore, we investigated whether neuroligins and neurexin are differentially regulated by sensory input using a behavioural model system with an advanced capacity for sensory processing, learning and memory, the honeybee.

Methodology/Principal Findings

Whole brain expression levels of neuroligin 1–5 (NLG1–5) and neurexin I (NrxI) were estimated by qRT-PCR analysis in three different behavioural paradigms: sensory deprivation, associative scent learning, and lateralised sensory input. Sensory deprived bees had a lower level of NLG1 expression, but a generally increased level of NLG2–5 and NrxI expression compared to hive bees. Bees that had undergone associative scent training had significantly increased levels of NrxI, NLG1 and NLG3 expression compared to untrained control bees. Bees that had lateralised sensory input after antennal amputation showed a specific increase in NLG1 expression compared to control bees, which only happened over time.

Conclusions/Significance

Our results suggest that (1) there is a lack of synaptic pruning during sensory deprivation; (2) NLG1 expression increases with sensory stimulation; (3) concomitant changes in gene expression suggests NrxI interacts with all neuroligins; (4) there is evidence for synaptic compensation after lateralised injury.     

RNA结合蛋白与非编码RNA调节关系的研究进展

近年来,越来越多的研究表明,RNA结合蛋白(RNA binding protein,RBP)与多种类型的非编码RNAs(noncoding RNA,ncRNAs)具有互相调节的关系,且调节机制形式多样。一方面,RBP可以调节ncRNA的生物合成、稳定性和功能;另一方面,ncRNA也可以影响RBP的功能和结构。同时,RBP和ncRNA的相互作用还在其他靶基因的调节上起着重要的作用,从而参与众多的生物过程,如组织发育、代谢性疾病、神经退行性疾病、抗病毒免疫和各种癌症等。该文就RBP与常见类型的ncRNAs,包括miRNA、lncRNA、circRNA的相互作用方式和调节机制的研究进展作一综述。     

Solubilisation of a Glutamate Binding Protein from Rat Brain

Rat brain synaptic plasma membranes were solubilised in either 1% Triton X-100 or potassium cholate and subjected to batch affinity adsorption on L-glutamate/bovine serum albumin reticulated glass fibre. The fibre was extensively washed, and bound proteins eluted with 0.1 mM L-glutamate in 0.1% detergent, followed by repeated dialysis to remove the glutamate from the eluted proteins. Aliquots of the dialysed extracts were assayed for L-[3H]glutamate binding activity in the presence or absence of 0.1 mM unlabelled L-glutamate (to define displaceable binding). Incubations were conducted at room temperature and terminated by rapid filtration through nitrocellulose membranes. Binding to solubilised fractions could be detected only following affinity chromatography. Binding was saturable and of relatively low affinity: KD = 1.0 and 1.8 microM for Triton X-100 and cholate extracts, respectively. The density of binding sites was remarkably high: approximately 18 nmol/mg protein for Triton X-100-solubilised preparations, and usually double this when cholate was employed. Analysis of structural requirements for inhibition of binding revealed that only a very restricted number of compounds were effective, i.e., L-glutamate, L-aspartate, and sulphur-containing amino acids. Binding was not inhibited significantly by any of the selective excitatory amino acid receptor agonists--quisqualate, N-methyl-D-aspartate, or kainate. The implication from this study is that the glutamate binding protein is similar if not identical to one previously isolated and probably is not related to the pharmacologically defined postsynaptic receptor subtypes, unless solubilisation of synaptic membranes resulted in major alterations to binding site characteristics. Since solubilisation with Triton X-100 is known to preserve synaptic junctional complexes, it seems likely that the origin of the glutamate binding protein may be extrajunctional, although its functional role is unknown.     

Tissue-Specific Processing of the Neuroendocrine Protein VGF

Abstract: VGF is a neuroendocrine-specific gene product that is up-regulated by nerve growth factor in the PC12 cell line. In rat neuroendocrine tissues two polypeptides of 90 and 80 kDa were detected by an antiserum to an N-terminal domain of VGF (from residues 4 to 240). In parallel, an antiserum directed against the C-terminal nonapeptide of VGF (from residues 609 to 617) revealed several additional posttranslational products. Peptides of apparent molecular sizes of 20, 18, and 10 kDa were prominent in nerve tissues and the hypophysis but absent in the adrenal medulla, and their relative abundance varied in distinct regions of the CNS. In PC12 cells VGF was proteolytically processed only after nerve growth factor treatment, and primary cultures of rat cerebellar granule cells accumulated the low-molecular-weight forms of VGF during in vitro maturation. In these cells the specific cleavages of VGF occurred in a postendoplasmic reticulum compartment; the processed forms were enriched in the secretory vesicles and were preferentially secreted upon cell membrane depolarization. Distinct differential distribution in the CNS and in vitro release of such posttranslational products indicate that these species may represent biologically relevant forms of VGF that play a role in neuronal communication.