Pre-mRNA processing enhancer (PPE) element increases the expression of an intronless thymidylate synthase gene but does not affect intron-dependent S phase regulation

Lee, Tina X.; Johnson, Lee F.

doi:10.1002/(sici)1097-4644(19980501)69:2<104::aid-jcb2>3.0.co;2-w

Cited by 4 publications

(1 citation statement)

References 40 publications

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“…Studies in a variety of different systems indicate that cDNA transgenes are expressed at lower levels than their genomic counterparts in vivo and that expression of a cDNA can be elevated by inclusion of an intron near the 5Ј end of the gene (10,30,31,37,38,46). The magnitude of this effect varies, depending on sequences present in the RNA and the promoter that is used to direct transgene expression (33,35). Furthermore, in a study that examined the factors that influence intron-dependent enhancement of gene expression, the strength of the 5Ј splice site was found to be important (31).…”

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confidence: 99%

Arginine-Rich Regions Mediate the RNA Binding and Regulatory Activities of the Protein Encoded by theDrosophila melanogaster suppressor of sableGene

Turnage

Brewer-Jensen

Wei

et al. 2000

Molecular and Cellular Biology

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The Drosophila melanogaster suppressor of sable gene, su(s), encodes a novel, 150-kDa nuclear RNA binding protein, SU(S), that negatively regulates RNA accumulation from mutant alleles of other genes that have transposon insertions in the 5 transcribed region. In this study, we delineated the RNA binding domain of SU(S) and evaluated its relevance to SU(S) function in vivo. As a result, we have defined two arginine-rich motifs (ARM1 and ARM2) that mediate the RNA binding activity of SU(S). ARM1 is required for in vitro high-affinity binding of SU(S) to small RNAs that were previously isolated by SELEX (binding site selection assay) and that contain a common consensus sequence. ARM1 is also required for the association of SU(S) with larval polytene chromosomes in vivo. ARM2 promotes binding of SU(S) to SELEX RNAs that lack the consensus sequence and apparently is neither necessary nor sufficient for the stable polytene chromosome association of SU(S). Use of the GAL4/UAS system to drive ectopic expression of su(s) cDNA transgenes revealed two previously unknown properties of SU(S). First, overexpression of SU(S) is lethal. Second, SU(S) negatively regulates expression of su(s) intronless cDNA transgenes, and the ARMs are required for this effect. Considering these and previous results, we propose that SU(S) binds to the 5 region of nascent transcripts and inhibits RNA production in a manner that can be overcome by splicing complex assembly.Eukaryotic protein-coding RNAs are typically transcribed as larger pre-mRNAs that are processed to a mature form. PremRNA processing is coupled to transcription (7,8,27,42) and involves a complex set of events including the addition of a 7-methylguanosine cap to the 5Ј end, splicing to remove internal introns, and cleavage/polyadenylation of the 3Ј end. Interactions between the cellular transcription and RNA processing apparatuses and between RNA processing components that assemble at various sites on the pre-mRNA are thought to facilitate the efficient production of mRNAs that are suitable substrates for translation. Incorrectly processed transcripts can be recognized as such and degraded (16,24,26,40).The Drosophila melanogaster suppressor of sable gene, su(s), encodes a protein involved in nuclear pre-mRNA metabolism. Loss-of-function su(s) mutations either suppress or enhance specific mutant alleles of a variety of unlinked genes (49). Some su(s) mutants also exhibit defects in viability and male fertility (52). Although both the su(s) gene and mutant alleles affected by su(s) have been cloned and characterized, the function of the su(s) gene product, SU(S), has been somewhat elusive. The enhanced alleles are associated with large, complex genes that cannot easily be analyzed in detail at a molecular level. More is known about the suppressed alleles, through molecular studies of vermilion (v), yellow (y), and purple (pr) (20)(21)(22)29). The su(s)-suppressible mutations have transposon insertions near the 5Ј end of the transcribed region that interrupt either the first exon...

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Section: Discussionmentioning

confidence: 99%

Arginine-Rich Regions Mediate the RNA Binding and Regulatory Activities of the Protein Encoded by theDrosophila melanogaster suppressor of sableGene

Turnage

Brewer-Jensen

Wei

et al. 2000

Molecular and Cellular Biology

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Inactivation of MED-1 elements in the TATA-less, initiator-less mouse thymidylate synthase promoter has no effect on promoter strength or the complex pattern of transcriptional start sites

Rudge

Johnson

1999

J. Cell. Biochem.

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The mouse thymidylate synthase (TS) promoter is a member of a family of promoters that lack a TATA box as well as an initiator element and that initiate transcription at many sites over a broad initiation window. An element (MED-1) downstream of the initiation window of almost all promoters of this family has been proposed to be important for promoter activity, as well as for multiple start site utilization. Two consensus MED-1 elements are located downstream of the initiation window of the TS promoter. To determine the role of the MED-1 elements in the TS promoter, one or both elements were inactivated by site-directed mutagenesis and the effects on promoter function were determined. We found that inactivation of the MED-1 elements had no measurable effect on promoter strength, the boundaries of the initiation window, or the pattern of transcriptional start sites. Furthermore, inactivation of the elements did not affect the ability of the TS promoter to direct S phase-specific expression of the gene in growth-stimulated cells. We conclude that the MED-1 element does not play a significant role in TS promoter function and therefore is not an essential component of all TATA-less promoters with complex transcriptional initiation patterns.

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Transcriptional control elements and complex initiation pattern of the TATA-less bidirectional human thymidylate synthase promoter

2000

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The nucleotide sequences that are important for transcription of the human thymidylate synthase gene were analyzed by deletion and site-directed mutagenesis of the promoter region. Deletion analyses from the 5' and 3' ends indicated the presence of multiple positive and negative elements. The promoter had approximately the same strength in the normal or inverted orientation. The region between 161 and 141 nt upstream of the translational start codon was found to be both necessary and sufficient for high-level promoter activity in both directions and was designated the essential promoter region. This region, which is highly conserved in human, mouse and rat TS promoters, contains potential binding sites for Ets, Sp1, and LSF transcription factors. Site directed mutagenesis of each of these elements led to large decreases in promoter strength. However, inactivation of potential Sp1 and E2F elements adjacent to the essential promoter region led to increases in promoter strength. The transcriptional start site pattern was analyzed by S1 nuclease protection assays of mRNA isolated from cells transiently transfected with TS minigenes. Multiple start sites were detected, most of which were between 160 and 120 nt upstream of the AUG codon.

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Pre-mRNA processing enhancer (PPE) element increases the expression of an intronless thymidylate synthase gene but does not affect intron-dependent S phase regulation

Cited by 4 publications

References 40 publications

Arginine-Rich Regions Mediate the RNA Binding and Regulatory Activities of the Protein Encoded by theDrosophila melanogaster suppressor of sableGene

Arginine-Rich Regions Mediate the RNA Binding and Regulatory Activities of the Protein Encoded by theDrosophila melanogaster suppressor of sableGene

Inactivation of MED-1 elements in the TATA-less, initiator-less mouse thymidylate synthase promoter has no effect on promoter strength or the complex pattern of transcriptional start sites

Transcriptional control elements and complex initiation pattern of the TATA-less bidirectional human thymidylate synthase promoter

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