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Instability of retroviral vectors with HIV-1-specific RT aptamers due to cryptic splice sites in the U6 promoter.

Braun SE, Shi X, Qiu G, Wong FE, Joshi PJ, Prasad VR, Johnson RP - AIDS Res Ther (2007)

Bottom Line: We did not observe inhibition of HIV-1 replication in these transduced populations.This deletion decreased transcriptional activity of the U6 promoter.The existence of a cryptic splice site in the U6 promoter when expressed in a retroviral vector in the reverse orientation generates deletions during packaging and may limit the utility of this promoter for expression of small RNA inhibitors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Immunology, New England Primate Research Center, Harvard Medical School, One Pine Hill Drive, Southborough, MA 01772, USA. stephen_braun@hms.harvard.edu

ABSTRACT

Background: Internal polymerase III promoters in retroviral vectors have been used extensively to express short RNA sequences, such as ribozymes, RNA aptamers or short interfering RNA inhibitors, in various positions and orientations. However, the stability of these promoters in the reverse orientation has not been rigorously evaluated.

Results: A series of retroviral vectors was generated carrying the U6+1 promoter with 3 different HIV-1 RT-specific RNA aptamers and one control aptamer, all in the reverse orientation. After shuttle packaging, the CD4+ cell line CEMx174 was transduced with each vector, selected for expression of GFP, and challenged with HIV-1. We did not observe inhibition of HIV-1 replication in these transduced populations. PCR amplification of the U6+1 promoter-RNA aptamer inhibitor cassette from transduced CEMx174 cells and RT-PCR amplification from transfected Phoenix (amphotropic) packaging cells showed two distinct products: a full-length product of the expected size as well as a truncated product. The sequence of the full-length PCR product was identical to the predicted amplicon sequence. However, sequencing of the truncated product revealed a 139 bp deletion in the U6 promoter. This deletion decreased transcriptional activity of the U6 promoter. Analysis of the deleted sequences from the U6 promoter in the antisense direction indicated consensus splice donor, splice acceptor and branch point sequences.

Conclusion: The existence of a cryptic splice site in the U6 promoter when expressed in a retroviral vector in the reverse orientation generates deletions during packaging and may limit the utility of this promoter for expression of small RNA inhibitors.

No MeSH data available.


Related in: MedlinePlus

Comparison of the antisense U6 promoter with splicing consensus sequences. A) Top row: the consensus splice donor and splice acceptor sequences with the intron sequences (red bold with a red bold line above) and the exon sequences (normal font with a thin line above). The necessity for the internal pyrmidine stretch is represented by the 10Y. The nucleotide-usage percentage at each position is noted as a subscript. M equals adenosine or cytosine, Y equals pyrimidines and R equals purine. Bottom row: the antisense U6 promoter sequences surrounding the 139 bp deletion with the sequences that remain (in normal font with a thin line below) and the sequences that were deleted (in red bold with a red bold line below). B) Top row: The consensus sequences for the internal branch point are represented with the nucleotide-usage percentage noted as a subscript. The distance of the invariant adenosine to the splice acceptor site is noted in parentheses. Potential branch sites: 5 potential branch sites in the antisense U6 promoter are indicated. Divergent sequences are shown in normal font and conserved sequences are shown in bold. All sequences are intronic and shown in red. The distances to the end of the deletion are noted in parentheses.
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Figure 5: Comparison of the antisense U6 promoter with splicing consensus sequences. A) Top row: the consensus splice donor and splice acceptor sequences with the intron sequences (red bold with a red bold line above) and the exon sequences (normal font with a thin line above). The necessity for the internal pyrmidine stretch is represented by the 10Y. The nucleotide-usage percentage at each position is noted as a subscript. M equals adenosine or cytosine, Y equals pyrimidines and R equals purine. Bottom row: the antisense U6 promoter sequences surrounding the 139 bp deletion with the sequences that remain (in normal font with a thin line below) and the sequences that were deleted (in red bold with a red bold line below). B) Top row: The consensus sequences for the internal branch point are represented with the nucleotide-usage percentage noted as a subscript. The distance of the invariant adenosine to the splice acceptor site is noted in parentheses. Potential branch sites: 5 potential branch sites in the antisense U6 promoter are indicated. Divergent sequences are shown in normal font and conserved sequences are shown in bold. All sequences are intronic and shown in red. The distances to the end of the deletion are noted in parentheses.

Mentions: To address whether inadvertent RNA splicing of the inhibitor cassette in the antisense orientation was responsible for generating the deletion, we compared the antisense sequence in the deletion of the U6 promoter to consensus splicing signals. As shown in Figure 5a, the GU and AG at the ends of most introns, along with the internal stretch of pyrimidines and other frequently occurring nucleotides, define the consensus sequences for splice donor and splice acceptor sites (reviewed in [14-16]). In most exons, the unspliced sequence is (A/C)AG at the donor site and G at the acceptor sites (Figure 5a) [14-16]. However, the sequences at the intron/exon junction are not sufficient to signal the presence of an intron. The branch point is another required sequence usually located 20 – 50 bases upstream of the acceptor [14-16]. The consensus sequence of the branch point is "CU(Pu)A(Py)", where adenosine is absolutely required for splicing (Figure 5b) [14-16]. Additional positive and negative recognition elements are also necessary [14-16]. Interestingly, the junctions of the deleted sequences in the U6 promoter contain the requisite GU and AG sequences and show very strong similarities to the splice donor splice acceptor consensus sequences with an internal pyrimidine stretch (Figure 5a). Because the branch point consensus sequences are more flexible, the antisense U6 promoter has potential branch sites 3, 15, 21, 25, and 46 bps upstream of the splice acceptor site (Figure 5b). All these sites contain the requisite adenosine. The few divergent sequences in these potential branch points are shown in normal font.


Instability of retroviral vectors with HIV-1-specific RT aptamers due to cryptic splice sites in the U6 promoter.

Braun SE, Shi X, Qiu G, Wong FE, Joshi PJ, Prasad VR, Johnson RP - AIDS Res Ther (2007)

Comparison of the antisense U6 promoter with splicing consensus sequences. A) Top row: the consensus splice donor and splice acceptor sequences with the intron sequences (red bold with a red bold line above) and the exon sequences (normal font with a thin line above). The necessity for the internal pyrmidine stretch is represented by the 10Y. The nucleotide-usage percentage at each position is noted as a subscript. M equals adenosine or cytosine, Y equals pyrimidines and R equals purine. Bottom row: the antisense U6 promoter sequences surrounding the 139 bp deletion with the sequences that remain (in normal font with a thin line below) and the sequences that were deleted (in red bold with a red bold line below). B) Top row: The consensus sequences for the internal branch point are represented with the nucleotide-usage percentage noted as a subscript. The distance of the invariant adenosine to the splice acceptor site is noted in parentheses. Potential branch sites: 5 potential branch sites in the antisense U6 promoter are indicated. Divergent sequences are shown in normal font and conserved sequences are shown in bold. All sequences are intronic and shown in red. The distances to the end of the deletion are noted in parentheses.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC2211285&req=5

Figure 5: Comparison of the antisense U6 promoter with splicing consensus sequences. A) Top row: the consensus splice donor and splice acceptor sequences with the intron sequences (red bold with a red bold line above) and the exon sequences (normal font with a thin line above). The necessity for the internal pyrmidine stretch is represented by the 10Y. The nucleotide-usage percentage at each position is noted as a subscript. M equals adenosine or cytosine, Y equals pyrimidines and R equals purine. Bottom row: the antisense U6 promoter sequences surrounding the 139 bp deletion with the sequences that remain (in normal font with a thin line below) and the sequences that were deleted (in red bold with a red bold line below). B) Top row: The consensus sequences for the internal branch point are represented with the nucleotide-usage percentage noted as a subscript. The distance of the invariant adenosine to the splice acceptor site is noted in parentheses. Potential branch sites: 5 potential branch sites in the antisense U6 promoter are indicated. Divergent sequences are shown in normal font and conserved sequences are shown in bold. All sequences are intronic and shown in red. The distances to the end of the deletion are noted in parentheses.
Mentions: To address whether inadvertent RNA splicing of the inhibitor cassette in the antisense orientation was responsible for generating the deletion, we compared the antisense sequence in the deletion of the U6 promoter to consensus splicing signals. As shown in Figure 5a, the GU and AG at the ends of most introns, along with the internal stretch of pyrimidines and other frequently occurring nucleotides, define the consensus sequences for splice donor and splice acceptor sites (reviewed in [14-16]). In most exons, the unspliced sequence is (A/C)AG at the donor site and G at the acceptor sites (Figure 5a) [14-16]. However, the sequences at the intron/exon junction are not sufficient to signal the presence of an intron. The branch point is another required sequence usually located 20 – 50 bases upstream of the acceptor [14-16]. The consensus sequence of the branch point is "CU(Pu)A(Py)", where adenosine is absolutely required for splicing (Figure 5b) [14-16]. Additional positive and negative recognition elements are also necessary [14-16]. Interestingly, the junctions of the deleted sequences in the U6 promoter contain the requisite GU and AG sequences and show very strong similarities to the splice donor splice acceptor consensus sequences with an internal pyrimidine stretch (Figure 5a). Because the branch point consensus sequences are more flexible, the antisense U6 promoter has potential branch sites 3, 15, 21, 25, and 46 bps upstream of the splice acceptor site (Figure 5b). All these sites contain the requisite adenosine. The few divergent sequences in these potential branch points are shown in normal font.

Bottom Line: We did not observe inhibition of HIV-1 replication in these transduced populations.This deletion decreased transcriptional activity of the U6 promoter.The existence of a cryptic splice site in the U6 promoter when expressed in a retroviral vector in the reverse orientation generates deletions during packaging and may limit the utility of this promoter for expression of small RNA inhibitors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Immunology, New England Primate Research Center, Harvard Medical School, One Pine Hill Drive, Southborough, MA 01772, USA. stephen_braun@hms.harvard.edu

ABSTRACT

Background: Internal polymerase III promoters in retroviral vectors have been used extensively to express short RNA sequences, such as ribozymes, RNA aptamers or short interfering RNA inhibitors, in various positions and orientations. However, the stability of these promoters in the reverse orientation has not been rigorously evaluated.

Results: A series of retroviral vectors was generated carrying the U6+1 promoter with 3 different HIV-1 RT-specific RNA aptamers and one control aptamer, all in the reverse orientation. After shuttle packaging, the CD4+ cell line CEMx174 was transduced with each vector, selected for expression of GFP, and challenged with HIV-1. We did not observe inhibition of HIV-1 replication in these transduced populations. PCR amplification of the U6+1 promoter-RNA aptamer inhibitor cassette from transduced CEMx174 cells and RT-PCR amplification from transfected Phoenix (amphotropic) packaging cells showed two distinct products: a full-length product of the expected size as well as a truncated product. The sequence of the full-length PCR product was identical to the predicted amplicon sequence. However, sequencing of the truncated product revealed a 139 bp deletion in the U6 promoter. This deletion decreased transcriptional activity of the U6 promoter. Analysis of the deleted sequences from the U6 promoter in the antisense direction indicated consensus splice donor, splice acceptor and branch point sequences.

Conclusion: The existence of a cryptic splice site in the U6 promoter when expressed in a retroviral vector in the reverse orientation generates deletions during packaging and may limit the utility of this promoter for expression of small RNA inhibitors.

No MeSH data available.


Related in: MedlinePlus