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.

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.

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.

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.

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.

scholarly journals 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 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.

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.

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