scholarly journals Two G-Rich Regulatory Elements Located Adjacent to and 440 Nucleotides Downstream of the Core Poly(A) Site of the Intronless Melanocortin Receptor 1 Gene Are Critical for Efficient 3′ End Processing

2006 ◽  
Vol 27 (5) ◽  
pp. 1568-1580 ◽  
Author(s):  
Martin Dalziel ◽  
Nuno Miguel Nunes ◽  
Andre Furger
Keyword(s):  
Receptor Gene ◽  
Regulatory Elements ◽  
Transcription Unit ◽  
Melanocortin Receptor ◽  
Processing Efficiency ◽  
Sequence Element ◽  
Optimal Processing ◽  
Sequence Elements ◽  
A Site ◽  
Nuclear Ribonucleoprotein

ABSTRACT Cleavage and polyadenylation is an essential processing reaction required for the maturation of pre-mRNAs into stable, export- and translation-competent mature mRNA molecules. This reaction requires the assembly of a multimeric protein complex onto a bipartite core sequence element consisting of an AAUAAA hexamer and a GU/U-rich downstream sequence element. In this study we have analyzed 3′ end processing of the human melanocortin 1 receptor gene (MC1R). The MC1R gene is an intron-free transcription unit, and its poly(A) site lacks a defined U/GU-rich element. We describe two G-rich sequence elements that are critical for efficient cleavage at the MC1R poly(A) site. The first element is located 30 nucleotides downstream of the cleavage site and acts as an essential closely positioned enhancer. The second G-rich region is positioned more than 440 nucleotides downstream of the MC1R processing site and is instrumental for optimal processing efficiency. Both G-rich sequences contain clusters of heterogeneous nuclear ribonucleoprotein binding motifs and act together to enhance cleavage at the MC1R poly(A) site.

scholarly journals Sequence elements upstream of the 3' cleavage site confer substrate strength to the adenovirus L1 and L3 polyadenylation sites.

1994 ◽  
Vol 14 (7) ◽  
pp. 4682-4693 ◽  
Author(s):  
J Prescott ◽  
E Falck-Pedersen
Keyword(s):  
Consensus Sequence ◽  
Transcription Unit ◽  
Upstream Region ◽  
Stable Complex ◽  
Processing Efficiency ◽  
Sequence Element ◽  
Sequence Elements ◽  
Late Transcription ◽  
A Site ◽  
Stable Processing

The adenovirus major late transcription unit is a well-characterized transcription unit which relies heavily on alternative pre-mRNA processing to generate distinct populations of mRNA during the early and late stages of viral infection. In the early stage of infection, two major late transcription unit mRNA transcripts are generated through use of the first (L1) of five available poly(A) sites (L1 through L5). This contrasts with the late stage of infection when as many as 45 distinct mRNAs are generated, with each of the five poly(A) sites being used. In previous work characterizing elements involved in alternative poly(A) site use, we showed that the L1 poly(A) site is processed less efficiently than the L3 poly(A) site both in vitro and in vivo. Because of the dramatic difference in processing efficiency and the role processing efficiency plays in production of steady-state levels of mRNA, we have identified the sequence elements that account for the differences in L1 and L3 poly(A) site processing efficiency. We have found that the element most likely to be responsible for poly(A) site strength, the GU/U-rich downstream element, plays a minor role in the different processing efficiencies observed for the L1 and L3 poly(A) sites. The sequence element most responsible for inefficient processing of the L1 poly(A) site includes the L1 AAUAAA consensus sequence and those sequences which immediately surround the consensus hexanucleotide. This region of the L1 poly(A) site contributes to an inability to form a stable processing complex with the downstream GU/U-rich element. In contrast to the L1 element, the L3 poly(A) site has a consensus hexanucleotide and surrounding sequences which can form a stable processing complex in cooperation with the downstream GU/U-rich element. The L3 poly(A) site is also aided by the presence of sequences upstream of the hexanucleotide which facilitate processing efficiency. The sequence UUCUUUUU, present in the L3 upstream region, is shown to enhance processing efficiency as well as stable complex formation (shown by increased binding of the 64-kDa cleavage stimulatory factor subunit) and acts as a binding site for heterogeneous nuclear ribonucleoprotein C proteins.

Sequence elements upstream of the 3' cleavage site confer substrate strength to the adenovirus L1 and L3 polyadenylation sites

1994 ◽  
Vol 14 (7) ◽  
pp. 4682-4693
Author(s):  
J Prescott ◽  
E Falck-Pedersen
Keyword(s):  
Consensus Sequence ◽  
Transcription Unit ◽  
Upstream Region ◽  
Stable Complex ◽  
Processing Efficiency ◽  
Sequence Element ◽  
Sequence Elements ◽  
Late Transcription ◽  
A Site ◽  
Stable Processing

The adenovirus major late transcription unit is a well-characterized transcription unit which relies heavily on alternative pre-mRNA processing to generate distinct populations of mRNA during the early and late stages of viral infection. In the early stage of infection, two major late transcription unit mRNA transcripts are generated through use of the first (L1) of five available poly(A) sites (L1 through L5). This contrasts with the late stage of infection when as many as 45 distinct mRNAs are generated, with each of the five poly(A) sites being used. In previous work characterizing elements involved in alternative poly(A) site use, we showed that the L1 poly(A) site is processed less efficiently than the L3 poly(A) site both in vitro and in vivo. Because of the dramatic difference in processing efficiency and the role processing efficiency plays in production of steady-state levels of mRNA, we have identified the sequence elements that account for the differences in L1 and L3 poly(A) site processing efficiency. We have found that the element most likely to be responsible for poly(A) site strength, the GU/U-rich downstream element, plays a minor role in the different processing efficiencies observed for the L1 and L3 poly(A) sites. The sequence element most responsible for inefficient processing of the L1 poly(A) site includes the L1 AAUAAA consensus sequence and those sequences which immediately surround the consensus hexanucleotide. This region of the L1 poly(A) site contributes to an inability to form a stable processing complex with the downstream GU/U-rich element. In contrast to the L1 element, the L3 poly(A) site has a consensus hexanucleotide and surrounding sequences which can form a stable processing complex in cooperation with the downstream GU/U-rich element. The L3 poly(A) site is also aided by the presence of sequences upstream of the hexanucleotide which facilitate processing efficiency. The sequence UUCUUUUU, present in the L3 upstream region, is shown to enhance processing efficiency as well as stable complex formation (shown by increased binding of the 64-kDa cleavage stimulatory factor subunit) and acts as a binding site for heterogeneous nuclear ribonucleoprotein C proteins.

3' RNA processing efficiency plays a primary role in generating termination-competent RNA polymerase II elongation complexes

1993 ◽  
Vol 13 (6) ◽  
pp. 3472-3480
Author(s):  
G Edwalds-Gilbert ◽  
J Prescott ◽  
E Falck-Pedersen
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Processing ◽  
Recombinant Adenovirus ◽  
Transcription Termination ◽  
Transcription Unit ◽  
Mrna Processing ◽  
Primary Role ◽  
Processing Efficiency ◽  
A Site

In several mammalian transcription units, a transcription termination mechanism in which efficient termination is dependent on the presence of an intact 3' RNA processing site has been identified. The mouse beta maj-globin transcription unit is one such example, in which an intact poly(A) site is required for efficient transcription termination. It is now evident that 3' mRNA processing sites are not always processed with the same efficiency. In this study, we characterized several pre-mRNAs as substrates for the 3' mRNA processing reaction of cleavage and polyadenylation. We then determined whether poly(A) sites which vary in processing efficiency support a poly(A) site-dependent termination event. The level of processing efficiency was determined in vitro by assays measuring the efficiency of the pre-mRNA cleavage event and in vivo by the level of poly(A) site-dependent mRNA and gene product expression generated in transient transfection assays. The beta maj globin pre-mRNA is very efficiently processed. This efficient processing correlates with its function in termination assays using recombinant adenovirus termination vectors in nuclear run-on assays. When the beta maj globin poly(A) site was replaced by the L1 poly(A) site of the adenovirus major late transcription unit (Ad-ml), which is a poor processing substrate, termination efficiency decreased dramatically. When the beta maj globin poly(A) site was replaced by the Ad-ml L3 poly(A) site, which is 10- to 20-fold more efficiently processed than the Ad-ml L1 poly(A) site, termination efficiency remained high. Termination is therefore dependent on the yield of the processing event. We then tested chimeric poly(A) sites containing the L3 core AAUAAA but varied downstream GU-rich elements. The change in downstream GU-rich elements affected processing efficiency in a manner which correlated with termination efficiency. These experiments provide evidence that the efficiency of 3' processing complex formation is directly correlated to the efficiency of RNA polymerase II termination at the 3' end of a mammalian transcription unit.

scholarly journals 3' RNA processing efficiency plays a primary role in generating termination-competent RNA polymerase II elongation complexes.

1993 ◽  
Vol 13 (6) ◽  
pp. 3472-3480 ◽  
Author(s):  
G Edwalds-Gilbert ◽  
J Prescott ◽  
E Falck-Pedersen
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Rna Processing ◽  
Recombinant Adenovirus ◽  
Transcription Termination ◽  
Transcription Unit ◽  
Mrna Processing ◽  
Primary Role ◽  
Processing Efficiency ◽  
A Site

In several mammalian transcription units, a transcription termination mechanism in which efficient termination is dependent on the presence of an intact 3' RNA processing site has been identified. The mouse beta maj-globin transcription unit is one such example, in which an intact poly(A) site is required for efficient transcription termination. It is now evident that 3' mRNA processing sites are not always processed with the same efficiency. In this study, we characterized several pre-mRNAs as substrates for the 3' mRNA processing reaction of cleavage and polyadenylation. We then determined whether poly(A) sites which vary in processing efficiency support a poly(A) site-dependent termination event. The level of processing efficiency was determined in vitro by assays measuring the efficiency of the pre-mRNA cleavage event and in vivo by the level of poly(A) site-dependent mRNA and gene product expression generated in transient transfection assays. The beta maj globin pre-mRNA is very efficiently processed. This efficient processing correlates with its function in termination assays using recombinant adenovirus termination vectors in nuclear run-on assays. When the beta maj globin poly(A) site was replaced by the L1 poly(A) site of the adenovirus major late transcription unit (Ad-ml), which is a poor processing substrate, termination efficiency decreased dramatically. When the beta maj globin poly(A) site was replaced by the Ad-ml L3 poly(A) site, which is 10- to 20-fold more efficiently processed than the Ad-ml L1 poly(A) site, termination efficiency remained high. Termination is therefore dependent on the yield of the processing event. We then tested chimeric poly(A) sites containing the L3 core AAUAAA but varied downstream GU-rich elements. The change in downstream GU-rich elements affected processing efficiency in a manner which correlated with termination efficiency. These experiments provide evidence that the efficiency of 3' processing complex formation is directly correlated to the efficiency of RNA polymerase II termination at the 3' end of a mammalian transcription unit.

scholarly journals Independent glucocorticoid induction and repression of two contiguous responsive genes.

1989 ◽  
Vol 9 (7) ◽  
pp. 3127-3131 ◽  
Author(s):  
J Charron ◽  
H Richard-Foy ◽  
D S Berard ◽  
G L Hager ◽  
J Drouin
Keyword(s):  
Hormonal Regulation ◽  
Inducible Promoter ◽  
Regulatory Elements ◽  
Transcription Unit ◽  
Regulation Of Transcription ◽  
Bovine Papillomavirus ◽  
Sequence Elements ◽  
Tumor Virus ◽  
Inducible Genes

Specific DNA sequence elements which contain binding sites for the glucocorticoid receptor mediate the action of glucocorticoid hormones on gene transcription. In glucocorticoid-inducible genes, these glucocorticoid-responsive elements behave as hormone-inducible enhancers of transcription. We have taken advantage of the bovine papillomavirus (BPV) system to test the stringency of glucocorticoid regulation of transcription. BPV episomes were constructed to contain two hormone-regulated transcription units in close proximity; one transcription unit is under control of a glucocorticoid-inducible promoter (mouse mammary tumor virus) while the other is under control of a glucocorticoid-inhibited promoter (pro-opiomelanocortin). Glucocorticoids independently regulated transcription of the two physically linked transcription units, irrespective of their relative orientation and of their proximity on the BPV episomes. This result contrasts with the so-called position-independent activity of enhancers and suggests that the multicomponent organization of eucaryotic promoters restricts the action of hormone-responsive regulatory elements to a specific transcription unit, thus accounting for the stringency of hormonal regulation observed in vivo.

Independent glucocorticoid induction and repression of two contiguous responsive genes

1989 ◽  
Vol 9 (7) ◽  
pp. 3127-3131
Author(s):  
J Charron ◽  
H Richard-Foy ◽  
D S Berard ◽  
G L Hager ◽  
J Drouin
Keyword(s):  
Hormonal Regulation ◽  
Inducible Promoter ◽  
Regulatory Elements ◽  
Transcription Unit ◽  
Regulation Of Transcription ◽  
Bovine Papillomavirus ◽  
Sequence Elements ◽  
Tumor Virus ◽  
Inducible Genes

Specific DNA sequence elements which contain binding sites for the glucocorticoid receptor mediate the action of glucocorticoid hormones on gene transcription. In glucocorticoid-inducible genes, these glucocorticoid-responsive elements behave as hormone-inducible enhancers of transcription. We have taken advantage of the bovine papillomavirus (BPV) system to test the stringency of glucocorticoid regulation of transcription. BPV episomes were constructed to contain two hormone-regulated transcription units in close proximity; one transcription unit is under control of a glucocorticoid-inducible promoter (mouse mammary tumor virus) while the other is under control of a glucocorticoid-inhibited promoter (pro-opiomelanocortin). Glucocorticoids independently regulated transcription of the two physically linked transcription units, irrespective of their relative orientation and of their proximity on the BPV episomes. This result contrasts with the so-called position-independent activity of enhancers and suggests that the multicomponent organization of eucaryotic promoters restricts the action of hormone-responsive regulatory elements to a specific transcription unit, thus accounting for the stringency of hormonal regulation observed in vivo.

scholarly journals Polyadenylation in Rice Tungro Bacilliform Virus:cis-Acting Signals and Regulation

2001 ◽  
Vol 75 (9) ◽  
pp. 4184-4194 ◽  
Author(s):  
Helen M. Rothnie ◽  
Gang Chen ◽  
Johannes Fütterer ◽  
Thomas Hohn
Keyword(s):  
Viral Genome ◽  
Cleavage Site ◽  
Expression System ◽  
Nicotiana Plumbaginifolia ◽  
Transcription Unit ◽  
Cauliflower Mosaic Virus ◽  
Rice Tungro Bacilliform Virus ◽  
Rich Region ◽  
Processing Efficiency ◽  
A Site

ABSTRACT The polyadenylation signal of rice tungro bacilliform virus (RTBV) was characterized by mutational and deletion analysis. Thecis-acting signals required to direct polyadenylation conformed to what is known for plant poly(A) signals in general and were very similar to those of the related cauliflower mosaic virus. Processing was directed by a canonical AAUAAA poly(A) signal, an upstream UG-rich region considerably enhanced processing efficiency, and sequences downstream of the cleavage site were not required. When present at the end of a transcription unit, thecis-acting signals for 3′-end processing were highly efficient in both monocot (rice) and dicot (Nicotiana plumbaginifolia) protoplasts. In a promoter-proximal position, as in the viral genome, the signal was also efficiently processed in rice protoplasts, giving rise to an abundant “short-stop” (SS-) RNA. The proportion of SS-RNA was considerably lower in N. plumbaginifolia protoplasts. In infected plants, SS-RNA was hardly detectable, suggesting either that SS-RNA is unstable in infected plants or that read-through of the promoter-proximal poly(A) site is very efficient. SS-RNA is readily detectable in transgenic rice plants (A. Klöti, C. Henrich, S. Bieri, X. He, G. Chen, P. K. Burkhardt, J. Wünn, P. Lucca, T. Hohn, I. Potrylus, and J. Fütterer, 1999. Plant Mol. Biol. 40:249–266), thus the absence of SS-RNA in infected plants can be attributed to poly(A) site bypass in the viral context to ensure production of the full-length pregenomic viral RNA. RTBV poly(A) site suppression thus depends both on context and the expression system; our results suggest that the circular viral minichromosome directs assembly of a transcription-processing complex with specific properties to effect read-through of the promoter-proximal poly(A) signal.

A Novel Ty1-Mediated Fragmentation Method for Native and Artificial Yeast Chromosomes Reveals That the Mouse Steel Gene is a Hotspot for Ty1 Integration

Genetics ◽  
1996 ◽  
Vol 143 (2) ◽  
pp. 673-683
Author(s):  
Jacob Z Dalgaard ◽  
Mukti Banerjee ◽  
M Joan Curcio
Keyword(s):  
Transcription Unit ◽  
Yeast Genome ◽  
Physical Analysis ◽  
Single Chromosome ◽  
Artificial Chromosomes ◽  
Unique Site ◽  
High Fraction ◽  
A Site ◽  
Yeast Artificial Chromosomes ◽  
Random Insertion

Abstract We have developed a powerful new tool for the physical analysis of genomes called Ty1-mediated chromosomal fragmentation and have used the method to map 24 retrotransposon insertions into two different mousederived yeast artificial chromosomes (YACs). Expression of a plasmid-encoded GAL1:Ty1 fusion element marked with the retrotransposition indicator gene, ade2AI, resulted in a high fraction of cells that sustained a single Ty1 insertion marked with ADE2. Strains in which Ty1ADE2 inserted into aYAC were identified by cosegregation of the ADE2 gene with the URA3-marked YAC. Ty1ADE2 elements also carried a site for the endonuclease I-DmoI, which we demonstrate is not present anywhere in the yeast genome. Consequently, I-DmoI cleaved a single chromosome or YAC at the unique site of Ty1ADE2 insertion, allowing rapid mapping of integration events. Our analyses showed that the frequency of Ty1ADE2 integration into YACs is equivalent to or higher than that expected based on random insertion. Remarkably, the 50-kb transcription unit of the mouse Steel locus was shown to be a highly significant hotspot for Ty1 integration. The accessibility of mammalian transcription units to Ty1 insertion stands in contrast to that of yeast transcription units.

scholarly journals Genomic structure of murine methylmalonyl-CoA mutase: evidence for genetic and epigenetic mechanisms determining enzyme activity

1993 ◽  
Vol 296 (3) ◽  
pp. 663-670 ◽  
Author(s):  
M F Wilkemeyer ◽  
E R Andrews ◽  
F D Ledley
Keyword(s):  
Enzyme Activity ◽  
Steady State ◽  
Transcription Initiation ◽  
Cultured Cells ◽  
Genomic Structure ◽  
Genetic Regulation ◽  
Regulatory Elements ◽  
Transcription Unit ◽  
Mrna Levels ◽  
Sequence Motifs

Methylmalonyl-CoA mutase (MCM) is a nuclear-encoded mitochondrial matrix enzyme. We have reported characterization of murine MCM and cloning of a murine MCM cDNA and now describe the murine Mut locus, its promoter and evidence for tissue-specific variation in MCM mRNA, enzyme and holo-enzyme levels. The Mut locus spans 30 kb and contains 13 exons constituting a unique transcription unit. A B1 repeat element was found in the 3′ untranslated region (exon 13). The transcription initiation site was identified and upstream sequences were shown to direct expression of a reporter gene in cultured cells. The promoter contains sequence motifs characteristic of: (1) TATA-less housekeeping promoters; (2) enhancer elements purportedly involved in co-ordinating expression of nuclear-encoded mitochondrial proteins; and (3) regulatory elements including CCAAT boxes, cyclic AMP-response elements and potential AP-2-binding sites. Northern blots demonstrate a greater than 10-fold variation in steady-state mRNA levels, which correlate with tissue levels of enzyme activity. However, the ratio of holoenzyme to total enzyme varies among different tissues, and there is no correlation between steady-state mRNA levels and holoenzyme activity. These results suggest that, although there may be regulation of MCM activity at the level of mRNA, the significance of genetic regulation is unclear owning to the presence of epigenetic regulation of holoenzyme formation.

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