In Silico Structural and Functional Characterization of the RSUME Splice Variants

RSUME (RWD-containing SUMO Enhancer) is a small protein that increases SUMO conjugation to proteins. To date, four splice variants that codify three RSUME isoforms have been described, which differ in their C-terminal end. Comparing the structure of the RSUME isoforms we found that, in addition to the previously described RWD domain in the N-terminal, all these RSUME variants also contain an intermediate domain. Only the longest RSUME isoform presents a C-terminal domain that is absent in the others. Given these differences, we used the shortest and longest RSUME variants for comparative studies. We found that the C-terminal domain is dispensable for the SUMO-conjugation enhancer properties of RSUME. We also demonstrate that these two RSUME variants are equally induced by hypoxia. The NF-κB signaling pathway is inhibited and the HIF-1 pathway is increased more efficiently by the longest RSUME, by means of a greater physical interaction of RSUME267 with the target proteins. In addition, the mRNA and protein levels of these isoforms differ in human glioma samples; while the shortest RSUME isoform is expressed in all the tumors analyzed, the longest variant is expressed in most but not all of them. The results presented here show a degree of redundancy of the RSUME variants on the SUMO pathway. However, the increased inhibition conferred by RSUME267 over the NF-κB signaling pathway, the increased activation over the HIF-1 pathway and the different expression of the RSUME isoforms suggest specific roles for each RSUME isoform which may be relevant in certain types of brain tumors that express RSUME, like human pituitary adenomas and gliomas.     

Characterization and Evolution of mariner Elements from Closely Related Species of Fruit Flies (Diptera: Tephritidae)

Mariner elements were amplified using the polymerase chain reaction from two species of tephritid flies, Ceratitis rosa and Trirhithrum coffeae. The sequences were ∼1.3 kb in length. None of these elements appeared to be functional, as in every case the open reading frame (ORF) was disrupted by the presence of frameshifts or stop codons. These elements, Crmar1 and Tcmar1, are very similar to the Ccmar1 element previously amplified from the closely related tephritid species C. capitata and are members of the mellifera subfamily of mariner elements. The phylogeny and pattern of divergence of these elements were examined in relation to the phylogeny of the host species. It is highly probable that the elements were present in the ancestral lineage prior to the divergence of the three species. The copy numbers of the elements within each species are very different, ranging from about 10 in T. coffeae to 5,000 in C. rosa. The possible mechanisms which determine the copy number of an element in the host genome are discussed. Received: 25 April 1997 / Accepted: 31 July 1997     

Functional Characterization of MC1R-TUBB3 Intergenic Splice Variants of the Human Melanocortin 1 Receptor

The melanocortin 1 receptor gene (MC1R) expressed in melanocytes is a major determinant of skin pigmentation. It encodes a Gs protein-coupled receptor activated by α-melanocyte stimulating hormone (αMSH). Human MC1R has an inefficient poly(A) site allowing intergenic splicing with its downstream neighbour Tubulin-β-III (TUBB3). Intergenic splicing produces two MC1R isoforms, designated Iso1 and Iso2, bearing the complete seven transmembrane helices from MC1R fused to TUBB3-derived C-terminal extensions, in-frame for Iso1 and out-of-frame for Iso2. It has been reported that exposure to ultraviolet radiation (UVR) might promote an isoform switch from canonical MC1R (MC1R-001) to the MC1R-TUBB3 chimeras, which might lead to novel phenotypes required for tanning. We expressed the Flag epitope-tagged intergenic isoforms in heterologous HEK293T cells and human melanoma cells, for functional characterization. Iso1 was expressed with the expected size. Iso2 yielded a doublet of Mr significantly lower than predicted, and impaired intracellular stability. Although Iso1- and Iso2 bound radiolabelled agonist with the same affinity as MC1R-001, their plasma membrane expression was strongly reduced. Decreased surface expression mostly resulted from aberrant forward trafficking, rather than high rates of endocytosis. Functional coupling of both isoforms to cAMP was lower than wild-type, but ERK activation upon binding of αMSH was unimpaired, suggesting imbalanced signaling from the splice variants. Heterodimerization of differentially labelled MC1R-001 with the splicing isoforms analyzed by co-immunoprecipitation was efficient and caused decreased surface expression of binding sites. Thus, UVR-induced MC1R isoforms might contribute to fine-tune the tanning response by modulating MC1R-001 availability and functional parameters.     

Identification and Characterization of Shiga Toxin Type 2 Variants in Escherichia coli Isolates from Animals, Food, and Humans

There is considerable heterogeneity among the Shiga toxin type 2 (Stx2) toxins elaborated by Shiga toxin-producing Escherichia coli (STEC). One such Stx2 variant, the Stx2d mucus-activatable toxin (Stx2dact), is rendered more toxic by the action of elastase present in intestinal mucus, which cleaves the last two amino acids of the A2 portion of the toxin A subunit. We screened 153 STEC isolates from food, animals, and humans for the gene encoding Stx2dact by using a novel one-step PCR procedure. This method targeted the region of stx_2dact that encodes the elastase recognition site. The presence of stx_2dact was confirmed by DNA sequencing of the complete toxin genes. Seven STEC isolates from cows (four isolates), meat (two isolates), and a human (one isolate) that carried the putative stx_2dact gene were identified; all were eae negative, and none was the O157:H7 serotype. Three of the isolates (CVM9322, CVM9557, and CVM9584) also carried stx₁, two (P1332 and P1334) carried stx₁ and stx_2c, and one (CL-15) carried stx_2c. One isolate, P1130, harbored only stx_2dact. The Vero cell cytotoxicities of supernatants from P1130 and stx₁ deletion mutants of CVM9322, CVM9557, and CVM9584 were increased 13- to 30-fold after treatment with porcine elastase. Thus, Stx2dact-producing strains, as detected by our one-step PCR method, can be isolated not only from humans, as previously documented, but also from food and animals. The latter finding has important public health implications based on a recent report from Europe of a link between disease severity and infection with STEC isolates that produce Stx2dact.     

Mouse Tissues Express Multiple Splice Variants of Prominin-1

Prominin-1, a heavily glycosylated pentaspan membrane protein, is mainly known for its function as a marker for (cancer) stem cells, although it can also be detected on differentiated cells. Mouse prominin-1 expression is heavily regulated by splicing in eight different variants. The function or the expression pattern of prominin-1 and its splice variants (SVs) is thus far unknown. In this study, we analyzed the expression of the prominin-1 splice variants on mRNA level in several mouse tissues and found a broad tissue expression of the majority of SVs, but a specific set of SVs had a much more restricted expression profile. For instance, the testis expressed only SV3 and SV7. Moreover, SV8 was solely detected in the eye. Intriguingly, prominin-1 knockout mice do not suffer from gross abnormalities, but do show signs of blindness, which suggest that SV8 has a specific function in this tissue. In addition, databases searches for putative promoter regions in the mouse prominin-1 gene revealed three potential promoter regions that could be linked to specific SVs. Interestingly, for both SV7 and SV8, a specific potential promoter region could be identified. To conclude, the majority of mouse prominin-1 splice variants are widely expressed in mouse tissues. However, specific expression of a few variants, likely driven by specific promoters, suggests distinct regulation and a potential important function for these variants in certain tissues.     

Sex-Specific Selection and Sex-Biased Gene Expression in Humans and Flies

Sexual dimorphism results from sex-biased gene expression, which evolves when selection acts differently on males and females. While there is an intimate connection between sex-biased gene expression and sex-specific selection, few empirical studies have studied this relationship directly. Here we compare the two on a genome-wide scale in humans and flies. We find a distinctive “Twin Peaks” pattern in humans that relates the strength of sex-specific selection, quantified by genetic divergence between male and female adults at autosomal loci, to the degree of sex-biased expression. Genes with intermediate degrees of sex-biased expression show evidence of ongoing sex-specific selection, while genes with either little or completely sex-biased expression do not. This pattern apparently results from differential viability selection in males and females acting in the current generation. The Twin Peaks pattern is also found in Drosophila using a different measure of sex-specific selection acting on fertility. We develop a simple model that successfully recapitulates the Twin Peaks. Our results suggest that many genes with intermediate sex-biased expression experience ongoing sex-specific selection in humans and flies.     

小鼠SmX5基因mRNA的选择性剪接

通过RT-PCR技术,发现小鼠SmX5基因的另外3种选择性剪接体,分别命名为SmX5a,b和c.小鼠SmX5a cDNA具有完整的内含子1,而SmX5b cDNA含有部分内含子2。SmX5c的保留片断和SmX5b在相同位置起始,但保留部分延伸经过余下的内含子2。余下的DNA序列在所有内含子-外显子边界处都符合剪接的“GT—AG”规则。RT-PCR的表达分析显示这3种异构体都是稳定的转录产物,均通过剪接体特异性引物的PCR反应在mRNA水平上得到证实。和SmX5相比较,剪接体的出现频率都很低。RT-PCR表达分析提示,XmX5和它的3种异构体存在于小鼠的脑、肾、睾丸、胸腺、肝、脾和心脏。SmX5a主要在胸腺中表达,而SmX5b和c在所有组织都可以检测到.SmX5不同选择剪接体及其组织特异的不同剪接方式的存在,表明SmX5的表达是复杂的,并且所有4种SmX5 mRNA水平的调控对于在不同细胞类型的mRNA前体的剪接机制维持具有重要作用。     

Two Splice Variants of Nopp140 in Drosophila melanogaster

The Nopp140 gene of Drosophila maps within 79A5 of chromosome 3. Alternative splicing yields two variants. DmNopp140 (654 residues) is the sequence homolog of vertebrate Nopp140. Its carboxy terminus is 64% identical to that of the prototypical rat Nopp140. DmNopp140-RGG (688 residues) is identical to DmNopp140 throughout its first 551 residues, but its carboxy terminus contains a glycine/arginine-rich domain that is often found in RNA-binding proteins such as vertebrate nucleolin. Both Drosophila variants localize to nucleoli in Drosophila Schneider II cells and Xenopus oocytes, specifically within the dense fibrillar components. In HeLa cells, DmNopp140-RGG localizes to intact nucleoli, whereas DmNopp140 partitions HeLa nucleoli into phase-light and phase-dark regions. The phase-light regions contain DmNopp140 and endogenous fibrillarin, whereas the phase-dark regions contain endogenous nucleolin. When coexpressed, both Drosophila variants colocalize to HeLa cell nucleoli. Both variants fail to localize to endogenous Cajal bodies in Xenopus oocyte nuclei and in HeLa cell nuclei. Endogenous HeLa coilin, however, accumulates around the periphery of phase-light regions in cells expressing DmNopp140. The carboxy truncation (DmNopp140DeltaRGG) also fails to localize to Cajal bodies, but it forms similar phase-light regions that peripherally accumulate endogenous coilin. Conversely, we see no unusual accumulation of coilin in cells expressing DmNopp140-RGG.     

Splice Variants of SmgGDS Control Small GTPase Prenylation and Membrane Localization

Ras and Rho small GTPases possessing a C-terminal polybasic region (PBR) are vital signaling proteins whose misregulation can lead to cancer. Signaling by these proteins depends on their ability to bind guanine nucleotides and their prenylation with a geranylgeranyl or farnesyl isoprenoid moiety and subsequent trafficking to cellular membranes. There is little previous evidence that cellular signals can restrain nonprenylated GTPases from entering the prenylation pathway, leading to the general belief that PBR-possessing GTPases are prenylated as soon as they are synthesized. Here, we present evidence that challenges this belief. We demonstrate that insertion of the dominant negative mutation to inhibit GDP/GTP exchange diminishes prenylation of Rap1A and RhoA, enhances prenylation of Rac1, and does not detectably alter prenylation of K-Ras. Our results indicate that the entrance and passage of these small GTPases through the prenylation pathway is regulated by two splice variants of SmgGDS, a protein that has been reported to promote GDP/GTP exchange by PBR-possessing GTPases and to be up-regulated in several forms of cancer. We show that the previously characterized 558-residue SmgGDS splice variant (SmgGDS-558) selectively associates with prenylated small GTPases and facilitates trafficking of Rap1A to the plasma membrane, whereas the less well characterized 607-residue SmgGDS splice variant (SmgGDS-607) associates with nonprenylated GTPases and regulates the entry of Rap1A, RhoA, and Rac1 into the prenylation pathway. These results indicate that guanine nucleotide exchange and interactions with SmgGDS splice variants can regulate the entrance and passage of PBR-possessing small GTPases through the prenylation pathway.     

An Extended Nomenclature for Mammalian V-ATPase Subunit Genes and Splice Variants

The vacuolar-type H⁺-ATPase (V-ATPase) is a multisubunit proton pump that is involved in both intra- and extracellular acidification processes throughout the body. Multiple homologs and splice variants of V-ATPase subunits are thought to explain its varied spatial and temporal expression pattern in different cell types. Recently subunit nomenclature was standardized with a total of 22 subunit variants identified. However this standardization did not accommodate the existence of splice variants and is therefore incomplete. Thus, we propose here an extension of subunit nomenclature along with a literature and sequence database scan for additional V-ATPase subunits. An additional 17 variants were pulled from a literature search while 4 uncharacterized potential subunit variants were found in sequence databases. These findings have been integrated with the current V-ATPase knowledge base to create a new V-ATPase subunit catalogue. It is envisioned this catalogue will form a new platform on which future studies into tissue- and organelle-specific V-ATPase expression, localization and function can be based.     

Expression of CD44 Splice Variants During Lymphocyte Activation and Tumor Progression

Recently, splice variants of CD44 have been described that confer metastatic potential to non-metastasizing rat pancreatic carcinoma and sarcoma cell lines. Using antibodies against variant CD44 (CD44v) sequences, we have examined the expression of variant CD44 glycoproteins on human lymphoid cells and tissues and in colorectal neoplasia. Lymphohematopoietic cells express low levels of CD44v glycoproteins. During the process of lymphocyte activation in vitro and in vivo, expression of CD44v glycoproteins is transiently upregulated. The reaction pattern of various antibodies indicates that these CD44 variants contain the domain encoded by exon v6, which is part of the variant that confers metastatic capability. In human colorectal neoplasia we observed overexpression of CD44 splice variants in all invasive carcinomas. Already at early stages of colorectal tumor progression exon v5 epitopes were overexpressed. Tumor progression was strongly related to expression of CD44 isoforms containing exon v6 encoded domains. The findings establish CD44 variants as tumor progression markers in colorectal cancer.     

Expression of CD44 Splice Variants During Lymphocyte Activation and Tumor Progression

Recently, splice variants of CD44 have been described that confer metastatic potential to non-metastasizing rat pancreatic carcinoma and sarcoma cell lines. Using antibodies against variant CD44 (CD44v) sequences, we have examined the expression of variant CD44 glycoproteins on human lymphoid cells and tissues and in colorectal neoplasia. Lymphohematopoietic cells express low levels of CD44v glycoproteins. During the process of lymphocyte activation in vitro and in vivo, expression of CD44v glycoproteins is transiently upregulated. The reaction pattern of various antibodies indicates that these CD44 variants contain the domain encoded by exon v6, which is part of the variant that confers metastatic capability. In human colorectal neoplasia we observed overexpression of CD44 splice variants in all invasive carcinomas. Already at early stages of colorectal tumor progression exon v5 epitopes were overexpressed. Tumor progression was strongly related to expression of CD44 isoforms containing exon v6 encoded domains. The findings establish CD44 variants as tumor progression markers in colorectal cancer.     

The Value of Patch-Choice Copying in Fruit Flies

Many animals copy the choices of others but the functional and mechanistic explanations for copying are still not fully resolved. We relied on novel behavioral protocols to quantify the value of patch-choice copying in fruit flies. In a titration experiment, we quantified how much nutritional value females were willing to trade for laying eggs on patches already occupied by larvae (social patches). Females were highly sensitive to nutritional quality, which was positively associated with their offspring success. Females, however, perceived social, low-nutrition patches (33% of the nutrients) as equally valuable as non-social, high-nutrition ones (100% of the nutrients). In follow-up experiments, we could not, however, either find informational benefits from copying others or detect what females'' offspring may gain from developing with older larvae. Because patch-choice copying in fruit flies is a robust phenomenon in spite of potential costs due to competition, we suggest that it is beneficial in natural settings, where fruit flies encounter complex dynamics of microbial communities, which include, in addition to the preferred yeast species they feed on, numerous harmful fungi and bacteria. We suggest that microbial ecology underlies many cases of copying in nature.     

糖尿病血管病变中的RAGE可变剪接物

该文简要回顾高级糖化终产物受体（RAGE）可变剪接物[全长RAGE、截去N端型RAGE和截去C端型RAGE（esRAEG）1的结构和功能;阐述RAGE可变剪接物由于在内皮细胞中的丰度、表达水平不同,使介导的生物学效应也不同;从而有助于我们了解细胞内RAGE可变剪接物对高级糖化终产物（AGE）反应的分子机制及糖尿病血管并发症易感性的个体差异。     

Splice Variants of Perlucin from Haliotis laevigata Modulate the Crystallisation of CaCO3

Perlucin is one of the proteins of the organic matrix of nacre (mother of pearl) playing an important role in biomineralisation. This nacreous layer can be predominately found in the mollusc lineages and is most intensively studied as a compound of the shell of the marine Australian abalone Haliotis laevigata. A more detailed analysis of Perlucin will elucidate some of the still unknown processes in the complex interplay of the organic/inorganic compounds involved in the formation of nacre as a very interesting composite material not only from a life science-based point of view. Within this study we discovered three unknown Perlucin splice variants of the Australian abalone H. laevigata. The amplified cDNAs vary from 562 to 815 base pairs and the resulting translation products differ predominantly in the absence or presence of a varying number of a 10 mer peptide C-terminal repeat. The splice variants could further be confirmed by matrix-assisted laser desorption ionisation time of flight mass spectrometry (MALDI-ToF MS) analysis as endogenous Perlucin, purified from decalcified abalone shell. Interestingly, we observed that the different variants expressed as maltose-binding protein (MBP) fusion proteins in E. coli showed strong differences in their influence on precipitating CaCO₃ and that these differences might be due to a splice variant-specific formation of large protein aggregates influenced by the number of the 10 mer peptide repeats. Our results are evidence for a more complex situation with respect to Perlucin functional regulation by demonstrating that Perlucin splice variants modulate the crystallisation of calcium carbonate. The identification of differentially behaving Perlucin variants may open a completely new perspective for the field of nacre biomineralisation.