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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.


Sequencing analysis of the truncated PCR product. A) Multiple sequence alignment of the full-length and the truncated PCR products from 70.15-transduced CEMx174 cells with the PCR product from plasmid template. The sequence of the full-length clones match the 654 bps predicted sequence from the plasmid template. The truncated product aligns with the predicted sequence except for a 139 bp deletion. The colored boxes indicate the transcription factor binding sequences: TATA box (red), the PSE (plum), and the sequences related to the octamer consensus (ATTTGCAT, blue), the protected SPH domain (light blue), while the grey boxes are non-functional octamer-related sequences [19, 20, 28]. The grey shading is the sequence of the HIV-1-specific RT aptamer 70.15 and the black box is the pol III termination signal. The bold sequences are the proposed splice donor and splice acceptor sequences. B) Schematic map of the predicted amplicon in forward orientation as described in Figure 1. The black arrows indicate the amplification primers. The boxes represent from left to right the 3' end of GFP, the Pol III termination sequence, a self-splicing ribozyme, the HIV-1-specific RT aptamer 70.15 (grey box), a self-splicing ribozyme, the human U6+1 promoter, and MMP 3' UTR sequences. The line represents linker sequences between the functional sequences. The spotted ovals represent the TATA box (red) and PSE (plum) in proximal promoter; the non-function octamer sequences (grey), the Staf-binding domain (light blue) and the two octamer-related sequences (blue) in the distal promoter all within the U6 promoter. A curved arrow represents the position and direction of the transcriptional start site. The black X represents the splicing deletion.
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Figure 4: Sequencing analysis of the truncated PCR product. A) Multiple sequence alignment of the full-length and the truncated PCR products from 70.15-transduced CEMx174 cells with the PCR product from plasmid template. The sequence of the full-length clones match the 654 bps predicted sequence from the plasmid template. The truncated product aligns with the predicted sequence except for a 139 bp deletion. The colored boxes indicate the transcription factor binding sequences: TATA box (red), the PSE (plum), and the sequences related to the octamer consensus (ATTTGCAT, blue), the protected SPH domain (light blue), while the grey boxes are non-functional octamer-related sequences [19, 20, 28]. The grey shading is the sequence of the HIV-1-specific RT aptamer 70.15 and the black box is the pol III termination signal. The bold sequences are the proposed splice donor and splice acceptor sequences. B) Schematic map of the predicted amplicon in forward orientation as described in Figure 1. The black arrows indicate the amplification primers. The boxes represent from left to right the 3' end of GFP, the Pol III termination sequence, a self-splicing ribozyme, the HIV-1-specific RT aptamer 70.15 (grey box), a self-splicing ribozyme, the human U6+1 promoter, and MMP 3' UTR sequences. The line represents linker sequences between the functional sequences. The spotted ovals represent the TATA box (red) and PSE (plum) in proximal promoter; the non-function octamer sequences (grey), the Staf-binding domain (light blue) and the two octamer-related sequences (blue) in the distal promoter all within the U6 promoter. A curved arrow represents the position and direction of the transcriptional start site. The black X represents the splicing deletion.

Mentions: We initially examined whether the deletion was caused by recombination of the short repeat elements in the flanking ribozyme domains because of misalignment during reverse transcription. To determine the nature of the deletion, we gel purified and cloned the truncated product, the full-length product, and the plasmid product for sequencing analysis. Individual clones were isolated and sequenced. The consensus sequences of the clones from the full-length PCR products and from the plasmid amplicon are identical to the predicted amplicon sequence (Figure 4a). In contrast, the sequence alignments from 20 of 21 clones from the truncated PCR product contain an identical 139 bp deletion in the predicated amplicon sequence (Figure 4a), while one clone contained the full-length sequence. When the deletion was compared to the predicted amplicon sequence, the deletion was mapped from bases -62 to -200 (relative to the transcriptional start site) in the middle of the U6 promoter (Figure 4a). These data demonstrate that the deletion in the inhibitor cassette did not occur in the sequences between the repeat elements in the self-splicing ribozymes but occurred in the promoter region during post-transcriptional processing of the mRNA, potentially from splicing of retroviral genomic mRNA.


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)

Sequencing analysis of the truncated PCR product. A) Multiple sequence alignment of the full-length and the truncated PCR products from 70.15-transduced CEMx174 cells with the PCR product from plasmid template. The sequence of the full-length clones match the 654 bps predicted sequence from the plasmid template. The truncated product aligns with the predicted sequence except for a 139 bp deletion. The colored boxes indicate the transcription factor binding sequences: TATA box (red), the PSE (plum), and the sequences related to the octamer consensus (ATTTGCAT, blue), the protected SPH domain (light blue), while the grey boxes are non-functional octamer-related sequences [19, 20, 28]. The grey shading is the sequence of the HIV-1-specific RT aptamer 70.15 and the black box is the pol III termination signal. The bold sequences are the proposed splice donor and splice acceptor sequences. B) Schematic map of the predicted amplicon in forward orientation as described in Figure 1. The black arrows indicate the amplification primers. The boxes represent from left to right the 3' end of GFP, the Pol III termination sequence, a self-splicing ribozyme, the HIV-1-specific RT aptamer 70.15 (grey box), a self-splicing ribozyme, the human U6+1 promoter, and MMP 3' UTR sequences. The line represents linker sequences between the functional sequences. The spotted ovals represent the TATA box (red) and PSE (plum) in proximal promoter; the non-function octamer sequences (grey), the Staf-binding domain (light blue) and the two octamer-related sequences (blue) in the distal promoter all within the U6 promoter. A curved arrow represents the position and direction of the transcriptional start site. The black X represents the splicing deletion.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 4: Sequencing analysis of the truncated PCR product. A) Multiple sequence alignment of the full-length and the truncated PCR products from 70.15-transduced CEMx174 cells with the PCR product from plasmid template. The sequence of the full-length clones match the 654 bps predicted sequence from the plasmid template. The truncated product aligns with the predicted sequence except for a 139 bp deletion. The colored boxes indicate the transcription factor binding sequences: TATA box (red), the PSE (plum), and the sequences related to the octamer consensus (ATTTGCAT, blue), the protected SPH domain (light blue), while the grey boxes are non-functional octamer-related sequences [19, 20, 28]. The grey shading is the sequence of the HIV-1-specific RT aptamer 70.15 and the black box is the pol III termination signal. The bold sequences are the proposed splice donor and splice acceptor sequences. B) Schematic map of the predicted amplicon in forward orientation as described in Figure 1. The black arrows indicate the amplification primers. The boxes represent from left to right the 3' end of GFP, the Pol III termination sequence, a self-splicing ribozyme, the HIV-1-specific RT aptamer 70.15 (grey box), a self-splicing ribozyme, the human U6+1 promoter, and MMP 3' UTR sequences. The line represents linker sequences between the functional sequences. The spotted ovals represent the TATA box (red) and PSE (plum) in proximal promoter; the non-function octamer sequences (grey), the Staf-binding domain (light blue) and the two octamer-related sequences (blue) in the distal promoter all within the U6 promoter. A curved arrow represents the position and direction of the transcriptional start site. The black X represents the splicing deletion.
Mentions: We initially examined whether the deletion was caused by recombination of the short repeat elements in the flanking ribozyme domains because of misalignment during reverse transcription. To determine the nature of the deletion, we gel purified and cloned the truncated product, the full-length product, and the plasmid product for sequencing analysis. Individual clones were isolated and sequenced. The consensus sequences of the clones from the full-length PCR products and from the plasmid amplicon are identical to the predicted amplicon sequence (Figure 4a). In contrast, the sequence alignments from 20 of 21 clones from the truncated PCR product contain an identical 139 bp deletion in the predicated amplicon sequence (Figure 4a), while one clone contained the full-length sequence. When the deletion was compared to the predicted amplicon sequence, the deletion was mapped from bases -62 to -200 (relative to the transcriptional start site) in the middle of the U6 promoter (Figure 4a). These data demonstrate that the deletion in the inhibitor cassette did not occur in the sequences between the repeat elements in the self-splicing ribozymes but occurred in the promoter region during post-transcriptional processing of the mRNA, potentially from splicing of retroviral genomic mRNA.

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.