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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.


Northern blot of wildtype and mutant U6 promoter activity. Plasmid DNA with the wildtype (wt) and mutant U6 promoters transcriptionally regulating the aptamer 70.15 were transfected into 293T cells. After 48 hours, total RNA was isolated, separated by electrophoresis, blotted and probed for expression of the aptamer 70.15 and of the U6 snRNA (endogenous control). Shown are 3 replicates for the aptamer and the U6 snRNA. The intensity of each band (x104 units) was quantified with the STORM 820 phosphorimager and is shown below the image. A relative ratio of the aptamer 70.15 signal to the endogenous U6 snRNA expression is shown. The fold decrease of mutant U6 promoter activity and Wt U6 promoter activity in regulating transcription of the aptamer 70.15 is indicated below the line for each replicate. The reduction in expression of the mutant U6 promoter was 0.50 ± 0.12 fold (mean ± standard deviation [SD]).
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Figure 7: Northern blot of wildtype and mutant U6 promoter activity. Plasmid DNA with the wildtype (wt) and mutant U6 promoters transcriptionally regulating the aptamer 70.15 were transfected into 293T cells. After 48 hours, total RNA was isolated, separated by electrophoresis, blotted and probed for expression of the aptamer 70.15 and of the U6 snRNA (endogenous control). Shown are 3 replicates for the aptamer and the U6 snRNA. The intensity of each band (x104 units) was quantified with the STORM 820 phosphorimager and is shown below the image. A relative ratio of the aptamer 70.15 signal to the endogenous U6 snRNA expression is shown. The fold decrease of mutant U6 promoter activity and Wt U6 promoter activity in regulating transcription of the aptamer 70.15 is indicated below the line for each replicate. The reduction in expression of the mutant U6 promoter was 0.50 ± 0.12 fold (mean ± standard deviation [SD]).

Mentions: Comparisons of the deletion with the functional domains of the U6 promoter revealed that the deletion removes 5 of 19 bps in the proximal sequence element (PSE) and two non-functional Oct-1 binding domains in the distal enhancer, but not the functional Oct-1 and SPH elements in the distal enhancer (Figure 4). The 139 bp deletion also changes the spacing between the enhancer element and the PSE within the U6 promoter to only 13 bps (Figure 4). Previous studies have shown that the promoter is transcriptionally active with limited distance between the PSE and the upstream enhancer elements [17]. To determine whether the deletion had an effect on the transcriptional activity of the U6 promoter, we used the pCRII plasmids containing full-length and truncated U6 promoter-70.15 amplicons to transiently transfect 239T cells and quantified expression of the 70.15 aptamer by Northern blot (Figure 7). Endogenous expression of the U6 snRNA gene was used to normalize samples (Figure 7). The mutation in the U6 promoter consistently reduced expression of the 70.15 aptamer compared to the wildtype U6 promoter (0.50 ± 0.12, mean ± SD) but did not completely eliminate expression (Figure 7). Thus, deletion of 139 bps within the U6 promoter, either through loss of a portion of the PSE and/or through alterations in the spatial arrangement of transcription factors, lowered U6 promoter activity.


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)

Northern blot of wildtype and mutant U6 promoter activity. Plasmid DNA with the wildtype (wt) and mutant U6 promoters transcriptionally regulating the aptamer 70.15 were transfected into 293T cells. After 48 hours, total RNA was isolated, separated by electrophoresis, blotted and probed for expression of the aptamer 70.15 and of the U6 snRNA (endogenous control). Shown are 3 replicates for the aptamer and the U6 snRNA. The intensity of each band (x104 units) was quantified with the STORM 820 phosphorimager and is shown below the image. A relative ratio of the aptamer 70.15 signal to the endogenous U6 snRNA expression is shown. The fold decrease of mutant U6 promoter activity and Wt U6 promoter activity in regulating transcription of the aptamer 70.15 is indicated below the line for each replicate. The reduction in expression of the mutant U6 promoter was 0.50 ± 0.12 fold (mean ± standard deviation [SD]).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2211285&req=5

Figure 7: Northern blot of wildtype and mutant U6 promoter activity. Plasmid DNA with the wildtype (wt) and mutant U6 promoters transcriptionally regulating the aptamer 70.15 were transfected into 293T cells. After 48 hours, total RNA was isolated, separated by electrophoresis, blotted and probed for expression of the aptamer 70.15 and of the U6 snRNA (endogenous control). Shown are 3 replicates for the aptamer and the U6 snRNA. The intensity of each band (x104 units) was quantified with the STORM 820 phosphorimager and is shown below the image. A relative ratio of the aptamer 70.15 signal to the endogenous U6 snRNA expression is shown. The fold decrease of mutant U6 promoter activity and Wt U6 promoter activity in regulating transcription of the aptamer 70.15 is indicated below the line for each replicate. The reduction in expression of the mutant U6 promoter was 0.50 ± 0.12 fold (mean ± standard deviation [SD]).
Mentions: Comparisons of the deletion with the functional domains of the U6 promoter revealed that the deletion removes 5 of 19 bps in the proximal sequence element (PSE) and two non-functional Oct-1 binding domains in the distal enhancer, but not the functional Oct-1 and SPH elements in the distal enhancer (Figure 4). The 139 bp deletion also changes the spacing between the enhancer element and the PSE within the U6 promoter to only 13 bps (Figure 4). Previous studies have shown that the promoter is transcriptionally active with limited distance between the PSE and the upstream enhancer elements [17]. To determine whether the deletion had an effect on the transcriptional activity of the U6 promoter, we used the pCRII plasmids containing full-length and truncated U6 promoter-70.15 amplicons to transiently transfect 239T cells and quantified expression of the 70.15 aptamer by Northern blot (Figure 7). Endogenous expression of the U6 snRNA gene was used to normalize samples (Figure 7). The mutation in the U6 promoter consistently reduced expression of the 70.15 aptamer compared to the wildtype U6 promoter (0.50 ± 0.12, mean ± SD) but did not completely eliminate expression (Figure 7). Thus, deletion of 139 bps within the U6 promoter, either through loss of a portion of the PSE and/or through alterations in the spatial arrangement of transcription factors, lowered U6 promoter activity.

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.