Splicing affects presentation of RNA dimerization signals in HIV-2 in vitro

Lanchy, Jean Marc; Szafran, Quenna N.; Lodmell, J. Stephen

doi:10.1093/nar/gkh800

Cited by 9 publications

(8 citation statements)

References 33 publications

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“…The 186 truncation lacked the entire TAR sequence as well as the poly(A) signal domain. The 198 truncation was similar to the 186 truncation with just twelve additional nucleotides corresponding to the C-box deleted 13. The 486 truncation lacked TAR, the poly(A) signal domain, the C-box, and the PBS domain.…”

Section: Resultsmentioning

confidence: 80%

“…A sense primer (sNHErod) containing a Nhe I site and an antisense primer (asBsoBIrod) containing a Bso BI site were used to amplify the first 1-569 nucleotides of the HIV-2 genomic RNA, ROD isolate (GenBank: M15390; genomic RNA sequence starts at 1) and the first 1-569 nucleotides minus the 5′UTR intron (61-202) of HIV-2 genomic RNA 13, respectively. Sense primers (sNHE186, sNHE198, sNHE486) containing a Nhe I site in combination with an antisense primer (asBsoBIrod) containing a Bso BI site were used to amplify 186-569, 198-569, and 486-569 of HIV-2 genomic RNA, ROD isolate, respectively.…”

Section: Methodsmentioning

confidence: 99%

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A 5′UTR-Spliced mRNA Isoform Is Specialized for Enhanced HIV-2 gag Translation

Strong

Lanchy

Dieng-Sarr

et al. 2009

Journal of Molecular Biology

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Full-length unspliced genomic RNA plays critical roles in HIV replication, serving both as mRNA for the synthesis of the key viral polyproteins Gag and Gag-Pol and as genomic RNA for encapsidation into assembling viral particles. We show that a second gag mRNA species is produced during HIV-2 replication in cell culture and in infected patients that differs from the genomic RNA molecule by the absence of an intron in the 5′ untranslated region (5′UTR). We developed a cotransfection system in which epitopically tagged Gag proteins can be traced back to their mRNA origins in the translation pool. We show that a disproportionate amount of Gag is translated from 5′ UTR intron-spliced mRNAs, demonstrating a role for the 5′UTR intron in the regulation of gag translation. To further characterize the effects of the HIV-2 5′UTR on translation, we fused wild type, spliced, or mutant leader RNA constructs to a luciferase reporter gene and assayed their translation in reticulocyte lysates. These assays confirmed that leaders lacking the 5′UTR intron increased translational efficiency compared to the unspliced leader. In addition, we found that removal or mutagenesis of the C-box, a pyrimidine-rich sequence located in the 5′UTR intron and previously shown to affect RNA dimerization, also strongly influenced translational efficiency. These results suggest that both the splicing of the 5′UTR intron and the C-box element have key roles in regulation of HIV-2 gag translation in vitro and in vivo.

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Section: Resultsmentioning

confidence: 80%

Section: Methodsmentioning

confidence: 99%

A 5′UTR-Spliced mRNA Isoform Is Specialized for Enhanced HIV-2 gag Translation

Strong

Lanchy

Dieng-Sarr

et al. 2009

Journal of Molecular Biology

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“…This model is supported by the ability of the MLV SD' spliced RNA to heterodimerize with the genomic RNA [ 36 ]. Note that HIV spliced RNAs were also able to dimerize in vitro [ 49 , 50 ]. It is still not clear how splicing contributes to dimerization.…”

Section: Resultsmentioning

confidence: 99%

Murine leukemia virus RNA dimerization is coupled to transcription and splicing processes

Maurel

Mougel

2010

Retrovirology

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Most of the cell biological aspects of retroviral genome dimerization remain unknown. Murine leukemia virus (MLV) constitutes a useful model to study when and where dimerization occurs within the cell. For instance, MLV produces a subgenomic RNA (called SD') that is co-packaged with the genomic RNA predominantly as FLSD' heterodimers. This SD' RNA is generated by splicing of the genomic RNA and also by direct transcription of a splice-associated retroelement of MLV (SDARE). We took advantage of these two SD' origins to study the effects of transcription and splicing events on RNA dimerization. Using genetic approaches coupled to capture of RNA heterodimer in virions, we determined heterodimerization frequencies in different cellular contexts. Several cell lines were stably established in which SD' RNA was produced by either splicing or transcription from SDARE. Moreover, SDARE was integrated into the host chromosome either concomitantly or sequentially with the genomic provirus. Our results showed that transcribed genomic and SD' RNAs preferentially formed heterodimers when their respective proviruses were integrated together. In contrast, heterodimerization was strongly affected when the two proviruses were integrated independently. Finally, dimerization was enhanced when the transcription sites were expected to be physically close. For the first time, we report that splicing and RNA dimerization appear to be coupled. Indeed, when the RNAs underwent splicing, the FLSD' dimerization reached a frequency similar to co-transcriptional heterodimerization. Altogether, our results indicate that randomness of heterodimerization increases when RNAs are co-expressed during either transcription or splicing. Our results strongly support the notion that dimerization occurs in the nucleus, at or near the transcription and splicing sites, at areas of high viral RNA concentration.

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“…Nevertheless, whatever these undefined sequences or their conformations, they do not affect the dimerization process, as all HIV-1 spliced RNA fragments dimerized efficiently. This study has to be compared with the one by Lanchy et al (2004), which showed that removing intronic sequence from HIV-2 RNA (as in tat, rev, and nef mRNAs) only locally affected the RNA conformation of RNAs. As the main packaging signal is located upstream of the SD site in HIV-2, these investigators suggested that the interaction between elements upstream and downstream of the SD site may help to select gRNA preferentially over spliced RNAs during packaging.…”

Section: Discussionmentioning

confidence: 99%

In vitro dimerization of human immunodeficiency virus type 1 (HIV-1) spliced RNAs

Sinck¹,

Richer²,

Howard³

et al. 2007

RNA

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The human immunodeficiency virus type 1 (HIV-1) packages its genomic RNA as a dimer of homologous RNA molecules that has to be selected among a multitude of cellular and viral RNAs. Interestingly, spliced viral mRNAs are packaged into viral particles with a relatively low efficiency despite the fact that they contain most of the extended packaging signal found in the 59 untranslated region of the genomic RNA, including the dimerization initiation site (DIS). As a consequence, HIV-1 spliced viral RNAs can theoretically homodimerize and heterodimerize with the genomic RNA, and thus they should directly compete with genomic RNA for packaging. To shed light on this issue, we investigated for the first time the in vitro dimerization properties of spliced HIV-1 RNAs. We found that singly spliced (env, vpr) and multispliced (tat, rev, and nef) RNA fragments are able to dimerize in vitro, and to efficiently form heterodimers with genomic RNA. Chemical probing experiments and inhibition of RNA dimerization by an antisense oligonucleotide directed against the DIS indicated that the DIS is structurally functional in spliced HIV-1 RNA, and that RNA dimerization occurs through a loop-loop interaction. In addition, by combining in vitro transcription and dimerization assays, we show that heterodimers can be efficiently formed only when the two RNA fragments are synthesized simultaneously, in the same environment. Together, our results support a model in which RNA dimerization would occur during transcription in the nucleus and could thus play a major role in splicing, transport, and localization of HIV-1 RNA.

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Splicing affects presentation of RNA dimerization signals in HIV-2 in vitro

Cited by 9 publications

References 33 publications

A 5′UTR-Spliced mRNA Isoform Is Specialized for Enhanced HIV-2 gag Translation

A 5′UTR-Spliced mRNA Isoform Is Specialized for Enhanced HIV-2 gag Translation

Murine leukemia virus RNA dimerization is coupled to transcription and splicing processes

In vitro dimerization of human immunodeficiency virus type 1 (HIV-1) spliced RNAs

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