hnRNP A1 controls HIV-1 mRNA splicing through cooperative binding to intron and exon splicing silencers in the context of a conserved secondary structure

Damgaard, Christian Kroun; Tange, Thomas Ø.; Kjems, Jørgen

doi:10.1017/s1355838202023075

Cited by 108 publications

(138 citation statements)

References 53 publications

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“…2B). The consensus secondary structure determined here agrees with earlier phylogenetic work based on a much smaller set of group M sequences (12,13). Folding of consensus sequences from individual subtypes further supports that the ability to form a stem loop structure is a phylogenetic feature of this region of env wherein a perfectly base-paired lower helix and purine-rich apical loop are the most conserved secondary structural elements (Fig.…”

Section: The Iss Element Folds Into a Phylogenetically Conserved Stemsupporting

confidence: 88%

“…1). Cellular proteins hnRNP A1 and SRSF1 (ASF/SF2) were identified as the trans repressor and activator of ssA7 by forming complexes with their respective silencer and enhancer elements (12,13). Interactions between hnRNP A1 and ssA7 effectively block early spliceosome assembly, whereas SRSF1 counteracts the block by stabilizing U2AF loading at the 3Ј splice site (5,7,8).…”

Section: Human Immunodeficiency Virus Type 1 (Hiv-1)mentioning

confidence: 99%

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Solution Structure of the HIV-1 Intron Splicing Silencer and Its Interactions with the UP1 Domain of Heterogeneous Nuclear Ribonucleoprotein (hnRNP) A1

Jain

Morgan

Rife

et al. 2016

Journal of Biological Chemistry

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Splicing patterns in human immunodeficiency virus type 1 (HIV-1) are maintained through cis regulatory elements that recruit antagonistic host RNA-binding proteins. The activity of the 3 acceptor site A7 is tightly regulated through a complex network of an intronic splicing silencer (ISS), a bipartite exonic splicing silencer (ESS3a/b), and an exonic splicing enhancer (ESE3). Because HIV-1 splicing depends on protein-RNA interactions, it is important to know the tertiary structures surrounding the splice sites. Herein, we present the NMR solution structure of the phylogenetically conserved ISS stem loop. ISS adopts a stable structure consisting of conserved UG wobble pairs, a folded 2X2 (GU/UA) internal loop, a UU bulge, and a flexible AGUGA apical loop. Calorimetric and biochemical titrations indicate that the UP1 domain of heterogeneous nuclear ribonucleoprotein A1 binds the ISS apical loop site-specifically and with nanomolar affinity. Collectively, this work provides additional insights into how HIV-1 uses a conserved RNA structure to commandeer a host RNA-binding protein.Human immunodeficiency virus type 1 (HIV-1) 2 requires controlled synthesis of its protein complement for persistent infection and successful virion production. Genome expression is tightly regulated at the levels of transcription, splicing, mRNA nuclear export, and translation (1). RNA polymerase II-dependent transcription yields a 9-kilobase (kb) polycistronic transcript that undergoes multiple rounds of alternative splicing to produce upward of 100 different viral mRNAs that are classified by their intron composition: unspliced, incompletely spliced (4 kb), and completely spliced (2 kb) (2, 3). During early phase HIV-1 replication, cytoplasmic levels of the 2-kb transcripts predominate to encode Tat, Rev, and Nef. Accumulation of unspliced and 4-kb transcripts coincides with the transition to late phase replication and expression of Gag, Gag/Pol, Vpr, Vif, and Env/Vpu. Thus, HIV-1 splicing pathways are essential components of the viral replication cycle and represent new targets for therapeutic intervention (3, 4).The identities of HIV-1 transcripts are determined by the combinatorial use of several 5Ј donor and 3Ј acceptor splice sites. Efforts to understand mechanisms of HIV-1 splicing reveal that all of the acceptor sites and splice donors D2-D4 are suboptimal due to non-consensus core splicing signals (4, 5). Proper spliced ratios are therefore established via auxiliary cis RNA features, collectively known as splicing regulatory elements. Splicing regulatory elements function either as enhancers or silencers of splicing by recruiting host proteins belonging to the serine-arginine-rich (SR) and hnRNP families, respectively. Splicing regulatory element location (intronic or exonic) influences how frequently a given splice site is utilized, thereby making it difficult to determine general rules of the mechanisms that regulate alternative splicing (6).Splicing from site D4 to A7 removes the Rev-responsive element, which leads to the accu...

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Section: The Iss Element Folds Into a Phylogenetically Conserved Stemsupporting

confidence: 88%

Section: Human Immunodeficiency Virus Type 1 (Hiv-1)mentioning

confidence: 99%

Solution Structure of the HIV-1 Intron Splicing Silencer and Its Interactions with the UP1 Domain of Heterogeneous Nuclear Ribonucleoprotein (hnRNP) A1

Jain

Morgan

Rife

et al. 2016

Journal of Biological Chemistry

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“…Cooperation between an hnRNP A1-dependent ESS and an intronic site has been described in HIV tat exon 3 splicing (11,32). However, in this case the intronic site is located within the polypyrimidine tract at the 3Ј splice site, which suggests that repression is due to competition between hnRNP A1 and U2AF binding to the polypyrimidine tract.…”

Section: Discussionmentioning

confidence: 92%

“…Intronic and exonic hnRNP A1 binding sites function together to suppress HIV tat exon 3 splicing (11,32). We therefore next searched for the presence of the consensus hnRNP A1 binding motif, UAGNNA/U (29, 33), in intron 6 and especially intron 7 of SMN1 E and SMN2 E ( Fig.…”

Section: Identification Of Consensus Hnrnp A1 Binding Sites In Intronmentioning

confidence: 99%

An intronic element contributes to splicing repression in spinal muscular atrophy

Kashima

Rao

Manley

2007

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

116

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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“…Splice enhancers and silencers throughout the viral genome have been implicated in splicing regulation. [63][64][65][66] We studied the potential regulatory role of RNA structure as the major 5' splice site, located in the untranslated leader region of HIV-1 RNA, is embedded within a semi-stable stem-loop structure, the splice donor or SD hairpin. Indeed, we demonstrated that further stabilization of this structure did interfere with virus replication and a splicing defect was apparent in reporter constructs.…”

confidence: 99%

HIV-1 as RNA evolution machine

Berkhout

2011

RNA Biology

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hnRNP A1 controls HIV-1 mRNA splicing through cooperative binding to intron and exon splicing silencers in the context of a conserved secondary structure

Cited by 108 publications

References 53 publications

Solution Structure of the HIV-1 Intron Splicing Silencer and Its Interactions with the UP1 Domain of Heterogeneous Nuclear Ribonucleoprotein (hnRNP) A1

Solution Structure of the HIV-1 Intron Splicing Silencer and Its Interactions with the UP1 Domain of Heterogeneous Nuclear Ribonucleoprotein (hnRNP) A1

An intronic element contributes to splicing repression in spinal muscular atrophy

HIV-1 as RNA evolution machine

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