人受精促进肽受体基因（TCP11b）的剪接、克隆和差别表达

运用同源比较和PCR法 ,从人睾丸组织中分离了人受精促进肽受体TCP11基因的一个新的剪切体TCP11b ,它编码 5 0 3个氨基酸的蛋白质 ,与TCP11a相比 ,在基因组的 5′端存在复杂的外显子剪接现象。运用荧光原位杂交 (FISH)方法 ,显示该基因定位到人染色体 6p2 1。Northern杂交及多组织RT PCR的结果显示该转录本在正常睾丸中表达 ,而其他组织、无精症患者及胎儿睾丸组织中未见该基因的表达。该结果结合mTcp 11功能的提示 ,TCP11b这种转录本对精子发生和人受精过程可能起重要作用。     

Sam68 Regulates S6K1 Alternative Splicing during Adipogenesis

The requirement for alternative splicing during adipogenesis is poorly understood. The Sam68 RNA binding protein is a known regulator of alternative splicing, and mice deficient for Sam68 exhibit adipogenesis defects due to defective mTOR signaling. Sam68 null preadipocytes were monitored for alternative splicing imbalances in components of the mTOR signaling pathway. Herein, we report that Sam68 regulates isoform expression of the ribosomal S6 kinase gene (Rps6kb1). Sam68-deficient adipocytes express Rps6kb1-002 and its encoded p31S6K1 protein, in contrast to wild-type adipocytes that do not express this isoform. Sam68 binds an RNA sequence encoded by Rps6kb1 intron 6 and prevents serine/arginine-rich splicing factor 1 (SRSF1)-mediated alternative splicing of Rps6kb1-002, as assessed by cross-linking and immunoprecipitation (CLIP) and minigene assays. Depletion of p31S6K1 with small interfering RNAs (siRNAs) partially restored adipogenesis of Sam68-deficient preadipocytes. The ectopic expression of p31S6K1 in wild-type 3T3-L1 cells resulted in adipogenesis differentiation defects, showing that p31S6K1 is an inhibitor of adipogenesis. Our findings indicate that Sam68 is required to prevent the expression of p31S6K1 in adipocytes for adipogenesis to occur.     

Light Regulates Plant Alternative Splicing through the Control of Transcriptional Elongation

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Novel Alternative Splicing Predicts a Truncated Isoform of the NMDA Receptor Subunit 1 (NR1) in Embryonic Rat Brain

The expression of mesencephalic brain derived neurotrophic factor (BDNF) has been shown to be regulated by dopaminergic neuronal functioning and glutamate receptors (GluRs). In turn, BDNF participates in the regulation of mesencephalic GluRs’ expression. In the present study we analyzed, using semi-quantitative RT-PCR, the effect of BDNF as well as of the GluRs agonists NMDA and trans-(±)-1-Amino-(1S,3R)-cyclopentane dicarboxylic acid (t-ACPD), on the expression levels of the NMDA GluR subunit 1 (NR1) mRNA, using rat cultured mesencephalic neurons. In the course of this study, a novel rat mRNA splice variant of NR1 was identified. This new NR1 mRNA isoform is characterized by the insertion of an 82 base pair intron containing an inframe stop codon, thus predicting the expression of a putative truncated protein of 465 amino acids. The RT-PCR and in situ hybridization reveals that the novel NR1 mRNA is expressed in various brain regions of the rat embryo, whereas no expression was detected in the adult rat brain. The modulation of the novel NR1 mRNA isoform by both BDNF and the metabotropic GluR agonist t-ACPD, suggests that the resulting putative NR1 truncated protein may be relevant in the regulatory network of glutamatergic neurotransmission in the developing central nervous system.     

Alternative Splicing of the RAGE Cytoplasmic Domain Regulates Cell Signaling and Function

The Receptor for Advanced Glycation End-products (RAGE) is a multi-ligand receptor present on most cell types. Upregulation of RAGE is seen in a number of pathological states including, inflammatory and vascular disease, dementia, diabetes and various cancers. We previously demonstrated that alternative splicing of the RAGE gene is an important mechanism which regulates RAGE signaling through the production of soluble ligand decoy isoforms. However, no studies have identified any alternative splice variants within the intracellular region of RAGE, a region critical for RAGE signaling. Herein, we have cloned and characterized a novel splice variant of RAGE that has a truncated intracellular domain (RAGEΔICD). RAGEΔICD is prevalent in both human and mouse tissues including lung, brain, heart and kidney. Expression of RAGEΔICD in C6 glioma cells impaired RAGE-ligand induced signaling through various MAP kinase pathways including ERK1/2, p38 and SAPK/JNK. Moreover, RAGEΔICD significantly affected tumor cell properties through altering cell migration, invasion, adhesion and viability in C6 glioma cells. Furthermore, C6 glioma cells expressing RAGEΔICD exhibited drastic inhibition on tumorigenesis in soft agar assays. Taken together, these data indicate that RAGEΔICD represents a novel endogenous mechanism to regulate RAGE signaling. Significantly, RAGEΔICD could play an important role in RAGE related disease states through down regulation of RAGE signaling.     

A Novel Short Isoform of Cytosolic PSD-95 Interactor (Cypin) Regulates Neuronal Development

The guanine deaminase cypin (cytosolic PSD-95 interactor) binds to PSD-95 (postsynaptic density protein 95) and regulates dendrite branching by promoting microtubule polymerization. Here, we identify a novel short isoform of cypin, termed cypinS, which is expressed in mouse and human, but not rat, tissues. Cypin and cypinS mRNA and protein levels peak at P7 and P14 in the mouse brain, suggesting a role for these isoforms during development. Interestingly, although cypinS lacks guanine deaminase activity, overexpression of cypinS increases dendrite branching. This increase occurs further away from soma than do increases resulting from overexpression of cypin. In contrast, overexpression of cypin, but not cypinS, decreases dendritic spine density and maturity. This suggests that changes to spines, but not to dendrites, may be dependent on guanine deaminase activity. Furthermore, overexpression of either cypin or cypinS increases miniature excitatory postsynaptic current (mEPSC) frequency, pointing to a presynaptic role for both isoforms. Interestingly, overexpression of cypinS results in a significantly greater increase in frequency than does overexpression of cypin. Thus, cypin and cypinS play distinct roles in neuronal development.     

A Novel,Universally Active C-terminal Protein Degradation Signal Generated by Alternative Splicing

Proteome integrity is crucial for cellular homeostasis and adaptation to stress conditions such as hypoxia. One mechanism for rapid adaptation of the proteome in response to changing environmental signals is alternative splicing. In addition to generating different protein isoforms, alternative splicing is also capable of controlling total protein levels by the regulated synthesis of non-productive mRNA isoforms. The hypoxia-induced isoform E of the tumor suppressor MAX is produced by retention and translation of the last intron. This leads to an alternative C-terminus that harbors a potent C-degron, the isoE degron. Strikingly, the isoE degron represents a universal protein degradation signal that is not only functional in mammalian cells, but also in yeast and even in bacteria. Essential for efficient protein decay is a conserved (F/W)xxW motif. Degradation of isoE tagged proteins is mediated by the proteasome in eukaryotes and Lon protease in bacteria. Thus, the isoE degron is a broadly applicable and highly efficient tool in protein analyses.     

鉴定9个新的RHD基因mRNA可变剪接体

为了研究各种RHD基因mRNA可变剪接体的基因结构, 应用逆转录聚合酶链反应(RT-PCR)检测正常人脐血样本RHD mRNA, 对RHD cDNA进行TA克隆和序列分析, 对各可变剪接体的剪接位点进行DNA序列分析, 并将RHD mRNA进行表达序列标签(ESTs)分析。结果在28个阳性克隆中, 除全长RHD cDNA外, 共检测到12种(包括9种新的)RHD可变剪接体, 发现外显子遗漏、5′和3′剪接位点变异3种剪接形式, 涉及外显子2~9, 其中6种新的剪接体同时存在RHD和RHCE基因同源杂交现象。ESTs分析还检索到内含子保留形式的剪接体。研究表明, RHD基因mRNA存在复杂的可变剪接机制, 除已报道的剪接体外, 检测到9种新的RHD可变剪接体, 并发现了可变剪接和同源杂交并存现象。     

mRNA前体的选择性剪接

人类基因组草图已基本完成 ,预测人类约有 350 0 0个编码蛋白质的基因 ,只是线虫或果蝇的 2倍[1] 。人类是怎样完成其复杂的生物功能 ?越来越多的证据表明选择性剪接在扩大蛋白质的多样性中发挥重要作用 ,并且有助于解释基因数目与生物复杂程度两者的不一致性。选择性剪接能够从一个基因产生多个转录本 ,从而产生远多于基因数目的蛋白质 ,完成机体的复杂功能及精细调节。1 .ｍＲＮＡ选择性剪接的普遍性目前根据ＥＳＴｓ分析 ,在人类 350 0 0个基因中大约有 40 %的基因具有选择性剪接的形式[1] 。尽管这个数目让人惊讶 ,但这个数目可能比实际…     

S6K1 Alternative Splicing Modulates Its Oncogenic Activity and Regulates mTORC1

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Highlights? The long S6K1 isoform functions as a tumor suppressor ? S6K1 short isoforms are upregulated in breast cancer and possess oncogenic properties ? S6K1 short isoforms bind mTORC1 and enhance cap-dependent translation ? Inhibition of mTORC1 can reverse the oncogenic properties of the S6K1 short isoforms