Multiple sclerosis is a demyelinating neurodegenerative disease with a strong genetic component. Previous genetic risk studies have failed to identify consistently linked regions or genes outside of the major histocompatibility complex on chromosome 6p. We describe allelic association of a polymorphism in the gene encoding the interleukin 7 receptor alpha chain (IL7R) as a significant risk factor for multiple sclerosis in four independent family-based or case-control data sets (overall P = 2.9 x 10(-7)). Further, the likely causal SNP, rs6897932, located within the alternatively spliced exon 6 of IL7R, has a functional effect on gene expression. The SNP influences the amount of soluble and membrane-bound isoforms of the protein by putatively disrupting an exonic splicing silencer.

Bioactive molecules can pass between microbiota and host to influence host cellular functions. However, general principles of interspecies communication have not been discovered. We show here in C. elegans that nitric oxide derived from resident bacteria promotes widespread S-nitrosylation of the host proteome. We further show that microbiota-dependent S-nitrosylation of C. elegans Argonaute protein (ALG-1)-at a site conserved and S-nitrosylated in mammalian Argonaute 2 (AGO2)-alters its function in controlling gene expression via microRNAs. By selectively eliminating nitric oxide generation by the microbiota or S-nitrosylation in ALG-1, we reveal unforeseen effects on host development. Thus, the microbiota can shape the post-translational landscape of the host proteome to regulate microRNA activity, gene expression, and host development. Our findings suggest a general mechanism by which the microbiota may control host cellular functions, as well as a new role for gasotransmitters.

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The ligand specificity of fibroblast growth factor receptor 2 (FGFR2) is determined by the alternative splicing of exons 8 (IIIb) or 9 (IIIc). Exon IIIb is included in epithelial cells, whereas exon IIIc is included in mesenchymal cells. Although a number of cis elements and trans factors have been identified that play a role in exon IIIb inclusion in epithelium, little is known about the activation of exon IIIc in mesenchyme. We report here the identification of a splicing enhancer required for IIIc inclusion. This 24-nucleotide (nt) downstream intronic splicing enhancer (DISE) is located within intron 9 immediately downstream of exon IIIc. DISE was able to activate the inclusion of heterologous exons rat FGFR2 IIIb and human ␤-globin exon 2 in cell lines from different tissues and species and also in HeLa cell nuclear extracts in vitro. DISE was capable of replacing the intronic activator sequence 1 (IAS1), a known IIIb splicing enhancer and vice versa. This fact, together with the requirement for DISE to be close to the 5-splice site and the ability of DISE to promote binding of U1 snRNP, suggested that IAS1 and DISE belong to the same class of cis-acting elements.Alternative splicing of a given pre-mRNA can lead to changes in the open reading frame of the mature mRNA, imparting tremendous diversity to the proteome (1-3). The protein isoforms encoded by alternative splicing can have significantly different activities. A salient example of this is the production of different isoforms of the fibroblast growth factor receptor-2 (FGFR2), 4 where isoform FGFR2(IIIb) has a high affinity for FGF7 or keratinocyte growth factor while isoform FGFR2 (IIIc) preferentially binds FGF2 (Basic FGF) (4, 5). These isoforms arise because of the alternative inclusion of exon 8 (IIIb) or exon 9 (IIIc). This alternative splicing event is regulated in a cell type-specific manner such that epithelial cells produce FGFR2(IIIb) and mesenchymal cells produce FGFR2(IIIc). This tissue-specific exon choice is the outcome of complex regulation involving multiple cis elements and trans factors (6 -8).cis-Acting elements within FGFR2 transcripts activate exon IIIb, repress IIIc, or do both simultaneously in epithelial cells (see Ref. 6 and references within). In contrast to this multifaceted regulation in epithelial cells, the only cis elements known to be active in mesenchymal cells are silencers of exon IIIb (7). Therefore, the choice of exon IIIc in these cells was presumed to be by default, i.e. mediated solely by the splice sites flanking the exon (8). More recently Mauger and Garcia-Blanco have shown that the factor SFRS1 (also known as ASF/SF2) activates exon IIIc by binding an exonic splicing enhancer (ESE) and antagonizing hnRNP H/F proteins that bind an overlapping silencer. 5In this article, we revise this simple model in light of the identification of an intronic enhancer element that was absolutely required for the efficient inclusion of the rat FGFR2 IIIc. This enhancer was localized to a phylogenetically conserved region o...

Synthesis of collagen, a major component of the extracellular matrix, is increased dramatically in fibrotic conditions such as scleroderma. This overaccumulation of collagen is associated with increased pyridinoline cross-links. These cross-links are derived by the action of the alternatively spliced long form of lysyl hydroxylase 2 (LH2), a collagen telopeptide LH. As LH2 (long) is reported to be overexpressed in scleroderma fibroblasts, the regulation of LH2 splicing suggests an important step in controlling fibrosis. Using an LH2 minigene, we have compared the regulation of the alternative splicing pattern of LH2, both endogenously and in the minigene, by the RNA-binding splicing proteins TIA-1 and TIAL1 (T-cell-restricted intracellular antigens). A decrease in the ratio of LH2 (long) to LH2 (short) was observed in fibroblasts from TIAL1 knockout mice, and in HEK293 cells knocked down for TIA-1 and TIAL1. As a corollary, overexpression of TIA-1/TIAL1 in HEK293 cells resulted in an increase in LH2 (long) minigene transcripts, accompanied by a decrease in LH2 (short). In scleroderma fibroblasts, a double TIA-1/TIAL1 knockdown reduced the ratio of LH2 (long) to LH2 (short) by over fivefold compared to controls. Identification of these TIA regulatory factors therefore suggests a tool to manipulate cellular LH2 levels in scleroderma so that potential intervention therapies may be identified.

Objective. Scleroderma (systemic sclerosis [SSc]) is a complex connective tissue disorder characterized by hardening and thickening of the skin. One hallmark of scleroderma is excessive accumulation of collagen accompanied by increased levels of pyridinoline collagen crosslinks derived from hydroxylysine residues in the collagen telopeptide domains. Lysyl hydroxylase 2 (LH2), an important alternatively spliced enzyme in collagen biosynthesis, acts as a collagen telopeptide hydroxylase. Changes in the pattern of LH2 alternative splicing, favoring increased inclusion of the alternatively spliced LH2 exon 13A, thereby increasing the levels of the long transcript of LH2 (LH2[long]), are linked to scleroderma disease. This study was undertaken to examine the role played by RNA binding protein Fox-2 in regulating exon 13A inclusion, which leads to the generation of scleroderma-associated LH2(long) messenger RNA (mRNA).Methods. Phylogenetic sequence analysis of introns flanking exon 13A was performed. A tetracyclineinducible system in T-Rex 293 cells was used to induce Fox-2 protein, and endogenous LH2(long) mRNA was determined by reverse transcriptase-polymerase chain reaction. An LH2 minigene was designed, validated, and used in Fox-2 overexpression and mutagenesis experiments. Knockdown of Fox-2 was performed in mouse embryonic fibroblasts and in fibroblasts from SSc patients.Results The biosynthesis and assembly of triple helical collagen fibrils is a multistep process that begins inside a cell but is completed in the extracellular matrix (1). One of the important groups of enzymes that play a key role in the collagen biosynthesis pathway is the lysyl hydroxylase (LH) family (also named procollagen-lysine, 2-oxoglutarate 5-dioxygenase [PLOD]) that includes LH1, LH2, and LH3. These enzymes catalyze the conversion of specific Lys residues on the nascent collagen chains to hydroxylysines (Hyl). The target sequences of LH are located in both the helical and non-helical telopeptide collagen domains (Gly-X-Lys and Ala/Ser-X-Lys, respectively) (2). The telopeptide Hyl, in turn, act as precursors that are responsible for the formation of intermolecular trifunctional pyridinoline (Pyr) crosslinks. These Pyr crosslinks are critical for the mechanical stability and tensile strength of collagen fibrils. Although each member of the LH family hydroxylates collagen Lys residues, only LH2 has been shown to act specifically on the telopeptide lysines of collagen molecules (3). LH2 is also the only member of the LH family that is known to be alternatively spliced (4). Its alternative splicing leads to the generation of 2

and W. Liesack. 1999. Comparative phylogenetic assignment of environmental sequences of genes encoding 16S rRNA and numerically abundant culturable bacteria from an anoxic rice paddy soil. Appl. Environ. Microbiol. 65 :5050-5058. 5. Ludwig, W. and O. Strunk. 1997. ARB-a software environment for sequence data (http://www.biol.chemie.tu-muenchen.de/ pub/ARB/documentation). 6. Moyer, C.L., J.M. Tiedje, F.C. Dobbs, and D.M. Karl. 1996. A computer-simulated restriction fragment length polymorphism analysis of bacterial small-subunit rRNA genes: efficacy of selected tetrameric restriction enzymes for studies of microbial diversity in nature. Appl. Environ. Microbiol.