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Hua Lou Karla M. Neugebauer Robert F. Gagel Susan M. Berget 《Molecular and cellular biology》1998,18(9):4977-4985
Although considerable information is currently available about the factors involved in constitutive vertebrate polyadenylation, the factors and mechanisms involved in facilitating communication between polyadenylation and splicing are largely unknown. Even less is known about the regulation of polyadenylation in genes in which 3′-terminal exons are alternatively recognized. Here we demonstrate that an SR protein, SRp20, affects recognition of an alternative 3′-terminal exon via an effect on the efficiency of binding of a polyadenylation factor to an alternative polyadenylation site. The gene under study codes for the peptides calcitonin and calcitonin gene-related peptide. Its pre-mRNA is alternatively processed by the tissue-specific inclusion or exclusion of an embedded 3′-terminal exon, exon 4, via factors binding to an intronic enhancer element that contains both 3′ and 5′ splice site consensus sequence elements. In cell types that preferentially exclude exon 4, addition of wild-type SRp20 enhances exon 4 inclusion via recognition of the intronic enhancer. In contrast, in cell types that preferentially include exon 4, addition of a mutant form of SRp20 containing the RNA-binding domain but missing the SR domain inhibits exon 4 inclusion. Inhibition is likely at the level of polyadenylation, because the mutant SRp20 inhibits binding of CstF to the exon 4 poly(A) site. This is the first demonstration that an SR protein can influence alternative polyadenylation and suggests that this family of proteins may play a role in recognition of 3′-terminal exons and perhaps in the communication between polyadenylation and splicing. 相似文献
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SR proteins are essential splicing factors involved in the use of both constitutive and alternative exons. We previously showed that the SR proteins SRp20 and ASF/SF2 have antagonistic activities on SRp20 pre-mRNA splicing. SRp20 activates exon 4 recognition in its pre-mRNA, whereas ASF/SF2 inhibits this recognition. In experiments aimed at testing the specificity of SRp20 and ASF/SF2 for exon 4 splicing regulation, we show here that this specificity lies in the RNA binding domains of SRp20 and ASF/SF2 and not in the RS domains. Surprisingly, a deletion of 14 amino acids at the end of ASF/SF2-RBD2 converts ASF/SF2 from an inhibitor to an activator of exon 4 splicing. We found that ASF3 also inhibits exon 4 recognition, thus acting similarly to ASF/SF2, while SC35 activates a cryptic 5' splice site downstream of exon 3 and, in doing so, represses exon 4 use. In contrast, Tra2 and the SR proteins 9G8 and SRp40 do not appear to affect exon 4 splicing. 相似文献
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DNA microarray experiments have generated large amount of gene expression measurements across different conditions. One crucial step in the analysis of these data is to detect differentially expressed genes. Some parametric methods, including the two-sample t-test (T-test) and variations of it, have been used. Alternatively, a class of non-parametric algorithms, such as the Wilcoxon rank sum test (WRST), significance analysis of microarrays (SAM) of Tusher et al. (2001), the empirical Bayesian (EB) method of Efron et al. (2001), etc., have been proposed. Most available popular methods are based on t-statistic. Due to the quality of the statistic that they used to describe the difference between groups of data, there are situations when these methods are inefficient, especially when the data follows multi-modal distributions. For example, some genes may display different expression patterns in the same cell type, say, tumor or normal, to form some subtypes. Most available methods are likely to miss these genes. We developed a new non-parametric method for selecting differentially expressed genes by relative entropy, called SDEGRE, to detect differentially expressed genes by combining relative entropy and kernel density estimation, which can detect all types of differences between two groups of samples. The significance of whether a gene is differentially expressed or not can be estimated by resampling-based permutations. We illustrate our method on two data sets from Golub et al. (1999) and Alon et al. (1999). Comparing the results with those of the T-test, the WRST and the SAM, we identified novel differentially expressed genes which are of biological significance through previous biological studies while they were not detected by the other three methods. The results also show that the genes selected by SDEGRE have a better capability to distinguish the two cell types. 相似文献
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Background
Clusters of genes co-expressed are known in prokaryotes (operons) and were recently described in several eukaryote organisms, including Human. According to some studies, these clusters consist of housekeeping genes, whereas other studies suggest that these clustered genes exhibit similar tissue specificity. Here we further explore the relationship between co-expression and chromosomal co-localization in the human genome by analyzing the expression status of the genes along the best-annotated chromosomes 20, 21 and 22. 相似文献7.
Identification of differentially expressed genes by mutually subtracted RNA fingerprinting 总被引:6,自引:0,他引:6
A mutually subtracted RNA fingerprinting (SuRF) method has been developed that allows efficient identification of differentially expressed sequence tags between two samples. Mutual subtractions of two RNA samples are achieved by first synthesizing cDNAs using oligo(dT) coupled with magnetic beads which are then reciprocally hybridized to starting RNA samples to remove common mRNAs between them. The second step involves differential fingerprinting of the subtracted RNA samples by polymerase chain reaction with specially designed degenerate primers. SuRF was applied to identify alteration in gene expression pertinent to osteogenic sarcoma which was achieved by employing the method between FOB (an immortalized fetal osteoblast) and MG63 (an osteosarcoma) cell lines. An estimated 10% of the total expressed genes in these two cell types were screened by the method. This analysis identified 96 differentially expressed sequences, none of which was identified repeatedly. A subset of these sequences was subsequently confirmed to have differential expression between the two cell types. Removal of common mRNAs prior to differential display should diminish redundant identification of abundant genes and increase the chance of identifying rare differentially expressed genes. 相似文献
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We have uncovered a novel function for two members of the SR protein family in mRNA export. Using UV cross-linking, transient transfection, and Xenopus oocyte microinjection, we find that the nucleocytoplasmic shuttling proteins SRp20 and 9G8 interact specifically with a 22-nt RNA element from the histone H2a gene to promote the export of intronless RNAs in both mammalian cells and Xenopus oocytes. Antibodies to SRp20 or 9G8 eliminate RNA binding and significantly inhibit the export of RNAs carrying the element from oocyte nuclei. Our observation that SRp20 and 9G8 can be UV cross-linked to polyadenylated RNA in both the nucleus and cytoplasm of HeLa cells suggests a more general role for these SR proteins in mRNA export. 相似文献
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生命活动的各个进程伴随着不同基因的选择性开启和关闭。如何有效地分离克隆各种差异表达的基因,成为分子生物学研究的一个努力方向,大量差异基因分离策略因之问世。本文扼要介绍了近年来发展的几种主要分离策略,并详细介绍一种新的差异基因分离方法--抑制差减杂交。 相似文献
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Delogu G Sanguinetti M Pusceddu C Bua A Brennan MJ Zanetti S Fadda G 《Microbes and infection / Institut Pasteur》2006,8(8):2061-2067
Characterization of PE_PGRS gene expression will help define the role of this protein family in the biology of Mycobacterium tuberculosis. In this report, quantitative real-time RT-PCR (QRT-PCR) was implemented to assess expression of three PE_PGRS genes (rv0746, rv1651c and rv1818c) under different experimental conditions. The three PE_PGRS genes showed a similar expression profile in axenic cultures, with a significant up-regulation occurring at late log and early stationary phases. rv1651c gene expression increased following intracellular growth in bone marrow-derived macrophages but not in type-II human pneumocytes, while rv0746 was induced in both in vitro systems. Following the infection of mice with M. tuberculosis, expression levels of rv1651c and rv0746 normalized to ftsZ and 16S rRNA were highest in the spleen tissue during the chronic stages of murine tuberculosis, with a >20- and >30-fold up-regulation, respectively. Levels of expression remained lower in the lung over the same time period. Expression of the rv1818c gene did not change significantly under different experimental conditions tested. The results of this study indicate that M. tuberculosis can differentially regulate expression of PE_PGRS genes and that genes such as rv0746 and rv1651c are significantly induced while M. tuberculosis persists in host cells and tissues. 相似文献
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Blastocyst formation is blocked in mouse embryos lacking the splicing factor SRp20. 总被引:14,自引:0,他引:14
SRp20 is a splicing factor belonging to the highly conserved family of SR proteins [1] [2] [3] [4], which have multiple roles in the regulation of constitutive and alternative splicing in vivo. It has been suggested that SR proteins are involved in bringing together the splice sites during spliceosome assembly [5]. SR proteins show partial redundancy, as each single SR protein can restore splicing activity to a splicing-deficient cytoplasmic extract (termed S-100 extract). Nevertheless, several studies demonstrate that individual SR proteins have different effects on the selection of specific alternative splice sites, and they recognize distinct RNA sequences [6] [7] [8] [9] [10] [11] [12]. Also, inactivation of two SR proteins, B52/SRp55 in Drosophila [13] or ASF/SF2 in the chicken cell line DT40 [14], is lethal, indicating the existence of nonredundant functions. Here, using Cre-loxP-mediated recombination in mice to inactivate the SRp20 gene, we found that it is essential for mouse development. Mutant preimplantation embryos failed to form blastocysts and died at the morula stage. Immunofluorescent staining showed that SRp20 is present in oocytes and early stages of embryonic development. This is the first report of mice deficient for a member of the SR protein family. Our experiments confirm that, although similar in structure, the SR proteins are not functionally redundant. 相似文献
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A great deal of recent research has focused on the challenging task of selecting differentially expressed genes from microarray data ("gene selection"). Numerous gene selection algorithms have been proposed in the literature, but it is often unclear exactly how these algorithms respond to conditions like small sample sizes or differing variances. Choosing an appropriate algorithm can therefore be difficult in many cases. In this paper we propose a theoretical analysis of gene selection, in which the probability of successfully selecting differentially expressed genes, using a given ranking function, is explicitly calculated in terms of population parameters. The theory developed is applicable to any ranking function which has a known sampling distribution, or one which can be approximated analytically. In contrast to methods based on simulation, the approach presented here is computationally efficient and can be used to examine the behavior of gene selection algorithms under a wide variety of conditions, even when the number of genes involved runs into the tens of thousands. The utility of our approach is illustrated by comparing three widely-used gene selection methods. 相似文献
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Identification of differentially expressed genes by restriction endonuclease-based gene expression fingerprinting. 总被引:8,自引:3,他引:5
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A novel method for identification of differentially expressed genes has been developed. It is based on the consecutive restriction digestions of 3' terminal cDNA fragments to produce a fingerprint of gene expression. cDNA molecules are synthesized using a biotinylated oligo(dT) primer, digested with a frequently cutting restriction endonuclease and the 3'-terminal restriction fragments are isolated using streptavidin microbeads. After amplification by PCR, cDNA fragments are immobilized again on streptavidin beads, radiolabeled and treated sequentially with a set of restriction endonucleases. The products of individual enzymatic reactions from two or more different RNA populations are resolved by polyacrylamide gel electrophoresis and compared to reveal differentially expressed genes. This strategy enabled us to identify and clone the fragments of five genes expressed differentially in murine thymus and spleen. One of the genes was found to encode terminal deoxynucleotidyl transferase; others are apparently previously unknown genes. 相似文献