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N J Proudfoot

Publications and source records attributed to N J Proudfoot.

At least 19 recordsLinked to original sources
PubMed2002

Definition of transcriptional promoters in the human beta globin locus control region.

Our previous studies on the human beta globin gene cluster revealed the presence of intergenic transcripts throughout the locus, and demonstrated that transcription of the locus control region (LCR) initiates within an ERV9 endogenous retroviral long-terminal repeat (LTR) upstream of DNase I hypersensitive site 5. We show, using a combination of assays, that there are additional sites of transcription initiation within the LCR at hypersensitive sites 2 and 3. We have defined sites of transcription initiation, which occurs at discrete positions in a direction towards the globin genes. In addition, we show that mutation of specific transcription factor binding sites within HS2 leads to a reduction in transcription levels from within this site. We propose that these initiation events within the LCR can account for the observed orientation dependence of LCR function, and contribute to the open chromatin configuration of the beta globin locus. In addition, transcription from within the LCR hypersensitive sites could compensate for the absence of the ERV9 LTR in many transgenic mice lines, which nevertheless regulate their globin clusters correctly.

Binding Sites↗
PubMed2001

Multiple transcript cleavage precedes polymerase release in termination by RNA polymerase II.

The requirement of poly(A) signals to elicit transcription termination of RNA polymerase II (pol II) is firmly established. However, little else is known about the actual process of pol II transcription termination. Evidence presented in this paper, based on analysis of nascent transcripts of the human beta- and epsilon-globin genes, demonstrates that pol II transcription termination involves two distinct and temporally separate events. The first event, termed pretermination cleavage (PTC), is mediated by sequence tracts located downstream of the poly(A) site which appear to promote heterogeneous cleavage of the nascent transcript. The second event, in which pol II disengages from the DNA template, requires that polymerase has transcribed both a PTC sequence tract and a functional poly(A) site.

3' Untranslated Regions↗
PubMed2001

Cleavage/polyadenylation factor IA associates with the carboxyl-terminal domain of RNA polymerase II in Saccharomyces cerevisiae.

The carboxyl-terminal domain (CTD) of the largest subunit of RNA polymerase II plays an important role in transcription and processing of the nascent transcript by interacting with both transcription and RNA processing factors. We show here that the cleavage/polyadenylation factor IA of Saccharomyces cerevisiae directly contacts CTD. First by affinity chromatography experiments with yeast extracts we demonstrate that the Rna15p, Rna14p, and Pcf11p subunits of this complex are associated with phosphorylated CTD. This interaction is confirmed for Rna15p by yeast two-hybrid analysis. Second, Pcf11p, but not Rna15p, is shown to directly contact phosphorylated CTD based on in vitro binding studies with recombinant proteins. These findings establish a direct interaction of cleavage/polyadenylation factor IA with the CTD. Furthermore, a quantitative analysis of transcription run-on performed on temperature-sensitive mutant strains reveals that the lack of either functional Rna14p or Pcf11p affects transcription termination more severely than the absence of a functional Rna15p. Moreover, these data reinforce the concept that CTD phosphorylation acts as a regulatory mechanism in the maturation of the primary transcript.

Amino Acid Sequence↗
PubMed2001

The retroviruses human immunodeficiency virus type 1 and Moloney murine leukemia virus adopt radically different strategies to regulate promoter-proximal polyadenylation.

Maximal gene expression in retroviruses requires that polyadenylation in the 5' long terminal repeat (LTR) is suppressed. In human immunodeficiency virus type 1 (HIV-1) the promoter-proximal poly(A) site is blocked by interaction of U1 snRNP with the closely positioned major splice donor site (MSD) 200 nucleotides downstream. Here we investigated whether the same mechanism applies to down-regulate 5' LTR polyadenylation in Moloney murine leukemia virus (MoMLV). Although the same molecular architecture is present in both viruses, the MoMLV poly(A) signal in the 5' LTR is active whether or not the MSD is mutated. This surprising difference between the two retroviruses is not due to their actual poly(A) signals or MSD sequences, since exchange of either element between the two viral sequences does not alter their ability to regulate 5' LTR poly(A) site use. Instead we demonstrate that sequence between the cap and AAUAAA is required for MSD-dependent poly(A) regulation in HIV-1, indicating a key role for this part of the LTR in poly(A) site suppression. We also show that the MoMLV poly(A) signal is an intrinsically weak RNA-processing signal. This suggests that in the absence of a poly(A) site suppression mechanism, MoMLV is forced to use a weak poly(A) signal.

3T3 Cells↗
PubMed2001

Intergenic transcription in the human beta-globin gene cluster.

Our previous studies on nascent transcription across the human beta-globin gene cluster revealed the presence of intergenic transcripts in addition to the expected genic transcripts. We now show that transcription into the beta-globin locus control region (LCR) begins within an ERV9 endogenous retroviral long terminal repeat upstream of DNase I hypersensitive site 5. However, in a transgenic mouse, which has the human beta-globin LCR but lacks the ERV9 LTR, transcription begins upstream of the transgenic locus. We postulate that in this transgenic mouse nearby endogenous mouse promoters are activated by the LCR. Intergenic transcription is also detected across the whole transgenic globin gene locus independently of the stage of erythroid development. Intergenic transcription in the beta-globin cluster is erythroid specific; however, it can be induced in nonerythroid cells by several means: by transinduction with a plasmid transcribing part of the cluster, by exogenous addition of transcription factors, and by treatment with the histone deacetylase inhibitor trichostatin A.

5' Untranslated Regions↗
PubMed2000

Transcriptional termination and coupled polyadenylation in vitro.

Using a coupled, in vitro transcription and polyadenylation system we have investigated the molecular mechanism of transcriptional termination by RNA polymerase II (PolII). We showed previously that specific G-rich sequences pause transcription and then activate polyadenylation. We show that physiological pause sites activate polyadenylation in our in vitro system. We also investigate the mechanism of PolII transcriptional termination, and show that these transcripts are either directly released from the transcription complex or are 3' end processed while still attached to the complex. We also show that 3' product (generated by cleavage/polyadenylation) remains associated with the transcription complex, but is rapidly degraded on it.

Complement C2↗
PubMed2000

Stem-loop 1 of the U1 snRNP plays a critical role in the suppression of HIV-1 polyadenylation.

The inactivity or occlusion of the HIV-1 poly(A) signal when in the 5' long terminal repeat (LTR) has been mechanistically investigated. First we show that neither the homologous HIV-1 promoter nor the close proximity of this RNA processing signal to the transcript initiation site is required for the occlusion effect. Instead we demonstrate that the major splice donor (MSD) site positioned about 200 bp downstream maintains the poly(A) site in an inactive state. Although mutation of MSD results in activation of the 5' LTR poly(A) signal, this effect can be suppressed by targeting U1 snRNAs near to the mutated MSD by base pairing. We show that hybrid U7-U1 snRNAs can also suppress the poly(A) signal and that this suppression is dependent on the U1 stem-loop 1. In particular the binding site for the U1 snRNP protein 70K that binds to the loop structure of stem-loop 1 is associated with poly(A) site occlusion. These experiments were carried out with an HIV-1 proviral construct and as such emphasize the physiological importance of this splice donor-poly(A) site interaction.

Base Sequence↗
PubMed2000

Recruitment of a basal polyadenylation factor by the upstream sequence element of the human lamin B2 polyadenylation signal.

We have investigated how the upstream sequence element (USE) of the lamin B2 poly(A) signal mediates efficient 3'-end formation. In vitro analysis demonstrates that this USE increases both the efficiency of 3'-end cleavage and the processivity of poly(A) addition. Cross-linking using selectively labeled synthetic RNAs confirms that cleavage stimulation factor interacts with the sequences downstream of the cleavage site, while electrophoresis mobility shift assays demonstrate that the USE directly stabilizes the binding of the cleavage and polyadenylation specificity factor to the poly(A) signal. Thus in common with other poly(A) signals, the lamin B2 USE directly enhances the binding of basal poly(A) factors. In addition, a novel 55-kDa protein binds to the USE and the core poly(A) signal and appears to inhibit cleavage. The binding activity of this factor appears to change during the cell cycle, being greatest in S phase, when the lamin B2 gene is transcribed.

3' Untranslated Regions↗
PubMed1999

Transcription of the human U2 snRNA genes continues beyond the 3' box in vivo.

The 3' box of the human class II snRNA genes is required for proper 3' processing of transcripts, but how it functions is unclear. Several lines of evidence suggest that termination of transcription occurs at the 3' box and the terminated transcript is then a substrate for processing. However, using nuclear run-on analysis of endogenous genes, we demonstrate that transcription continues for at least 250 nucleotides beyond the 3' box of the U2 genes. Although in vivo footprinting analysis of both the U1 and U2 genes detects no protein-DNA contacts directly over the 3' box, a series of G residues immediately downstream from the 3' box of the U1 gene are clearly protected from methylation by dimethylsulfate. In conjunction with the 3' box of the U1 gene, this in vivo footprinted region causes termination of transcription of transiently transfected U2 constructs, whereas a 3' box alone does not. Taken together, these results indicate that the 3' box is not an efficient transcriptional terminator but may act as a processing element that is functional in the nascent RNA.

Base Sequence↗
PubMed1999

Specific transcriptional pausing activates polyadenylation in a coupled in vitro system.

We have developed a coupled in vitro transcription-polyadenylation system to investigate RNA polymerase II (Pol II) termination, which depends on active polyadenylation of the nascent RNA. Specific G-rich sequences originally identified as binding sites for the transcription factor MAZ both pause Pol II and activate polyadenylation of an upstream poly(A) signal. They do not affect polyadenylation efficiency in an uncoupled cleavage assay. In contrast, pausing of Pol II elongation induced by a high-affinity DNA-binding protein does not activate polyadenylation, indicating that G-rich MAZ sequences have a specific effect on polyadenylation. They also promote intrinsic pausing of purified Pol II, indicating a general role in the modulation of cotranscriptional RNA processing events.

Cell Nucleus↗
PubMed1999

Terminal exon definition occurs cotranscriptionally and promotes termination of RNA polymerase II.

Analysis of nascent transcription from the human epsilon- and beta-globin genes shows that transcriptional termination occurs within 1.5 kb of the poly(A) site and is dependent on the presence of functional poly(A) signals. Even so, transcripts that have not been cleaved at the poly(A) site are detected up to the termination region, suggesting that there is a kinetic lag between transcription over the poly(A) signal and its effect on transcriptional termination. Surprisingly, mutation of the splice acceptor (SA) of the beta-globin IVS2 also abolishes transcriptional termination. Our results emphasize the interconnection of transcription and RNA processing by showing that the enhancement of 3' end processing by the terminal splice acceptor occurs cotranscriptionally.

Cell Nucleus↗
PubMed1999

EM visualization of transcription by RNA polymerase II: downstream termination requires a poly(A) signal but not transcript cleavage.

We have used EM visualization of active genes on plasmid vectors in Xenopus oocyte nuclei to investigate the relationship between poly(A) signals and RNA polymerase II transcription termination. Although a functional poly(A) signal is required for efficient termination, cotranscriptional RNA cleavage at the poly(A) site is not. Furthermore, the phenomena of termination and cotranscriptional RNA cleavage can be uncoupled, and the efficiency of both varies independently on different copies of the same plasmid template in the same oocyte nucleus. The combined observations are consistent with a scenario in which there is template-specific addition to Pol II (presumably at the promoter) of elongation and/or RNA processing factors, which are altered upon passage through a poly(A) signal, resulting in termination and, in some cases, cotranscriptional RNA cleavage.

Animals↗
PubMed1999

Definition of transcriptional pause elements in fission yeast.

Downstream elements (DSEs) with transcriptional pausing activity play an important role in transcription termination of RNA polymerase II. We have defined two such DSEs in Schizosaccharomyces pombe, one for the ura4 gene and a new one in the 3'-end region of the nmt2 gene. Although these DSEs do not have sequence homology, both are orientation specific and are composed of multiple and redundant sequence elements that work together to achieve full pausing activity. Previous studies on the nmt1 and nmt2 genes revealed that transcription extends several kilobases past the genes' poly(A) sites. We show that the insertion of either DSE immediately downstream of the nmt1 poly(A) site induces more immediate termination. nmt2 termination efficiency can be increased by moving the DSE closer to the poly(A) site. These results suggest that DSEs may be a common feature in yeast genes.

Base Sequence↗
PubMed1998

Co-transcriptional commitment to alternative splice site selection.

Production of mRNA in eukaryotic cells involves not only transcription but also various processing reactions such as splicing. Recent experiments have indicated that there are direct physical connections between components of the transcription and processing machinery, supporting previous suggestions that pre-mRNA splicing occurs co-transcriptionally. Here we have used a novel functional approach to demonstrate co-transcriptional regulation of alternative splicing. Exon 3 of the alpha-tropomyosin gene is specifically repressed in smooth muscle cells. By delaying synthesis of an essential downstream inhibitory element, we show that the decision to splice or repress exon 3 occurs during a limited window of opportunity following transcription, indicating that splice site selection proceeds rapidly after transcription.

Alternative Splicing↗
PubMed1998

Poly(A) signals control both transcriptional termination and initiation between the tandem GAL10 and GAL7 genes of Saccharomyces cerevisiae.

We have investigated transcriptional interactions between the GAL10 and GAL7 genes of Saccharomyces cerevisiae. Both genes are part of the galactose (GAL) gene cluster which is transcriptionally activated to high levels in the presence of galactose. Since GAL7 is positioned downstream of GAL10 and both genes are expressed co-ordinately at high levels, the possibility that GAL10 transcription influences GAL7 was analysed. Using transcriptional run-on assays, we show that high levels of polymerase are found in the 600 bp GAL10-7 intergenic region that accumulate over the GAL7 promoter. Furthermore, GAL7 transcription is enhanced when the GAL10 upstream activating sequence (UASG) is deleted, indicating that interference between GAL10 and GAL7 is likely to occur in the chromosomal locus. Deletions in the GAL10 poly(A) signal result in complete inactivation of the GAL7 promoter and cause a dramatic increase in bi-cistronic GAL10-7 mRNA, predominantly utilizing the downstream, GAL7 poly(A) site. These data demonstrate a pivotal role for the GAL10 poly(A) site in allowing the simultaneous expression of GAL10 and GAL7. In effect, this RNA processing signal has a direct influence on both transcriptional termination and initiation.

DNA-Directed RNA Polymerases↗
PubMed1998

The upstream sequence element of the C2 complement poly(A) signal activates mRNA 3' end formation by two distinct mechanisms.

The poly(A) signal of the C2 complement gene is unusual in that it possesses an upstream sequence element (USE) required for full activity in vivo. We describe here in vitro experiments demonstrating that this USE enhances both the cleavage and poly(A) addition reactions. We also show that the C2 USE can be cross-linked efficiently to a 55-kD protein that we identify as the polypyrimidine tract-binding protein (PTB), implicated previously in modulation of pre-mRNA splicing. Mutation of the PTB-binding site significantly reduces the efficiency of the C2 poly(A) site both in vivo and in vitro. Furthermore, addition of PTB to reconstituted processing reactions enhances cleavage at the C2 poly(A) site, indicating that PTB has a direct role in recognition of this signal. The C2 USE, however, also increases the affinity of general polyadenylation factors independently for the C2 poly(A) signal as detected by enhanced binding of cleavage-stimulaton factor (CstF). Strikingly, this leads to a novel CstF-dependant enhancement of the poly(A) synthesis phase of the reaction. These studies both emphasize the interconnection between splicing and polyadenylation and indicate an unexpected flexibility in the organization of mammalian poly(A) sites.

Binding Sites↗
PubMed1998

Nascent transcription from the nmt1 and nmt2 genes of Schizosaccharomyces pombe overlaps neighbouring genes.

We have determined the extent of the primary transcription unit for the two highly expressed genes nmt1 and nmt2 of Schizosaccharomyces pombe. Transcription run-on analysis in permeabilized yeast cells was employed to map polymerase density across the 3'-flanking region of these two genes. Surprisingly, polymerases were detected 4.3 kb beyond the nmt1 polyadenylation [poly(A)] site and 2.4 kb beyond the nmt2 poly(A) site, which in each case have transcribed through an entire convergent downstream transcription unit. However, the steady-state levels of both downstream genes were unaffected by the high level of nmt1 or nmt2 nascent transcription. Analysis of nmt1 and nmt2 RNA 3' end formation signals indicates that efficient termination of transcription requires not only a poly(A) signal but also additional pause elements. The absence of such pause elements close to the poly(A) sites of these genes may account for their extended nascent transcripts.

Base Sequence↗