A sequence predicted to form a stem–loop is proposed to be required for formation of an RNA–protein complex involving the 3'UTR of β-subunit F0F1-ATPase mRNA

ATP-synthase assembly requires coordinated control of ATP mRNA translation; this may e.g. occur through the formation of mRNA–protein complexes. In this study we aim to identify sequences in the 3'UTR of the β-subunit F₁-ATPase mRNA necessary for RNA–protein complex formation. We examined the interaction between a brain cytoplasmic protein extract and in vitro-synthesized β-subunit 3'UTR probes containing successive accumulative 5'- and 3'-deletions, as well as single subregion deletions, with or without poly(A) tail. Using electrophoretic mobility shift assays we found that two major RNA–protein complexes (here called RPC1 and RPC2) were formed with the full-length 3'UTR. The RPC2 complex formation was fully dependent on the presence of both the poly(A) tail and one subregion directly adjacent to it. For RPC1 complex formation, a 3'UTR sequence stretch (experimentally divided into three subregions) adjacent to but not including the poly(A) tail was necessary. This sequence stretch includes a conserved 40-nucleotide region that, according to the structure prediction program mfold, is able to fold into a characteristic stem–loop structure. Since the formation of the RPC1 complex was not dependent on a conventional sequence motif in the 3'UTR of the β-subunit mRNA but rather on the presence of the predicted stem–loop-forming region as such, we hypothetize that this RNA region, by forming a stem–loop in the 3'UTR β-subunit mRNA, is necessary for formation of the RNA–protein complex.     

Molecular cloning,characterisation and expression of the translationally controlled tumor protein gene in rock bream (Oplegnathus fasciatus)

Translationally controlled tumor protein (TCTP) is an important immune regulator that has been implicated in a number of cellular processes, including cell growth, cell cycle progression, apoptosis regulation and protection of cells against various environmental stresses. In this study, we cloned and characterised TCTP from rock bream (Oplegnathus fasciatus), which is an economically important species in the Korean aquaculture industry. The full-length rock bream TCTP (RbTCTP) cDNA was of 1,041 bp and contained an open reading frame of 513 bp, which encoded 170 amino acids. The 5′ untranslated region (UTR) was 90 bp, while the 3′ UTR was 438 bp, containing a polyadenylation signal. RbTCTP showed 76, 75 and 74 % amino acid sequence identities to those of tilapia (Oreochromis niloticus), orange-spotted grouper (Epinephelus coioides) and Japanese sea perch (Lateolabrax japonicus), respectively. The positions of microtubule binding region, Ca⁺ binding region and TCTP signature regions in RbTCTP were similar to other fish species and mammals. RbTCTP mRNA expression level was highest in the gill compared to other tissues. The level of RbTCTP mRNA expression was significantly regulated by injection of red seabream iridovirus, Streptococcus iniae and Edwardsiella tarda.     

miR-9 在卵巢癌细胞上皮间质转化中的初步研究

目的:研究miR-9在卵巢癌细胞上皮间质转化(EMT)中的作用。方法:上调或者下调miR-9后,在RNA水平上通过RT-qPCR检测卵巢癌细胞系SKOV3和A2780中上皮指标E-cadherin表达变化;在蛋白水平,通过western blotting方法检测2株细胞系中上皮指标E-cadherin和间质指标vimentin蛋白表达变化。生物信息学预测可能靶向E-cadherin 3'UTR的miR NA,双荧光素酶报告系统进一步验证miR-9靶向结合E-cadherin的3'UTR区。结果:上调miR-9后,卵巢癌细胞系中E-cadherin表达受到明显抑制,vimentin表达明显增加;反之,下调miR-9后,E-cadherin表达明显增高,vimentin表达明显降低。通过生物信息学预测发现miR-9可以直接靶向E-cadherin的3'UTR区,荧光素酶报告系统验证预测结果正确。结论:miR-9促进卵巢癌细胞上皮间质转化。     

Mos 3' UTR regulatory differences underlie species-specific temporal patterns of Mos mRNA cytoplasmic polyadenylation and translational recruitment during oocyte maturation

The Mos proto-oncogene is a critical regulator of vertebrate oocyte maturation. The maturation-dependent translation of Mos protein correlates with the cytoplasmic polyadenylation of the maternal Mos mRNA. However, the precise temporal requirements for Mos protein function differ between oocytes of model mammalian species and oocytes of the frog Xenopus laevis. Despite the advances in model organisms, it is not known if the translation of the human Mos mRNA is also regulated by cytoplasmic polyadenylation or what regulatory elements may be involved. We report that the human Mos 3' untranslated region (3' UTR) contains a functional cytoplasmic polyadenylation element (CPE) and demonstrate that the endogenous Mos mRNA undergoes maturation-dependent cytoplasmic polyadenylation in human oocytes. The human Mos 3' UTR interacts with the human CPE-binding protein and exerts translational control on a reporter mRNA in the heterologous Xenopus oocyte system. Unlike the Xenopus Mos mRNA, which is translationally activated by an early acting Musashi/polyadenylation response element (PRE)-directed control mechanism, the translational activation of the human Mos 3' UTR is dependent on a late acting CPE-dependent process. Taken together, our findings suggest a fundamental difference in the 3' UTR regulatory mechanisms controlling the temporal induction of maternal Mos mRNA polyadenylation and translational activation during Xenopus and mammalian oocyte maturation.     

乳腺癌细胞中HuR调控PDFC的分子机制研究

目的:探讨乳腺癌细胞中HuR调控PDGFC的分子机制。方法:通过软件预测分析,乳腺癌细胞中PDGFC 3'UTR的HuR结合位点;在RNA免疫共沉淀实验中,加入PDGFC刺激后检测HuR与PDGFC mRNA的相互作用;通过构建PDGFC 3'UTR五个截短体,荧光素酶报告基因实验检测HuR调控PDGFC的结合位点。结果:软件预测分析发现,PDGFC 3'UTR可能存在五个HuR结合位点;RNA免疫共沉淀实验中,当加入PDGFC刺激后,HuR与PDGFC mRNA出现免疫共沉淀,证明HuR和PDGFC之间的直接相互作用;PDGFC mRNA3'UTR报告基因系统检测显示,第2个和第4个位点可与HuR结合调控PDGFC。结论:本研究揭示了乳腺癌细胞中HuR通过与PDGFC mRNA 3'UTR结合调控PDGFC的分子机制,为乳腺癌的临床诊断和治疗提供了新的思路。