The Rev protein is able to transport to the cytoplasm small nucleolar RNAs containing a Rev binding element

Buonomo, Sara B.C.; Michienzi, Alessandro; Angelis, Fernanda Gabriella De; Bozzoni, Irene

doi:10.1017/s1355838299990064

Cited by 24 publications

(25 citation statements)

References 50 publications

(3 reference statements)

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“…U16 is a member of the C͞D box snoRNA family that is primarily involved in the 2Ј O-methylation of specific rRNA residues (42). We chose the U16 snoRNA because it has been extensively studied (43) and has been successfully used for the nucleolar localization of a ribozyme (30) and a Rev binding element decoy (31,44). To insert the TAR element in the U16, we replaced the U16 apical loop with a minimal, functional TAR sequence (Fig.…”

Section: Resultsmentioning

confidence: 99%

A nucleolar TAR decoy inhibitor of HIV-1 replication

Michienzi

Zaia

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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The Tat protein is a key regulator of HIV-1 replication (1). Its major function is to transactivate RNA polymerase (pol) II transcription from the viral LTR promoter. The binding of Tat to a transactivation response (TAR) element in the 5Ј UTR of the viral RNAs stimulates the processivity of RNA pol II, greatly increasing HIV-1 RNA transcription (2-6). In the absence of Tat the transcription complex is able to initiate transcription from the LTR, but elongates very inefficiently (3). The transactivation activity of Tat is mediated by the interaction with a positive transcription elongation factor, P-TEFb (7-9), composed of CDK9 and cyclin T1 (10-16). The binding of Tat with cyclin T1 increases its affinity for the TAR element and induces the cooperative binding of the P-TEFb complex to TAR (13). CDK 9 can phosphorylate the carboxyl-terminal domain of the largest subunit of RNA pol II, stimulating its processivity (10,16,17). Because Tat affects one of the earliest stages of HIV-1 gene expression and may be involved in other critical steps in virus replication (reverse transcription for example, ref. 18), it has been considered a good candidate for therapeutic intervention against HIV-1. Among the different gene therapy strategies that have been tested to block Tat activity in human cells one of the most successful is the use of small RNA molecules that work as ''decoys.'' These RNAs mimic the specific RNA binding element for Tat, subsequently leading to its titration. TAR RNA or in vitro-evolved Tat binding aptamers have been previously used as decoys for Tat and have resulted in inhibitory effects on HIV-1 replication (19-23).In addition to its known localization in the nucleoplasm, Tat has been shown to have nucleolar localization properties (24-28). The functional role of Tat nucleolar trafficking is unclear, but it may associate with cyclin T1 in this compartment (29). We have previously used strategies for localizing an anti-HIV ribozyme as well as a Rev binding element in the nucleolus and demonstrated that both of these strategies inhibit HIV-1 replication (30, 31). To test whether Tat nucleolar localization is functionally important, we have used a similar strategy to direct a TAR element into this compartment. We demonstrate here that human T-lymphoblastoid CEM cells stably expressing a nucleolar-localized TAR element are highly resistant to HIV-1 infection. Using in situ hybridization analyses we also show that a Tat-enhanced GFP (EGFP) fusion protein colocalizes with the nucleolar-localized TAR element. The present observations taken together with the nucleolar localization properties of HIV-1 Rev (32-34), as well as some HIV RNAs (30,35,36), represent an additional paradigm for the role of the nucleolus in HIV-1 replication. The potent inhibition of HIV-1 replication mediated by the nucleolar-localized TAR decoy also suggests a strategy for genetic therapy of HIV-1 infection. Materials and MethodsPlasmid Constructs. The U16TAR DNA was prepared synthetically by PCR (37) using the primers A, B, C, D, E...

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Section: Resultsmentioning

confidence: 99%

A nucleolar TAR decoy inhibitor of HIV-1 replication

Michienzi

Zaia

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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“…Many properties of RNA, if appropriately modified, can represent powerful tools for controlling gene expression, at the posttranscriptional level, in a sequence-specific way (17,18,28,29). In human, this approach finds interesting applications in the gene therapy of inherited and acquired genetic disorders where one wants to repress or modify the expression of specific genes.…”

Section: Discussionmentioning

confidence: 99%

“…Even if such kind of control can be accomplished by the use of synthetic oligonucleotides (10-13), a significant drawback to this approach is caused by the fact that oligonucleotides would require periodic administrations. To circumvent this problem, over the last few years a lot of effort has been devoted to the production of several vectors able to express in vivo, in a stable fashion, large amounts of chimeric RNAs containing therapeutic sequences (17,29,(33)(34)(35).…”

Section: Discussionmentioning

confidence: 99%

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Chimeric snRNA molecules carrying antisense sequences against the splice junctions of exon 51 of the dystrophin pre-mRNA induce exon skipping and restoration of a dystrophin synthesis in Δ48-50 DMD cells

Angelis

Sthandier

Berarducci

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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119

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Deletions and point mutations in the dystrophin gene cause either the severe progressive myopathy Duchenne muscular dystrophy (DMD) or the milder Becker muscular dystrophy, depending on whether the translational reading frame is lost or maintained. Because internal in-frame deletions in the protein produce only mild myopathic symptoms, it should be possible, by preventing the inclusion of specific mutated exon(s) in the mature dystrophin mRNA, to restore a partially corrected phenotype. Such control has been previously accomplished by the use of synthetic oligonucleotides; nevertheless, a significant drawback to this approach is caused by the fact that oligonucleotides would require periodic administrations. To circumvent this problem, we have produced several constructs able to express in vivo, in a stable fashion, large amounts of chimeric RNAs containing antisense sequences. In this paper we show that antisense molecules against exon 51 splice junctions are able to direct skipping of this exon in the human DMD deletion 48 -50 and to rescue dystrophin synthesis. We also show that the highest skipping activity was found when antisense constructs against the 5 and 3 splice sites are coexpressed in the same cell. D uchenne muscular dystrophy (DMD) is an X-linked recessive disorder that affects 1 in every 3,500 males. It is characterized by the absence of the cytoskeletal dystrophin (427-kDa protein) that in turn produces a severe and progressive muscle deterioration. Most of the DMD mutations consist in deletions and point mutations in the 2.5-Mb dystrophin gene that introduce stop codons and consequently premature translation termination. A milder myopathy is the Becker muscular dystrophy; in this case, deletions inside the gene produce in frame mRNAs and consequently shorter but semifunctional dystrophin proteins (1). A third of DMD cases are the results of a de novo mutation (2, 3), therefore the disease can never be eliminated through genetic screening and counseling. For this reason, many efforts are now being devoted to the development of a treatment for this disorder, and several strategies have been designed that might provide an insight into finding a cure. One of these strategies involves the transplantation of normal myoblasts into the muscle tissues that lack this protein (4, 5), whereas the other strategy tries to restore correct expression of the dystrophin through a gene therapy approach. In this direction, several groups have tried to deliver full-length or mini cDNA copies of dystrophin into cells with the mutated gene (6-8). Even if this approach is very promising, several problems still remain to be solved, such as size capacity and transducing activity of the vector and immune response to the ''therapeutic'' gene (9).Another powerful approach of gene therapy is based on the fact that internal in-frame deletions in the protein produce only mild myopathic symptoms; therefore, it should be possible, by preventing the inclusion of specific mutated exon(s) in the mature dystrophin mRNA, to restore ...

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“…Likewise, the mechanism by which mature ribosomal subunits are exported from nucleoli to the cytoplasm is not clearly elucidated (Aitchison and Rout, 2000;Kuersten et al, 2001). Furthermore, it was recently proposed that nucleoli may also play a crucial role in several other cellular processes such as the control of cell cycle, aging, and possibly mRNA export (Bond and Wold, 1993;Kadowaki et al, 1994b;Johnson et al, 1998;Pederson, 1998;Buonomo et al, 1999;Olson et al, 2000;Pederson and Politz, 2000;Visintin and Amon, 2000).…”

Section: Introductionmentioning

confidence: 99%

Functional Proteomic Analysis of Human Nucleolus

Scherl

Couté

Déon

et al. 2002

MBoC

443

378

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The notion of a “plurifunctional” nucleolus is now well established. However, molecular mechanisms underlying the biological processes occurring within this nuclear domain remain only partially understood. As a first step in elucidating these mechanisms we have carried out a proteomic analysis to draw up a list of proteins present within nucleoli of HeLa cells. This analysis allowed the identification of 213 different nucleolar proteins. This catalog complements that of the 271 proteins obtained recently by others, giving a total of ∼350 different nucleolar proteins. Functional classification of these proteins allowed outlining several biological processes taking place within nucleoli. Bioinformatic analyses permitted the assignment of hypothetical functions for 43 proteins for which no functional information is available. Notably, a role in ribosome biogenesis was proposed for 31 proteins. More generally, this functional classification reinforces the plurifunctional nature of nucleoli and provides convincing evidence that nucleoli may play a central role in the control of gene expression. Finally, this analysis supports the recent demonstration of a coupling of transcription and translation in higher eukaryotes.

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The Rev protein is able to transport to the cytoplasm small nucleolar RNAs containing a Rev binding element

Cited by 24 publications

References 50 publications

A nucleolar TAR decoy inhibitor of HIV-1 replication

A nucleolar TAR decoy inhibitor of HIV-1 replication

Chimeric snRNA molecules carrying antisense sequences against the splice junctions of exon 51 of the dystrophin pre-mRNA induce exon skipping and restoration of a dystrophin synthesis in Δ48-50 DMD cells

Functional Proteomic Analysis of Human Nucleolus

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