scholarly journals Functionally Significant Secondary Structure of the Simian Virus 40 Late Polyadenylation Signal

2000 ◽  
Vol 20 (8) ◽  
pp. 2926-2932 ◽  
Author(s):  
Holly Hans ◽  
James C. Alwine
Keyword(s):  
Secondary Structure ◽  
Rna Processing ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Polyadenylation Signal ◽  
Analysis Techniques ◽  
Downstream Region ◽  
Polyadenylation Signals ◽  
Structural Region

ABSTRACT The structure of the highly efficient simian virus 40 late polyadenylation signal (LPA signal) is more complex than those of most known mammalian polyadenylation signals. It contains efficiency elements both upstream and downstream of the AAUAAA region, and the downstream region contains three defined elements (two U-rich elements and one G-rich element) instead of the single U- or GU-rich element found in most polyadenylation signals. Since many reports have indicated that the secondary structure in RNA may play a significant role in RNA processing, we have used nuclease structure analysis techniques to determine the secondary structure of the LPA signal. We find that the LPA signal has a functionally significant secondary structure. Much of the region upstream of AAUAAA is sensitive to single-strand-specific nucleases. The region downstream of AAUAAA has both double- and single-stranded characteristics. Both U-rich elements are predominately sensitive to the double-strand-specific nuclease RNase V1, while the G-rich element is primarily single stranded. The U-rich element closest to AAUAAA contains four distinct RNase V1-sensitive regions, which we have designated structural region 1 (SR1), SR2, SR3, and SR4. Linker scanning mutants in the downstream region were analyzed both for structure and for function by in vitro cleavage analyses. These data show that the ability of the downstream region, particularly SR3, to form double-stranded structures correlates with efficient in vitro cleavage. We discuss the possibility that secondary structure downstream of the AAUAAA may be important for the functions of polyadenylation signals in general.

scholarly journals Efficiency of utilization of the simian virus 40 late polyadenylation site: effects of upstream sequences.

1989 ◽  
Vol 9 (10) ◽  
pp. 4248-4258 ◽  
Author(s):  
S Carswell ◽  
J C Alwine
Keyword(s):  
Steady State ◽  
Rna Stability ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Polyadenylation Signal ◽  
Polyadenylation Site ◽  
Expression Vectors ◽  
Mrna Levels ◽  
The Difference ◽  
Polyadenylation Signals

The late polyadenylation signal of simian virus 40 functions with greater efficiency than the early polyadenylation signal, in turn affecting steady-state mRNA levels. Two chloramphenicol acetyltransferase (CAT) transient expression vectors, pL-EPA and pL-LPA, that differ only in their polyadenylation signals were constructed by using the early and late polyadenylation signals, respectively. In transfections of Cos, CV-1P, or HeLa cells and subsequent Northern blot analysis of CAT-specific RNA, approximately five times more steady-state CAT mRNA was produced in transfections with pL-LPA than with pL-EPA. The basis for this difference was not related to the specific promoter used or to RNA stability. Overall, the difference in steady-state mRNA levels derived from the two plasmids appeared to be attributable to intrinsic properties of the two polyadenylation signals, resulting in distinctly different cleavage and polyadenylation efficiencies. Additionally, we found that the utilization of the late polyadenylation site was dramatically reduced by deletion of sequences between 48 and 29 nucleotides 5' of the AAUAAA hexanucleotide. This reduction of mRNA levels was shown not to be caused by altered stability of mutant precursor RNAs or mRNAs, suggesting that these upstream sequences constitute an element of the late polyadenylation signal and may cause, at least to some extent, the greater efficiency of utilization of the late polyadenylation site.

scholarly journals Patterns of polyadenylation site selection in gene constructs containing multiple polyadenylation signals.

1988 ◽  
Vol 8 (11) ◽  
pp. 4829-4839 ◽  
Author(s):  
R M Denome ◽  
C N Cole
Keyword(s):  
Simian Virus 40 ◽  
Simian Virus ◽  
Polyadenylation Signal ◽  
Coding Region ◽  
S1 Nuclease ◽  
Cytoplasmic Rna ◽  
Sv40 Origin ◽  
Sv40 Infection ◽  
Simplex Virus ◽  
Polyadenylation Signals

We have constructed a series of plasmids containing multiple polyadenylation signals downstream of the herpes simplex virus type 1 (HSV) thymidine kinase (tk)-coding region. The signals used were from the simian virus 40 (SV40) late gene, the HSV tk gene, and an AATAAA-containing segment of the SV40 early region. This last fragment signals polyadenylation poorly in our constructs and not at all during SV40 infection. All plasmids contained the SV40 origin of replication. Plasmids were transfected into Cos-1 cells; after 48 h, cytoplasmic RNA was isolated and the quantity and 3'-end structure of tk mRNAs was analyzed by using S1 nuclease protection assays. In all constructs, all polyadenylation signals were used. Increasing the number of poly(A) signals 3' to the tk-coding region did not affect the total amount of polyadenylated RNA produced, even with the weakest signal. Increasing the distance between two signals caused an increase in the use of the 5' signal and a decrease in the use of the 3' signal. Changing the distance between the 5' cap and first signal did not affect signal use. Analyses of cytoplasmic mRNA stability, nuclear RNA distribution, and transcription in the polyadenylation signal region indicated that the distribution of tk RNAs ending at different poly(A) sites was the result of poly(A) signal choice, not other aspects of RNA metabolism. Four possible mechanisms of polyadenylation signal recognition are discussed.

Definition of the upstream efficiency element of the simian virus 40 late polyadenylation signal by using in vitro analyses

1992 ◽  
Vol 12 (12) ◽  
pp. 5386-5393 ◽  
Author(s):  
N Schek ◽  
C Cooke ◽  
J C Alwine
Keyword(s):  
Consensus Sequence ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Polyadenylation Signal ◽  
Upstream Region ◽  
Core Element ◽  
The Core ◽  
Core Elements ◽  
Upstream Element

The polyadenylation signal for the late mRNAs of simian virus 40 is known to have sequence elements located both upstream and downstream of the AAUAAA which affect efficiency of utilization of the signal. The upstream efficiency element has been previously characterized by using deletion mutations and transfection analyses. Those studies suggested that the upstream element lies between 13 and 48 nucleotides upstream of the AAUAAA. We have utilized in vitro cleavage and polyadenylation reactions to further define the upstream element. 32P-labeled substrate RNAs were prepared by in vitro transcription from wild-type templates as well as from mutant templates having deletions and linker substitutions in the upstream region. Analysis of these substrates defined the upstream region as sequences between 13 and 51 nucleotides upstream of the AAUAAA, in good agreement with the in vivo results. Within this region, three core elements with the consensus sequence AUUUGURA were identified and were specifically mutated by linker substitution. These core elements were found to contain the active components of the upstream efficiency element. Using substrates with both single and double linker substitution mutations of core elements, we observed that the core elements function in a distance-dependent manner. In mutants containing only one core element, the effect on efficiency increases as the distance between the element and the AAUAAA decreases. In addition, when core elements are present in multiple copies, the effect is additive. The core element consensus sequence, which bears homology to the Sm protein complex-binding site in human U1 RNA, is also found within the upstream elements of the ground squirrel hepatitis B and cauliflower mosaic virus polyadenylation signals (R. Russnak, Nucleic Acids Res. 19:6449-6456, 1991; H. Sanfacon, P. Brodmann, and T. Hohn, Genes Dev. 5:141-149, 1991), suggesting functional conservation of this element between mammals and plants.

scholarly journals An RNA-binding protein specifically interacts with a functionally important domain of the downstream element of the simian virus 40 late polyadenylation signal.

1991 ◽  
Vol 11 (10) ◽  
pp. 5312-5320 ◽  
Author(s):  
Z W Qian ◽  
J Wilusz
Keyword(s):  
Binding Site ◽  
Rna Binding ◽  
Specific Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Polyadenylation Signal ◽  
Band Shift

We have identified an RNA-binding protein which interacts with the downstream element of the simian virus 40 late polyadenylation signal in a sequence-specific manner. A partially purified 50-kDa protein, which we have named DSEF-1, retains RNA-binding specificity as assayed by band shift and UV cross-linking analyses. RNA footprinting assays, using end-labeled RNA ladder fragments in conjunction with native gel electrophoresis, have identified the DSEF-1 binding site as 5'-GGGGGAGGUGUGGG-3'. This 14-base sequence serves as an efficient DSEF-1 binding site when placed within a GEM4 polylinker-derived RNA. Finally, the DSEF-1 binding site restored efficient in vitro 3' end processing to derivatives of the simian virus 40 late polyadenylation signal in which it substituted for the entire downstream region. DSEF-1, therefore, may be a sequence-specific binding factor which regulates the efficiency of polyadenylation site usage.

scholarly journals A uridylate tract mediates efficient heterogeneous nuclear ribonucleoprotein C protein-RNA cross-linking and functionally substitutes for the downstream element of the polyadenylation signal.

1990 ◽  
Vol 10 (12) ◽  
pp. 6397-6407 ◽  
Author(s):  
J Wilusz ◽  
T Shenk
Keyword(s):  
Spatial Relationship ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Polyadenylation Signal ◽  
Cross Linking ◽  
Heterogeneous Nuclear Ribonucleoprotein ◽  
Hnrnp C ◽  
C Proteins ◽  
Nuclear Ribonucleoprotein

Every RNA added to an in vitro polyadenylation extract became stably associated with both the heterogeneous nuclear ribonucleoprotein (hnRNP) A and C proteins, as assayed by immunoprecipitation analysis using specific monoclonal antibodies. UV-cross-linking analysis, however, which assays the specific spatial relationship of certain amino acids and RNA bases, indicated that the hnRNP C proteins, but not the A proteins, were associated with downstream sequences of the simian virus 40 late polyadenylation signal in a sequence-mediated manner. A tract of five consecutive uridylate residues was required for this interaction. The insertion of a five-base U tract into a pGEM4 polylinker-derived transcript was sufficient to direct sequence-specific cross-linking of the C proteins to RNA. Finally, the five-base uridylate tract restored efficient in vitro processing to several independent poly(A) signals in which it substituted for downstream element sequences. The role of the downstream element in polyadenylation efficiency, therefore, may be mediated by sequence-directed alignment or phasing of an hnRNP complex.

Sequences downstream of AAUAAA signals affect pre-mRNA cleavage and polyadenylation in vitro both directly and indirectly

1989 ◽  
Vol 9 (4) ◽  
pp. 1759-1771
Author(s):  
L C Ryner ◽  
Y Takagaki ◽  
J L Manley
Keyword(s):  
Cleavage Site ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Cleavage Reaction ◽  
Mrna Cleavage ◽  
Nuclear Extracts ◽  
Cleavage And Polyadenylation ◽  
Polyadenylation Signals

To investigate the role of sequences lying downstream of the conserved AAUAAA hexanucleotide in pre-mRNA cleavage and polyadenylation, deletions or substitutions were constructed in polyadenylation signals from simian virus 40 and adenovirus, and their effects were assayed in both crude and fractionated HeLa cell nuclear extracts. As expected, these sequences influenced the efficiency of both cleavage and polyadenylation as well as the accuracy of the cleavage reaction. Sequences near or upstream of the actual site of poly(A) addition appeared to specify a unique cleavage site, since their deletion resulted, in some cases, in heterogeneous cleavage. Furthermore, the sequences that allowed the simian virus 40 late pre-RNA to be cleaved preferentially by partially purified cleavage activity were also those at the cleavage site itself. Interestingly, sequences downstream of the cleavage site interacted with factors not directly involved in catalyzing cleavage and polyadenylation, since the effects of deletions were substantially diminished when partially purified components were used in assays. In addition, these sequences contained elements that could affect 3'-end formation both positively and negatively.

scholarly journals Secondary Structure as a Functional Feature in the Downstream Region of Mammalian Polyadenylation Signals

2004 ◽  
Vol 24 (7) ◽  
pp. 2789-2796 ◽  
Author(s):  
Chunxiao Wu ◽  
James C. Alwine
Keyword(s):  
Secondary Structure ◽  
Secondary Structures ◽  
Loop Size ◽  
Alternative Structures ◽  
Cleavage Efficiency ◽  
Downstream Region ◽  
Wide Range ◽  
Functional Features ◽  
Polyadenylation Signals

ABSTRACT Secondary structure within the downstream region of mammalian polyadenylation signals has been proposed to perform important functions. The simian virus 40 late polyadenylation signal (SVLPA) forms alternate secondary structures in equilibrium. Their formation correlates with cleavage-polyadenylation efficiency (H. Hans and J. C. Alwine, Mol. Cell. Biol. 20:2926-2932, 2000; M. I. Zarudnaya, I. M. Kolomiets, A. L. Potyahaylo, and D. M. Hovorun, Nucleic Acids Res. 3:1375-1386, 2003), and oligonucleotides that disrupt the secondary structure inhibit in vitro cleavage. To define the important features of downstream secondary structure, we first minimized the SVLPA by deletion, forming a downstream region with fewer, and more stable, stem-loop structures. Specific mutagenesis showed that both stem stability and loop size are important functional features of the downstream region. Stabilization of the stem, thus minimizing alternative structures, decreased cleavage efficiency both in vitro and in vivo. This was most deleterious when the stem was stabilized at the base of the loop, constraining loop size by inhibiting breathing of the stem. The significance of loop size was supported by mutants that showed increased cleavage efficiency with increased loop size and vice versa. A loop of at least 12 nucleotides promoted cleavage; U richness in the loop also promoted cleavage and was particularly important when the stem was stabilized. A mutation designed to eliminate downstream secondary structure still formed many relatively weak alternative structures in equilibrium and retained function. The data suggest that although the downstream region is very important, its structure is quite malleable and is able to tolerate significant mutation within a wide range of primary and secondary structural features. We propose that this malleability is due to the enhanced ability of GU- and U-rich downstream elements to easily form secondary structures with surrounding sequences.

scholarly journals Sequences downstream of AAUAAA signals affect pre-mRNA cleavage and polyadenylation in vitro both directly and indirectly.

1989 ◽  
Vol 9 (4) ◽  
pp. 1759-1771 ◽  
Author(s):  
L C Ryner ◽  
Y Takagaki ◽  
J L Manley
Keyword(s):  
Cleavage Site ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Cleavage Reaction ◽  
Mrna Cleavage ◽  
Nuclear Extracts ◽  
Cleavage And Polyadenylation ◽  
Polyadenylation Signals

To investigate the role of sequences lying downstream of the conserved AAUAAA hexanucleotide in pre-mRNA cleavage and polyadenylation, deletions or substitutions were constructed in polyadenylation signals from simian virus 40 and adenovirus, and their effects were assayed in both crude and fractionated HeLa cell nuclear extracts. As expected, these sequences influenced the efficiency of both cleavage and polyadenylation as well as the accuracy of the cleavage reaction. Sequences near or upstream of the actual site of poly(A) addition appeared to specify a unique cleavage site, since their deletion resulted, in some cases, in heterogeneous cleavage. Furthermore, the sequences that allowed the simian virus 40 late pre-RNA to be cleaved preferentially by partially purified cleavage activity were also those at the cleavage site itself. Interestingly, sequences downstream of the cleavage site interacted with factors not directly involved in catalyzing cleavage and polyadenylation, since the effects of deletions were substantially diminished when partially purified components were used in assays. In addition, these sequences contained elements that could affect 3'-end formation both positively and negatively.

scholarly journals Definition of the upstream efficiency element of the simian virus 40 late polyadenylation signal by using in vitro analyses.

1992 ◽  
Vol 12 (12) ◽  
pp. 5386-5393 ◽  
Author(s):  
N Schek ◽  
C Cooke ◽  
J C Alwine
Keyword(s):  
Consensus Sequence ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Polyadenylation Signal ◽  
Upstream Region ◽  
Core Element ◽  
The Core ◽  
Core Elements ◽  
Upstream Element

The polyadenylation signal for the late mRNAs of simian virus 40 is known to have sequence elements located both upstream and downstream of the AAUAAA which affect efficiency of utilization of the signal. The upstream efficiency element has been previously characterized by using deletion mutations and transfection analyses. Those studies suggested that the upstream element lies between 13 and 48 nucleotides upstream of the AAUAAA. We have utilized in vitro cleavage and polyadenylation reactions to further define the upstream element. 32P-labeled substrate RNAs were prepared by in vitro transcription from wild-type templates as well as from mutant templates having deletions and linker substitutions in the upstream region. Analysis of these substrates defined the upstream region as sequences between 13 and 51 nucleotides upstream of the AAUAAA, in good agreement with the in vivo results. Within this region, three core elements with the consensus sequence AUUUGURA were identified and were specifically mutated by linker substitution. These core elements were found to contain the active components of the upstream efficiency element. Using substrates with both single and double linker substitution mutations of core elements, we observed that the core elements function in a distance-dependent manner. In mutants containing only one core element, the effect on efficiency increases as the distance between the element and the AAUAAA decreases. In addition, when core elements are present in multiple copies, the effect is additive. The core element consensus sequence, which bears homology to the Sm protein complex-binding site in human U1 RNA, is also found within the upstream elements of the ground squirrel hepatitis B and cauliflower mosaic virus polyadenylation signals (R. Russnak, Nucleic Acids Res. 19:6449-6456, 1991; H. Sanfacon, P. Brodmann, and T. Hohn, Genes Dev. 5:141-149, 1991), suggesting functional conservation of this element between mammals and plants.

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