The precision and complexity of intron removal during pre-mRNA splicing still amazes even 26 years after the discovery that the coding information of metazoan genes is interrupted by introns (Berget et al. 1977;Chow et al. 1977). Adding to this amazement is the recent realization that most human genes express more than one mRNA by alternative splicing, a process by which functionally diverse protein isoforms can be expressed according to different regulatory programs. Given that the vast majority of human genes contain introns and that most pre-mRNAs undergo alternative splicing, it is not surprising that disruption of normal splicing patterns can cause or modify human disease. The purpose of this review is to highlight the different mechanisms by which disruption of pre-mRNA splicing play a role in human disease. Several excellent reviews provide detailed information on splicing and the regulation of splicing (Burge et al. 1999;Hastings and Krainer 2001; Black 2003). The potential role of splicing as a modifier of human disease has also recently been reviewed (NissimRafinia and Kerem 2002). Constitutive splicing and the basal splicing machineryThe typical human gene contains an average of 8 exons. Internal exons average 145 nucleotides (nt) in length, and introns average more than 10 times this size and can be much larger (Lander et al. 2001). Exons are defined by rather short and degenerate classical splice-site sequences at the intron/exon borders (5Ј splice site, 3Ј splice site, and branch site; Fig. 1A). Components of the basal splicing machinery bind to the classical splice-site sequences and promote assembly of the multicomponent splicing complex known as the spliceosome. The spliceosome performs the two primary functions of splicing: recognition of the intron/exon boundaries and catalysis of the cut-and-paste reactions that remove introns and join exons. The spliceosome is made up of five small nuclear ribonucleoproteins (snRNPs) and >100 proteins. Each snRNP is composed of a single uridinerich small nuclear RNA (snRNA) and multiple proteins. The U1 snRNP binds the 5Ј splice site, and the U2 snRNP binds the branch site via RNA:RNA interactions between the snRNA and the pre-mRNA (Fig. 1B). Spliceosome assembly is highly dynamic in that complex rearrangements of RNA:RNA, RNA:protein, and protein:protein interactions take place within the spliceosome. Coinciding with these internal rearrangements, both splice sites are recognized multiple times by interactions with different components during the course of spliceosome assembly (for example, see Burge et al. 1999;Du and Rosbash 2002;Lallena et al. 2002;Liu 2002). The catalytic component is likely to be U6 snRNP, which joins the spliceosome as a U4/U6 · U5 tri-snRNP (Villa et al. 2002).A splicing error that adds or removes even 1 nt will disrupt the open reading frame of an mRNA; yet exons are correctly spliced from within tens of thousands of intronic nucleotides. This remarkable precision is, in part, built into the mechanism of intron removal because once...

Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder caused by a CTG expansion in the 3' UTR of the dystrophia myotonica protein kinase (DMPK) gene. It has been hypothesized that the pathogenesis in DM1 is triggered by a toxic gain of function of the expanded DMPK RNA. This expanded RNA is retained in nuclear foci where it sequesters and induces alterations in the levels of RNA-binding proteins (RNA-BP). To model DM1 and study the implication of RNA-BP in CUG-induced toxicity, we have generated a Drosophila DM1 model expressing a non-coding mRNA containing 480 interrupted CUG repeats; i.e. [(CUG)20CUCGA]24. This (iCUG)480 transcript accumulates in nuclear foci and its expression leads to muscle wasting and degeneration in Drosophila. We also report that altering the levels of two RNA-BP known to be involved in DM1 pathogenesis, MBNL1 and CUGBP1, modify the (iCUG)480 degenerative phenotypes. Expanded CUG-induced toxicity in Drosophila is suppressed when MBNL1 expression levels are increased, and enhanced when MBNL1 levels are reduced. In addition, (iCUG)480 also causes a decrease in the levels of soluble MBNL1 that is sequestered in the CUG-containing nuclear foci. In contrast, increasing the levels of CUGBP1 worsens (iCUG)480-induced degeneration even though CUGBP1 distribution is not altered by the expression of the expanded triplet repeat. Our data supports a mechanism for DM1 pathogenesis in which decreased levels of MBNL and increased levels of CUGBP mediate the RNA-induced toxicity observed in DM1. Perhaps more importantly, they also provide proof of the principle that CUG-induced muscle toxicity can be suppressed.

ETR-3 (also know as BRUNOL3, NAPOR, and CUGBP2) is one of six members of the CELF (CUG-BP1-and ETR-3-like factor) family of splicing regulators. ETR-3 regulates splicing by direct binding to the pre-mRNA. We performed systematic evolution of ligands by exponential enrichment (SELEX) to identify the preferred binding sequence of ETR-3. After five rounds of SELEX, ETR-3 selected UG-rich sequences, in particular UG repeats and UGUU motifs. Either of these selected motifs was able to restore ETR-3 binding and responsiveness to a nonresponsive splicing reporter in vivo. Moreover, this effect was not specific to ETR-3 since minigenes containing either of the two motifs were responsive to two other CELF proteins (CUG-BP1 and CELF4), indicating that different members of the CELF family can mediate their effects via a common binding site. Using the SELEX-identified motifs to search the human genome, we identified several possible new ETR-3 targets. We created minigenes for two of these genes, the CFTR and MTMR1 genes, and confirmed that ETR-3 regulates their splicing patterns. For the CFTR minigene this regulation was demonstrated to be dependent on the presence of the putative binding site identified in our screen. These results validate this approach to search for new targets for RNA processing proteins.The fact that the large number of proteins that comprise the human proteome are generated from a much smaller number of genes has highlighted the role of posttranscriptional events such as alternative splicing and editing in the regulation of gene expression. These regulatory events allow the production of multiple mRNA species from a single gene, thus increasing the coding potential of the human genome (25,36,41).CUG-binding protein 1 (CUG-BP1) and ELAV-type RNA binding protein 3 (ETR-3, also known as BRUNOL3, NAPOR, and CUGBP2)-like factor (CELF) proteins (also known as bruno-like or BRUNOL) are a family of related RNA binding proteins (see http://www.bcm.edu/pathology/cooper/files/celf _brunol_napor.htm for a description of nomenclature). They have been implicated in the regulation of splicing, editing, and translation and therefore are likely to play a major role in the generation of proteomic diversity (1,19,37). The human genome encodes six CELF proteins (CUG-BP1, ETR-3, and CELF3 through CELF6), all having similar architectures: two RNA recognition motifs (RRMs) near the N terminus, a third RRM near the C terminus, and a 160-to 230-amino-acid divergent domain between RRM2 and RRM3 (15,23,24). This divergent domain is unique to the CELF proteins and in ETR-3 and CELF4 has been shown to contain one or more activation modules necessary for splicing activity (45).The CELF proteins have been demonstrated to regulate alternative splicing events for several variable regions (cardiac troponin T [cTNT] exon 5, insulin receptor [IR] exon 11, chloride channel 1 [ClC1] intron 2, NMDAR-1 exons 5 and 21, and the ␣-actinin muscle-specific exon) (16,23,40,42,50). The mechanism by which the CELF proteins regulate splicing is stil...

A Bacillus licheniformis strain, 189, isolated from a hot spring environment in the Azores, Portugal, strongly inhibited growth of Gram-positive bacteria. It produced a peptide antibiotic at 50 degrees C. The antibiotic was purified and biochemically characterized. It was highly resistant to several proteolytic enzymes. Additionally, it retained its antimicrobial activity after incubation at pH values between 3.5 and 8; it was thermostable, retaining about 85% and 20% of its activity after 6 h at 50 degrees C and 100 degrees C, respectively. Its molecular mass determined by mass spectrometry was 3249.7 Da.