Sex-specific splicing and polyadenylation of dsx pre-mRNA requires a sequence that binds specifically to tra-2 protein in vitro

Hedley, Mary Lynne; Maniatis, Tom

doi:10.1016/0092-8674(91)90090-l

Cited by 231 publications

(185 citation statements)

References 38 publications

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“…The sequence motif involved in nonsense-mediated mRNA decay is another example of a short RNA repeat involved in posttranscriptional regulation. Short RNA sequences that modulate both mRNA turnover and splicing have been identified (10,27,33,34,36,37,63,68,73,74,77). As with the results presented here, sequences flanking these motifs can influence their activity.…”

Section: Discussionmentioning

confidence: 66%

Identification and Characterization of a Sequence Motif Involved in Nonsense-Mediated mRNA Decay

Zhang

Ruiz-Echevarría²,

Quan

et al. 1995

Molecular and Cellular Biology

123

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In both prokaryotes and eukaryotes, nonsense mutations in a gene can enhance the decay rate or reduce the abundance of the mRNA transcribed from that gene, and we call this process nonsense-mediated mRNA decay. We have been investigating the cis-acting sequences involved in this decay pathway. Previous experiments have demonstrated that, in addition to a nonsense codon, specific sequences 3 of a nonsense mutation, which have been defined as downstream elements, are required for mRNA destabilization. The results presented here identify a sequence motif (TGYYGATGYYYYY, where Y stands for either T or C) that can predict regions in genes that, when positioned 3 of a nonsense codon, promote rapid decay of its mRNA. Sequences harboring two copies of the motif from five regions in the PGK1, ADE3, and HIS4 genes were able to function as downstream elements. In addition, four copies of this motif can function as an independent downstream element. The sequences flanking the motif played a more significant role in modulating its activity when fewer copies of the sequence motif were present. Our results indicate the sequences 5 of the motif can modulate its activity by maintaining a certain distance between the sequence motif and the termination codon. We also suggest that the sequences 3 of the motif modulate the activity of the downstream element by forming RNA secondary structures. Consistent with this view, a stem-loop structure positioned 3 of the sequence motif can enhance the activity of the downstream element. This sequence motif is one of the few elements that have been identified that can predict regions in genes that can be involved in mRNA turnover. The role of these sequences in mRNA decay is discussed.The rates at which mRNAs decay play an important role in regulating gene expression. mRNA half-lives can vary from each other by as much as 100-fold (28,55,59,62,64), and this variation determines, in part, the cytoplasmic abundance of these RNAs. In addition, the decay rate of an mRNA can be modulated, and its half-life can vary depending on the cell type or the environmental situation of the cell (e.g., hormones, stress, cell cycle, etc. [3,55,62]). The goal of our work is to understand the cellular mechanisms that determine the stability of mRNAs.We are studying mRNA decay in the yeast Saccharomyces cerevisiae. Our results, as well as work from other laboratories, strongly indicate that the processes of mRNA turnover and translation are intimately linked and that understanding of this relationship is critical in elucidating the mechanism of mRNA decay (2, 5, 9, 11, 15, 21, 23, 29-31, 41, 53, 55-59, 76). The role of translation in determination of mRNA decay rates is direct, and, for certain instability elements identified in protein coding regions and 3Ј untranslated regions (3Ј-UTRs), translation of the protein coding region must occur in order for them to function (2,11,15,21,23,29,41,53,60,66,76).One clear example of the relationship between translation and mRNA decay is the observation that nonsense mutati...

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Section: Discussionmentioning

confidence: 66%

Identification and Characterization of a Sequence Motif Involved in Nonsense-Mediated mRNA Decay

Zhang

Ruiz-Echevarría²,

Quan

et al. 1995

Molecular and Cellular Biology

123

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“…In the absence of functional TRA-2, the dsx male-specific mRNA, encoding an alternative protein, is produced by default. The mechanism by which TRA and TRA-2 affect dsx splicing has now been studied in some detail (12)(13)(14)(15)(16)(17). In vitro studies suggest that these proteins both bind directly to the dsx splicing enhancer, a 300-nt region of the dsx pre-mRNA located in the femalespecific exon.…”

Section: Introductionmentioning

confidence: 99%

A human homologue of the Drosophila sex determination factor transformer-2 has conserved splicing regulatory functions.

Dauwalder

Amaya-Manzanares

Mattox

1996

Proc. Natl. Acad. Sci. U.S.A.

112

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Regulation of gene expression through alternative pre-mRNA splicing appears to occur in all metazoans, but most of our knowledge about splicing regulators derives from studies on genetically identified factors from Drosophila.Among the best studied of these is the transformer-2 (TRA-2) protein which, in combination with the transformer (TRA) protein, directs sex-specific splicing of pre-mRNA from the sex determination gene doublesex (dsx). Here we report the identification of htra-2a, a human homologue of tra-2. Two alternative types of htra-2a cDNA clones were identified that encode different protein isoforms with striking organizational similarity to Drosophila tra-2 proteins. When expressed in flies, one hTRA-2a isoform partially replaces the function of Drosophila TRA-2, affecting both female sexual differentiation and alternative splicing of dsx pre-mRNA. Like Drosophila TRA-2, the ability of hTRA-2a to regulate d&x is femalespecific and depends on the presence of the dsx splicing enhancer. These results demonstrate that htra-2a has conserved a striking degree of functional specificity during evolution and leads us to suggest that, although they are likely to serve different roles in development, the tra-2 products of flies and humans have similar molecular functions.Despite the fact that a large fraction of identified cellular pre-mRNAs undergo alternative splicing, few vertebrate factors have been identified that affect splicing patterns. Of those factors so far studied, the evidence for a role in the regulation of splicing is best for the SR proteins, a family of RNA binding proteins that contain extensive regions rich in arginine and serine (RS domains) (for review, see ref. 1). Several lines of evidence suggest these domains may facilitate interactions with other RNA binding proteins. While SR proteins are known to play vital roles during constitutive pre-mRNA splicing both in the initiation of spliceosome assembly and in interactions between small nuclear ribonucleoproteins they have also been shown to affect the selection of alternative 5' splice sites in a variety of artificial and natural substrates in a concentration dependent manner. In addition, several SR proteins are known to interact with the purine-rich splicing enhancer elements found in some vertebrate exons. While these observations strongly suggest a role for SR proteins in regulating splicing, there is still little direct evidence that vertebrate SR proteins normally direct the developmentally specific alternative splicing of any particular cellular pre-mRNAs in vivo.Proteins with established roles in developmental regulation of splicing have been identified in Drosophila through genetic analysis (2-7). Many of these proteins form a cascade of splicing factors that directs sexual differentiation in the fly. Two SR-related proteins, encoded by the transformer (tra) and transformer-2 (tra-2) genes, play a central role in this pathway (8). However, unlike the vertebrate SR proteins described above, these SR-related splicing factors...

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“…The best characterized example of regulated alternative splicing is the inclusion of the female-specific exon 4 of the doublesex (dsx) gene in Drosophila. Inclusion of this exon requires the activation of a weak 3Ј splice site (31,32) and is controlled by three distinct cis regulatory sequences: the dsx repeat elements (31,(33)(34)(35), a purine-rich exon splicing enhancer (36), and a sequence just upstream of the 3Ј splice site (37). Of the three types of elements, only the repeat elements are recognized by a female-specific factor.…”

Section: Discussionmentioning

confidence: 99%

Binding of PurH to a Muscle-specific Splicing Enhancer Functionally Correlates with Exon Inclusion in Vivo

Ryan¹,

Charlet-B.

Cooper

2000

Journal of Biological Chemistry

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Regulated alternative splicing of avian cardiac troponin T (cTNT) pre-mRNA requires multiple intronic elements called muscle-specific splicing enhancers (MSEs) that flank the alternative exon 5 and promote musclespecific exon inclusion. To understand the function of the MSEs in muscle-specific splicing, we sought to identify trans-acting factors that bind to these elements. MSE3, which is located 66 -81 nucleotides downstream of exon 5, assembles a complex that is both sequenceand muscle-specific. Purification and characterization of the MSE3 complex identified one component as 5-aminoimidazole-4-carboxamide ribonucleotideformyltransferase/IMP cyclohydrolase (PurH), an enzyme involved in de novo purine synthesis. Recombinant human PurH protein directly binds MSE3 RNA and PurH is the primary determinant of sequence-specific binding in the native complex. Furthermore, we show a direct correlation between the in vitro binding affinity of both the MSE3 complex and recombinant PurH with functional activation of exon inclusion in vivo. Together, these results strongly suggest that PurH performs a second function as a component of a complex that regulates MSE3-dependent exon inclusion.Alternative splicing allows single genes to express multiple mRNAs. Splice site selection is often regulated in a cell-specific manner resulting in regulated expression of different protein isoforms (1-3). Genetic and biochemical studies in Drosophila have identified specific cis elements and trans-acting factors which mediate cell-specific splicing events (4). In vertebrates, cis elements that mediate cell-specific splicing events have been identified (5-12). Factors that bind to some of these elements have also been identified (13-18), but it remains unclear how these factors mediate cell-specific splicing events.We are using the chicken cardiac troponin T (cTNT) 1 gene to investigate the mechanisms of regulated splicing in striated muscle. cTNT expression is restricted to embryonic skeletal muscle and to embryonic and adult cardiac muscle. Exon 5 undergoes developmentally regulated splicing such that inclusion predominates in embryonic skeletal and cardiac muscle and skipping predominates in the adult (19). We have previously identified four cis-acting elements in the introns flanking exon 5 that function as muscle-specific splicing enhancers (MSEs) by transient transfection analysis of cTNT minigenes (5, 6). The MSEs are necessary for higher levels of exon inclusion in muscle cells than in fibroblast cells. Mutation of these elements causes exon skipping in muscle cells but has little effect on splicing in fibroblasts. These results have defined exon skipping as the default splicing pattern and indicate that exon inclusion requires positive-acting trans-factors present in muscle.The four MSEs are designated 1 to 4. MSE1 is located in intron 4, immediately upstream of exon 5. MSEs 2, 3, and 4 are located in the first 130 nucleotides of intron 5 (5). MSE3 includes nucleotides 66 -81 of intron 5 and was originally identified because of i...

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Sex-specific splicing and polyadenylation of dsx pre-mRNA requires a sequence that binds specifically to tra-2 protein in vitro

Cited by 231 publications

References 38 publications

Identification and Characterization of a Sequence Motif Involved in Nonsense-Mediated mRNA Decay

Identification and Characterization of a Sequence Motif Involved in Nonsense-Mediated mRNA Decay

A human homologue of the Drosophila sex determination factor transformer-2 has conserved splicing regulatory functions.

Binding of PurH to a Muscle-specific Splicing Enhancer Functionally Correlates with Exon Inclusion in Vivo

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