Presence of Exon Splicing Silencers within Human Immunodeficiency Virus Type 1 <i>tat</i> Exon 2 and <i>tat-rev</i> Exon 3: Evidence for Inhibition Mediated by Cellular Factors

Amendt, Brad A.; Si, Zhihai; Stoltzfus, C. Martin

doi:10.1128/mcb.15.8.4606

Cited by 156 publications

(157 citation statements)

References 65 publications

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“…Among these, the abundant hnRNP A1 protein has been extensively characterized. The first hnRNP A1-dependent ESS was identified in studies of HIV tat exon 2 repression (7)(8)(9). This ESS was found to bind hnRNP A1, and mutations disrupting the ESS prevented hnRNP A1 binding and allowed enhanced exon 2 splicing.…”

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confidence: 99%

An intronic element contributes to splicing repression in spinal muscular atrophy

Kashima

Rao

Manley

2007

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

115

109

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The neurodegenerative disease spinal muscular atrophy is caused by mutation of the survival motor neuron 1 (SMN1) gene. SMN2 is a nearly identical copy of SMN1 that is unable to prevent disease, because most SMN2 transcripts lack exon 7 and thus produce a nonfunctional protein. A key cause of inefficient SMN2 exon 7 splicing is a single nucleotide difference between SMN1 and SMN2 within exon 7. We previously provided evidence that this base change suppresses exon 7 splicing by creating an inhibitory element, a heterogeneous nuclear ribonucleoprotein (hnRNP) A1-dependent exonic splicing silencer. We now find that another rare nucleotide difference between SMN1 and SMN2, in intron 7, potentially creates a second SMN2-specific hnRNP A1 binding site. Remarkably, this single base change does indeed create a highaffinity hnRNP A1 binding site, and base substitutions that disrupt it restore exon 7 inclusion in vivo and prevent hnRNP A1 binding in vitro. We propose that interactions between hnRNP A1 molecules bound to the exonic and intronic sites cooperate to exclude exon 7 and discuss the significance of this exclusion with respect to SMN expression and splicing control more generally.alternative splicing ͉ exonic splicing silencer ͉ hnRNP A1 ͉ intronic splicing silencer A lternative splicing of mRNA precursors is an important mechanism that regulates gene expression and generates increased protein diversity from a limited number of genes. Indeed, expression of 70% or more of human genes is now estimated to involve alternative splicing (1, 2). In higher eukaryotes, alternative splicing plays an essential role in diverse cellular functions, contributing to such basic processes as cell growth, differentiation, and cell death (3, 4). Several types of cis-acting RNA elements and trans-acting protein factors help to regulate alternative splicing and do so by diverse mechanisms. In general, however, non-spliceosomal proteins that participate in regulating alternative splicing associate with regulatory elements in exons and/or introns. Serine/arginine-rich proteins (5) are typically involved in positive regulation of splicing, stimulating splicing by interacting with exonic splicing enhancer elements (ESEs) or intronic splicing enhancer elements. In contrast, negative regulation is promoted most frequently by heterogeneous nuclear ribonucleoproteins (hnRNPs) (6), which function by binding sequences known as exonic splicing silencers (ESSs) or intronic splicing silencers.Several hnRNP proteins have been identified as key splicing repressors. Among these, the abundant hnRNP A1 protein has been extensively characterized. The first hnRNP A1-dependent ESS was identified in studies of HIV tat exon 2 repression (7-9). This ESS was found to bind hnRNP A1, and mutations disrupting the ESS prevented hnRNP A1 binding and allowed enhanced exon 2 splicing. Several mechanisms have been proposed to explain hnRNP A1-mediated splicing repression. In one, hnRNP A1 binds to an ESS and through direct protein-protein interactions, recruits more ...

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confidence: 99%

An intronic element contributes to splicing repression in spinal muscular atrophy

Kashima

Rao

Manley

2007

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

115

109

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“…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)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21). Several studies have shown that HIV-1 acceptor sites are suboptimal as follows.…”

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confidence: 99%

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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“…ESS2 and ESS2p are located within tat exon 2 (3,14), ESS3 is located within tat exon 3 (10,(15)(16)(17)(18), ESSV is located in the vif coding region (19), and an intronic splicing silencer (ISS) is located in the intron upstream of tat exon 3 (13). In a previous study we showed that hnRNPs of the A/B family bind ESS2 to repress splicing of the upstream intron (34).…”

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confidence: 99%

SC35 and Heterogeneous Nuclear Ribonucleoprotein A/B Proteins Bind to a Juxtaposed Exonic Splicing Enhancer/Exonic Splicing Silencer Element to Regulate HIV-1 tat Exon 2 Splicing

Zahler

Damgaard

Kjems

et al. 2004

Journal of Biological Chemistry

128

139

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Splicing of the human immunodeficiency virus, type 1, primary transcript is highly regulated. Maintaining the proper equilibrium among spliced, unspliced, and partially spliced isoforms is essential for the replication of the virus. Here we characterize a complex cis-acting element located in tat exon 2 that is required for the splicing regulation of the upstream intron. An exonic splicing enhancer (ESE) and an exonic splicing silencer (ESS) are both located within the regulatory element. Heterogeneous nuclear ribonucleoprotein (hnRNP) A/B proteins bind the ESS to repress splicing, whereas the SR protein SC35 binds the ESE to activate it. We show that the SC35 and the hnRNP A1 binding sites overlap within the juxtaposed ESE/ESS. We propose that hnRNP A1 binding to the ESS inhibits splicing of the upstream intron by directly masking the SC35 binding site.The human immunodeficiency virus, type 1 (HIV-1), 1 primary RNA transcript is alternatively spliced into more than 40 different mRNAs, which can be divided into three main classes. The 2-kb class of multiply spliced messages encodes the regulatory proteins Tat, Rev, and Nef; the partially spliced 4-kb class of mRNAs encodes the Env, Vpu, Vif, and Vpr proteins; and the 9.2-kb unspliced transcript encodes the Gag and Pol proteins and provides RNA genomes for packaging into virions (1, 2). Alteration of the complex splicing pattern generating the viral mRNAs can dramatically affect HIV-1 infectivity and pathogenesis (1,(3)(4)(5). The study of HIV-1 splicing gives us helpful insights into the general mechanisms regulating the alternative splicing of cellular and viral messages. HIV-1 utilizes the cellular splicing machinery to properly regulate production of its primary transcripts through at least four alternative 5Ј splice sites (ss) and eight alternative 3Ј ss.Several of the HIV-1 viral sequences that regulate splicing have been characterized. These include suboptimal 3Ј ss sequences characterized by weak polypyrimidine tracts, non-consensus branch points (6 -9), exonic splicing enhancers (ESEs) (10 -13), and exonic splicing silencers (ESS) (3, 10, 14 -19). ESEs and ESS are splicing regulatory sequences found in exons (15, 20 -24). These regulatory sequences often bind transacting factors that interact with the basal splicing machinery. The factors that have been characterized as binding to HIV-1 regulatory elements belong to either the arginine-serine-rich (SR) protein family (25) or the heterogeneous nuclear ribonucleoprotein (hnRNP) family (26). SR proteins mainly participate in positive regulation of splicing, whereas members of the hnRNP family are often involved in negative regulation. However, proteins from both families have been implicated in positive as well as negative splicing regulation (26, 27). The mechanisms by which ESEs, ESS, and the factors they bind modulate splicing are still elusive.The binding properties of hnRNP A1 to different nucleic acid substrates have been thoroughly investigated (26). Although a minimal recognition sequence (UAGG) has...

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Presence of Exon Splicing Silencers within Human Immunodeficiency Virus Type 1 tat Exon 2 and tat-rev Exon 3: Evidence for Inhibition Mediated by Cellular Factors

Cited by 156 publications

References 65 publications

An intronic element contributes to splicing repression in spinal muscular atrophy

An intronic element contributes to splicing repression in spinal muscular atrophy

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

SC35 and Heterogeneous Nuclear Ribonucleoprotein A/B Proteins Bind to a Juxtaposed Exonic Splicing Enhancer/Exonic Splicing Silencer Element to Regulate HIV-1 tat Exon 2 Splicing

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