The D1-A2 and D2-A2 Pairs of Splice Sites from Human Immunodeficiency Virus Type 1 Are Highly Efficientin Vitro,in Spite of an Unusual Branch Site

Damier, Laurence; Domenjoud, Lionel; Branlant, Christiane

doi:10.1006/bbrc.1997.7091

Cited by 23 publications

(30 citation statements)

References 37 publications

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“…Plasmids Used in This Study-pLD-C2, pLD-C3, and pLD-L3-U1 constructs, used for production of the C2, C3, and L3-U1 transcripts, were described previously (11,23,24). To build the plasmid pLD-C1, two DNA fragments were generated by PCR amplifications of plasmid pBRU3 (42) containing the HIV-1 BRU/LAI complete cDNA (GenBank TM accession number K02013).…”

Section: Methodsmentioning

confidence: 99%

“…To build the plasmid pLD-C1, two DNA fragments were generated by PCR amplifications of plasmid pBRU3 (42) containing the HIV-1 BRU/LAI complete cDNA (GenBank TM accession number K02013). Amplifications were performed as described previously (23). The DNA fragment encoding the RNA region from position 4313 to 4545 (43) was amplified with the following: (i) a sense primer O-756 (5Ј-TATTCTAGATCTCAGGCTGAACATC-3Ј) containing the HIV-1 BRU RNA sequence from positions 4313 to 4325 (underlined) and a BglII restriction site (italic); and (ii) an antisense primer O-764 (5Ј-CGGAATTCCTTTCCAGAGGAGCT-3Ј) complementary to the HIV-1 BRU RNA from positions 4530 to 4545 (underlined) and containing an EcoRI restriction site (italic).…”

Section: Methodsmentioning

confidence: 99%

“…They were used as templates for the production of capped, uniformly labeled RNAs by the T7 or SP6 RNA polymerase in the presence of [␣-32 P]UTP. In vitro splicing assays were performed in HeLa cell nuclear extract (from the Computer Cell Culture Center S.A., Belgium), pure cytoplasmic S100 extract prepared as described previously (35), or mixtures of S100 and nuclear extracts (4:4 l and 7:1 l), in 22-l assays using classical conditions for in vitro splicing reaction (23). The duration of incubation at 30°C was for 2 h and 30 min.…”

Section: Methodsmentioning

confidence: 99%

“…(i) Their polypyrimidine tracts are short and interrupted by purines (3,5,9). (ii) Their branch point sequences are not canonical, and in some cases, a residue other than an adenosine is used as the branch site (22,23). (iii) Their accessibility is limited by their sequestering in stable secondary structures (24).…”

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Differential Effects of the SR Proteins 9G8, SC35, ASF/SF2, and SRp40 on the Utilization of the A1 to A5 Splicing Sites of HIV-1 RNA

Ropers¹,

Ayadi²,

Gattoni

et al. 2004

Journal of Biological Chemistry

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Splicing is a crucial step for human immunodeficiency virus, type 1 (HIV-1) multiplication; eight acceptor sites are used in competition to produce the vif, vpu, vpr, nef, env, tat, and rev mRNAs. The effects of SR proteins have only been investigated on a limited number of HIV-1 splicing sites by using small HIV-1 RNA pieces. To understand how SR proteins influence the use of HIV-1 splicing sites, we tested the effects of overproduction of individual SR proteins in HeLa cells on the splicing pattern of an HIV-1 RNA that contained all the splicing sites. The steady state levels of the HIV-1 mRNAs produced were quantified by reverse transcriptase-PCR. For interpretation of the data, transcripts containing one or several of the HIV-1 acceptor sites were spliced in vitro in the presence or the absence of one of the tested SR proteins. Both in vivo and in vitro, acceptor sites A2 and A3 were found to be strongly and specifically regulated by SR proteins. ASF/SF2 strongly activates site A2 and to a lesser extent site A1. As a result, upon ASF/SF2 overexpression, the vpr mRNA steady state level is specifically increased. SC35 and SRp40, but not 9G8, strongly activate site A3, and their overexpression ex vivo induces a dramatic accumulation of the tat mRNA, to the detriment of most of the other viral mRNAs. Here we showed by Western blot analysis that the Nef protein synthesis is strongly decreased by overexpression of SC35, SRp40, and ASF/SF2. Finally, activation by ASF/ SF2 and 9G8 was found to be independent of the RS domain. This is the first investigation of the effects of variations of individual SR protein concentrations that is performed ex vivo on an RNA containing a complex set of splicing sites.Splicing plays a key role for production of the HIV-1 1 retroviral proteins. By using the integrated proviral genome as the template, RNA polymerase II of the infected cell produces long primary transcripts that are all identical. Some of these transcripts are transported to the cytoplasm in an intact form to serve as genomes for new virions or as messenger RNAs for the production of the Gag-Pol protein precursor. The other transcripts undergo alternative splicing to produce mRNAs for the auxiliary and regulatory proteins and the Env precursor protein. Production of mRNAs encoding HIV-1 proteins depends on the alternative utilization of four 5Ј-splice sites D1 to D4 (1) and eight 3Ј-splice sites (A1, A2, A3, A4a, -b, -c, A5, and A7) (1). An additional 3Ј-splice site (A6) was only found in one HIV-1 strain (2). Donor sites D1, D2, and D3 can be coupled to any of the A1 to A5 acceptor sites, whereas donor site D4 is exclusively coupled to site A7 and to site A6 when this site is present (1, 2). The combination of these various sites gives rise to at least 35 different mRNAs (1). Although the relative efficiencies of the HIV-1 donor sites seem to depend mainly upon their complementarity to the U1 snRNA 5Ј-terminal sequence (3, 4), efficiencies of HIV-1 acceptor sites depend upon the presence of cis-regulatory elements (5...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Differential Effects of the SR Proteins 9G8, SC35, ASF/SF2, and SRp40 on the Utilization of the A1 to A5 Splicing Sites of HIV-1 RNA

Ropers¹,

Ayadi²,

Gattoni

et al. 2004

Journal of Biological Chemistry

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“…In contrast to the major HIV-1 5Ј splice sites, which are strong, the 3Ј splice sites are weak (33). These are characterized by the presence of nonconsensus polypyrimidine tracts and branch point sites other than the eukaryotic consensus adenosine residue (2,13,17,33,43). In addition, exon sequences downstream of the 3Ј splice sites enhance or inhibit splicing at the 3Ј splice sites (2,3,39,42,51).…”

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The Exon Splicing Silencer in Human Immunodeficiency Virus Type 1 Tat Exon 3 Is Bipartite and Acts Early in Spliceosome Assembly

Rauch

Stoltzfus

1998

Molecular and Cellular Biology

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Inefficient splicing of human immunodeficiency virus type 1 (HIV-1) RNA is necessary to preserve unspliced and singly spliced viral RNAs for transport to the cytoplasm by the Rev-dependent pathway. Signals within the HIV-1 genome that control the rate of splicing include weak 3 splice sites, exon splicing enhancers (ESE), and exon splicing silencers (ESS). We have previously shown that an ESS present within tat exon 2 (ESS2) and a suboptimal 3 splice site together act to inhibit splicing at the 3 splice site flanking tat exon 2. This occurs at an early step in spliceosome assembly. Splicing at the 3 splice site flanking tat exon 3 is regulated by a bipartite element composed of an ESE and an ESS (ESS3). Here we show that ESS3 is composed of two smaller elements (AGAUCC and UUAG) that can inhibit splicing independently. We also show that ESS3 is more active in the context of a heterologous suboptimal splice site than of an optimal 3 splice site. ESS3 inhibits splicing by blocking the formation of a functional spliceosome at an early step, since A complexes are not detected in the presence of ESS3. Competitor RNAs containing either ESS2 or ESS3 relieve inhibition of splicing of substrates containing ESS3 or ESS2. This suggests that a common cellular factor(s) may be required for the inhibition of tat mRNA splicing mediated by ESS2 and ESS3.Human immunodeficiency virus type 1 (HIV-1) is a complex retrovirus whose RNA splicing is dependent on the host cell splicing machinery (12, 50). HIV-1 pre-mRNA contains five 5Ј splice sites and nine 3Ј splice sites which are used to generate more than 30 different mRNA species by alternative splicing. In addition, a pool of unspliced RNA must be maintained to serve as genomic RNA and as mRNA for the gag and pol gene products (18,21,32,34,35,38). To achieve the balance between spliced and unspliced RNAs, splicing of HIV-1 RNA is inefficient, a feature which is shared with all other retroviruses (4,7,11,20,22,(45)(46)(47)52). In contrast to the major HIV-1 5Ј splice sites, which are strong, the 3Ј splice sites are weak (33). These are characterized by the presence of nonconsensus polypyrimidine tracts and branch point sites other than the eukaryotic consensus adenosine residue (2,13,17,33,43). In addition, exon sequences downstream of the 3Ј splice sites enhance or inhibit splicing at the 3Ј splice sites (2,3,39,42,51). Furthermore, the virus-encoded protein Rev plays an essential role in facilitating the transport of unspliced and singly spliced mRNAs, thus preventing the viral RNA from splicing to completion (19, 37; for a review, see reference 12).HIV-1 tat mRNA is formed by the splicing of two coding exons (exon 2 and exon 3) and one or more upstream noncoding exons. Exon 3 is also joined to rev exon 2 and is therefore referred to as tat/rev exon 3 (34). The 3Ј splice sites flanking exon 2 and exon 3 (3Ј splice sites 3 and 7, respectively [ Fig. 1]) contain suboptimal polypyrimidine tracts, and the 3Ј splice site flanking exon 3 has been reported to contain a nonconsensus bra...

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Teetering on the Edge

Balachandran

Liang

Cochrane

2018

Retrovirus-Cell Interactions

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The D1-A2 and D2-A2 Pairs of Splice Sites from Human Immunodeficiency Virus Type 1 Are Highly Efficientin Vitro,in Spite of an Unusual Branch Site

Cited by 23 publications

References 37 publications

Differential Effects of the SR Proteins 9G8, SC35, ASF/SF2, and SRp40 on the Utilization of the A1 to A5 Splicing Sites of HIV-1 RNA

Differential Effects of the SR Proteins 9G8, SC35, ASF/SF2, and SRp40 on the Utilization of the A1 to A5 Splicing Sites of HIV-1 RNA

The Exon Splicing Silencer in Human Immunodeficiency Virus Type 1 Tat Exon 3 Is Bipartite and Acts Early in Spliceosome Assembly

Teetering on the Edge

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