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.

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.

Alternative poly(A) site utilization during adenovirus infection coincides with a decrease in the activity of a poly(A) site processing factor

1993 ◽  
Vol 13 (4) ◽  
pp. 2411-2419
Author(s):  
K P Mann ◽  
E A Weiss ◽  
J R Nevins
Keyword(s):  
Early Phase ◽  
Late Phase ◽  
Transcription Unit ◽  
Adenovirus Infection ◽  
Processing Factor ◽  
Proximal Half ◽  
Rate Determining Step ◽  
Frequency Of Use ◽  
Late Transcription ◽  
A Site

The recognition and processing of a pre-mRNA to create a poly(A) addition site, a necessary step in mRNA biogenesis, can also be a regulatory event in instances in which the frequency of use of a poly(A) site varies. One such case is found during the course of an adenovirus infection. Five poly(A) sites are utilized within the major late transcription unit to produce more than 20 distinct mRNAs during the late phase of infection. The proximal half of the major late transcription unit is also expressed during the early phase of a viral infection. During this early phase of expression, the L1 poly(A) site is used three times more frequently than the L3 poly(A) site. In contrast, the L3 site is used three times more frequently than the L1 site during the late phase of infection. Recent experiments have suggested that the recognition of the poly(A) site GU-rich downstream element by the CF1 processing factor may be a rate-determining step in poly(A) site selection. We demonstrate that the interaction of CF1 with the L1 poly(A) site is less stable than the interaction of CF1 with the L3 poly(A) site. We also find that there is a substantial decrease in the level of CF1 activity when an adenovirus infection proceeds to the late phase. We suggest that this reduction in CF1 activity, coupled with the relative instability of the interaction with the L1 poly(A) site, contributes to the reduced use of the L1 poly(A) site during the late stage of an adenovirus infection.

scholarly journals Different Mechanisms for Pseudouridine Formation in Yeast 5S and 5.8S rRNAs

2008 ◽  
Vol 28 (10) ◽  
pp. 3089-3100 ◽  
Author(s):  
Wayne A. Decatur ◽  
Murray N. Schnare
Keyword(s):  
Large Subunit ◽  
Consensus Sequence ◽  
5S Rrna ◽  
Stem Loop ◽  
Conserved Sequence ◽  
5.8S Rrna ◽  
Sequence Elements ◽  
The Individual ◽  
A Site ◽  
Large Subunit Rrna

ABSTRACT The selection of sites for pseudouridylation in eukaryotic cytoplasmic rRNA occurs by the base pairing of the rRNA with specific guide sequences within the RNA components of box H/ACA small nucleolar ribonucleoproteins (snoRNPs). Forty-four of the 46 pseudouridines (Ψs) in the cytoplasmic rRNA of Saccharomyces cerevisiae have been assigned to guide snoRNAs. Here, we examine the mechanism of Ψ formation in 5S and 5.8S rRNA in which the unassigned Ψs occur. We show that while the formation of the Ψ in 5.8S rRNA is associated with snoRNP activity, the pseudouridylation of 5S rRNA is not. The position of the Ψ in 5.8S rRNA is guided by snoRNA snR43 by using conserved sequence elements that also function to guide pseudouridylation elsewhere in the large-subunit rRNA; an internal stem-loop that is not part of typical yeast snoRNAs also is conserved in snR43. The multisubstrate synthase Pus7 catalyzes the formation of the Ψ in 5S rRNA at a site that conforms to the 7-nucleotide consensus sequence present in other substrates of Pus7. The different mechanisms involved in 5S and 5.8S rRNA pseudouridylation, as well as the multiple specificities of the individual trans factors concerned, suggest possible roles in linking ribosome production to other processes, such as splicing and tRNA synthesis.

scholarly journals Sequences regulating temporal poly(A) site switching in the adenovirus major late transcription unit.

1991 ◽  
Vol 11 (12) ◽  
pp. 5977-5984 ◽  
Author(s):  
J D DeZazzo ◽  
E Falck-Pedersen ◽  
M J Imperiale
Keyword(s):  
Regulatory Element ◽  
Transcription Unit ◽  
Late Infection ◽  
Cis Acting ◽  
Temporal Regulation ◽  
Deletion Mutagenesis ◽  
Late Transcription ◽  
A Site ◽  
Adenovirus Recombinant ◽  
Selection Of

Temporal regulation of poly(A) site choice occurs in an adenovirus recombinant encoding a miniature version of the major late transcription unit with two poly(A) sites, L1 and L3. Using deletion mutagenesis, we have looked directly for cis-acting elements regulating poly(A) site choice in this recombinant. From this work, we draw two main conclusions. First, elements other than the AAUAAA and downstream sequences of the L1 poly(A) site are required for temporal regulation of poly(A) site choice during infection. Second, these regions function in two distinct modes during infection. The two regions enhance selection of the L1 poly(A) site in an additive manner during an early infection, but deletion of either element abolishes the switch in poly(A) site choice during a late infection. This work documents the first example of a regulatory element downstream of a core poly(A) region.

scholarly journals XACT-seq comprehensively defines the promoter-position and promoter-sequence determinants for initial-transcription pausing

2019 ◽  
Author(s):  
Jared T. Winkelman ◽  
Chirangini Pukhrambam ◽  
Irina O. Vvedenskaya ◽  
Yuanchao Zhang ◽  
Deanne M. Taylor ◽  
...  
Keyword(s):  
Active Center ◽  
Transcription Elongation ◽  
Consensus Sequence ◽  
Promoter Sequence ◽  
Sequence Element ◽  
Promoter Sequences ◽  
Nucleotide Addition ◽  
Sequence Elements ◽  
Nucleotide Resolution

AbstractPausing by RNA polymerase (RNAP) during transcription elongation, in which a translocating RNAP uses a “stepping” mechanism, has been studied extensively, but pausing by RNAP during initial transcription, in which a promoter-anchored RNAP uses a “scrunching” mechanism, has not. We report a method that directly defines RNAP-active-center position relative to DNA in vivo with single-nucleotide resolution (XACT-seq; crosslink-between-active-center-and-template sequencing). We apply this method to detect and quantify pausing in initial transcription at 411 (∼4,000,000) promoter sequences in vivo, in Escherichia coli. The results show initial-transcription pausing can occur in each nucleotide addition during initial transcription, particularly the first 4-5 nucleotide additions. The results further show initial-transcription pausing occurs at sequences that resemble the consensus sequence element for transcription-elongation pausing. Our findings define the positional and sequence determinants for initial-transcription pausing and establish initial-transcription pausing is hard-coded by sequence elements similar to those for transcription-elongation pausing.

Splice site choice in a complex transcription unit containing multiple inefficient polyadenylation signals

1991 ◽  
Vol 11 (10) ◽  
pp. 5291-5300
Author(s):  
Y Luo ◽  
G G Carmichael
Keyword(s):  
Splice Site ◽  
Transcription Unit ◽  
Model System ◽  
Late Transcription ◽  
Mouse Cells ◽  
A Site ◽  
L1 And L2 ◽  
The Relationship ◽  
Cytoplasmic Rnas ◽  
Polyadenylation Signals

The relationship between polyadenylation and splicing was investigated in a model system consisting of two tandem but nonidentical polyomavirus late transcription units. This model system exploits the polyomavirus late transcription termination and polyadenylation signals, which are sufficiently weak to allow the production of many multigenome-length primary transcripts with repeating introns, exons, and poly(A) sites. This double-genome construct contains exons of two types, those bordered by 3' and 5' splice sites (L1 and L2) and those bordered by a 3' splice site and a poly(A) site (V1 and V2). The L1 and L2 exons are distinguishable from one another but retain identical flanking RNA processing signals, as is the case for the V1 and V2 exons. Analysis of cytoplasmic RNAs obtained from mouse cells transfected with this construct and its derivatives revealed the following. (i) V1 and V2 exons are often skipped during pre-mRNA processing, while L1 and L2 exons are not skipped. (ii) No messages contain internal, unused polyadenylation signals. (iii) Poly(A) site choice is not required for the selection of an upstream 3' splice site. (iv) When two tandem poly(A) sites are placed downstream of a 3' splice site, the first poly(A) site is chosen almost exclusively, even though transcription can proceed past both sites. (v) Placing a 3' splice site between these two tandem poly(A) sites allows the more distal site to be chosen. These and other available data are most consistent with a model in which terminal exons are produced by the coordinate selection and use of a 3' splice site with the nearest available downstream poly(A) site.

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.

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