Control of HIV-1 env RNA splicing and transport: investigating the role of hnRNP A1 in exon splicing silencer (ESS3a) function

Asai, Kengo; Platt, Craig D.; Cochrane, Alan

doi:10.1016/s0042-6822(03)00400-8

Cited by 29 publications

(33 citation statements)

References 51 publications

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“…Previously, we demonstrated that the ESS3 within the terminal exon of HIV-1 not only inhibits use of the adjacent 3Ј ss but also blocks Rev-mediated export of the unspliced viral RNA to the cytoplasm (Pongoski et al 2002;Asai et al 2003). This block to Rev-mediated export was not due solely to the inhibition of splicing because replacement of ESS3 with high affinity hnRNP A1-binding sites was found to yield a comparable reduction in splicing but did not inhibit RNA transport (Asai et al 2003).…”

Section: Resultsmentioning

confidence: 91%

“…This block to Rev-mediated export was not due solely to the inhibition of splicing because replacement of ESS3 with high affinity hnRNP A1-binding sites was found to yield a comparable reduction in splicing but did not inhibit RNA transport (Asai et al 2003). Consequently, to identify the basis for the ESS3-induced block to unspliced RNA transport, our focus shifted to events other than splicing that are implicated in controlling RNA nuclear/cytoplasmic transport.…”

Section: Resultsmentioning

confidence: 99%

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A novel function for Sam68: Enhancement of HIV-1 RNA 3′ end processing

McLaren

Asai²,

Cochrane³

2004

RNA

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Both cis elements and host cell proteins can significantly affect HIV-1 RNA processing and viral gene expression. Previously, we determined that the exon splicing silencer (ESS3) within the terminal exon of HIV-1 not only reduces use of the adjacent 3 splice site but also prevents Rev-induced export of the unspliced viral RNA to the cytoplasm. In this report, we demonstrate that loss of unspliced viral RNA export is correlated with the inhibition of 3 end processing by the ESS3. Furthermore, we find that the host factor Sam68, a stimulator of HIV-1 protein expression, is able to reverse the block to viral RNA export mediated by the ESS3. The reversal is associated with a stimulation of 3 end processing of the unspliced viral RNA. Our findings identify a novel activity for the ESS3 and Sam68 in regulating HIV-1 RNA polyadenylation. Furthermore, the observations provide an explanation for how Sam68, an exclusively nuclear protein, modulates cytoplasmic utilization of the affected RNAs. Our finding that Sam68 is also able to enhance 3 end processing of a heterologous RNA raises the possibility that it may play a similar role in regulating host gene expression.

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Section: Resultsmentioning

confidence: 91%

Section: Resultsmentioning

confidence: 99%

A novel function for Sam68: Enhancement of HIV-1 RNA 3′ end processing

McLaren

Asai²,

Cochrane³

2004

RNA

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“…The Western Lightning Chemiluminescence Reagent Plus kit (PerkinElmer Life Sciences) was used, according to the manufacturer's instructions. For cell fractionation studies, the nuclear and cytoplasmic fractions were prepared as previously described prior to Western analysis (39). For IRES activity determinations, cells were harvested with passive lysis buffer (Promega, Madison, WI), and 20 g of cellular protein was used to assess IRES activity with the dual-luciferase reporter assay system (Promega) according to the manufactur-er's instructions.…”

Section: Methodsmentioning

confidence: 99%

Human Immunodeficiency Virus Type 1 (HIV-1) Induces the Cytoplasmic Retention of Heterogeneous Nuclear Ribonucleoprotein A1 by Disrupting Nuclear Import

Monette

Ajamian

López-Lastra

et al. 2009

Journal of Biological Chemistry

143

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Human immunodeficiency virus type 1 (HIV-1) co-opts host proteins and cellular machineries to its advantage at every step of the replication cycle. Here we show that HIV-1 enhances heterogeneous nuclear ribonucleoprotein (hnRNP) A1 expression and promotes the relocalization of hnRNP A1 to the cytoplasm. The latter was dependent on the nuclear export of the unspliced viral genomic RNA (vRNA) and to alterations in the abundance and localization of the FG-repeat nuclear pore glycoprotein p62. hnRNP A1 and vRNA remain colocalized in the cytoplasm supporting a post-nuclear function during the late stages of HIV-1 replication. Consistently, we show that hnRNP A1 acts as an internal ribosomal entry site trans-acting factor up-regulating internal ribosome entry site-mediated translation initiation of the HIV-1 vRNA. The up-regulation and cytoplasmic retention of hnRNP A1 by HIV-1 would ensure abundant expression of viral structural proteins in cells infected with HIV-1.During the late stages of the HIV-1 3 replication cycle, the full-length HIV-1 viral RNA (vRNA) must be exported from the nucleus and both translated and packaged into new viral particles (1). The orchestration of these events is directed by a diversity of viral and host proteins that interact with each other and with the vRNA to form HIV-1 ribonucleoprotein (RNP) complexes that originate in the nucleus and persist in the cytoplasm (2). Investigations into the composition and functions of the HIV-1 RNP will reveal new information about innate immunity as well as identify new potential therapeutic targets (3-6).The heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is a predominantly nuclear protein engaged in a number of cellular and viral RNPs for RNA-processing activities, including splicing regulation, nuclear export, microRNA processing, mRNA stability, telomere maintenance, and IRES-mediated translation initiation (7-12). What enables hnRNP A1 to have such broad functions in RNA metabolism is its ability to bind both nuclear and cytoplasmic RNAs (10). In addition to its well characterized role in nuclear RNA processing, hnRNP A1 binds to purine-rich sequences of mRNAs for mRNA turnover and translation (13,14). Close examination of the HIV-1 vRNA reveals many AG-and AU-rich sequences closely resembling hnRNP A1 binding motifs (15). In fact, hnRNP A1 binds to a number of sequences on the HIV-1 vRNA such as the cis-acting repressive sequences, instability elements, and exonic splicing silencer elements, and indeed, hnRNP A1 is implicated in the fate of HIV-1 RNA, including splicing regulation, nucleocytoplasmic export, and cytoplasmic stability (16 -21).Our previous work demonstrated that hnRNP A1 efficiently immunoprecipitated with the HIV-1 vRNA (22) and that siRNA-mediated knockdown of hnRNP A1 caused a dramatic decrease in HIV-1 structural protein, pr55Gag (herein termed Gag) expression and virus production with little effect on steady-state levels of the three HIV-1 RNA species (i.e. 9-, 4-, and 2-kb RNAs) (23). In this work, we show that HIV-1 ...

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“…Retroviruses use many general SREs to regulate splicing of their RNA (for review, see Stoltzfus and Madsen 2006) and also use RNA elements to regulate nuclear export of unspliced RNA (for reviews, see Pollard and Malim 1998;Cullen 2003). How HIV RNAs balance between the two pathways is not completely clear, but some common elements might serve as a functional bridge between the two pathways by participating in the regulation of both splicing and RNA export (e.g., an ESS recognized by hnRNP A1 [Asai et al 2003] and the Rev response elements [Pongoski et al 2002]). …”

Section: Elements That Regulate Intron Retentionmentioning

confidence: 99%

Splicing regulation: From a parts list of regulatory elements to an integrated splicing code

Wang¹,

Burge²

2008

RNA

881

761

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Alternative splicing of pre-mRNAs is a major contributor to both proteomic diversity and control of gene expression levels. Splicing is tightly regulated in different tissues and developmental stages, and its disruption can lead to a wide range of human diseases. An important long-term goal in the splicing field is to determine a set of rules or ''code'' for splicing that will enable prediction of the splicing pattern of any primary transcript from its sequence. Outside of the core splice site motifs, the bulk of the information required for splicing is thought to be contained in exonic and intronic cis-regulatory elements that function by recruitment of sequence-specific RNA-binding protein factors that either activate or repress the use of adjacent splice sites. Here, we summarize the current state of knowledge of splicing cis-regulatory elements and their context-dependent effects on splicing, emphasizing recent global/genome-wide studies and open questions.Keywords: context dependence; pre-mRNA splicing; splicing code; splicing factor; splicing regulation PRE-MRNA SPLICING Because human genes typically contain multiple introns, the process of pre-mRNA splicing is an essential step in the expression of most genes. A majority of human genes undergo alternative splicing (AS), generating multiple splicing isoforms containing different combinations of exons (Johnson et al. 2003). The effects of AS on protein products can be dramatic, e.g., producing soluble versus membranebound forms of the Fas receptor that have opposing effects on apoptosis (Cascino et al. 1995), or producing isoforms of the Drosophila fruitless protein that act to specify sexual orientation (Demir and Dickson 2005). The major forms of AS are summarized in Figure 1A. Splicing regulation has been comprehensively described in several recent reviews (Black 2003;Konarska and Query 2005;Matlin et al. 2005;Blencowe 2006;House and Lynch 2008). Here, we briefly summarize some aspects of splicing specificity and regulation before turning to our main topic of splicing regulatory elements and the rules governing their activity.The sequential phosphodiester transfer reactions involved in splicing are catalyzed by large ribonucleoprotein complexes known as spliceosomes. Containing more than 100 core proteins and five small nuclear RNAs (snRNAs U1, U2, U4, U5, and U6), spliceosomes may be the most complex machines in the cell (Zhou et al. 2002;Jurica and Moore 2003;Nilsen 2003). In addition to these core factors, additional regulatory proteins participate in the splicing of particular pre-mRNAs. Splicing of most introns is thought to occur cotranscriptionally with fairly extensive interactions between splicing factors and the core transcription machinery ( CORE SPLICING SIGNALSThree sites, the 59 splice site (59ss), the 39 splice site (39ss), and the branch point sequence (BPS), participate in the splicing reaction and are present in every intron, and thus are known as the core splicing signals. These signals are recognized multiple times during spl...

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Control of HIV-1 env RNA splicing and transport: investigating the role of hnRNP A1 in exon splicing silencer (ESS3a) function

Cited by 29 publications

References 51 publications

A novel function for Sam68: Enhancement of HIV-1 RNA 3′ end processing

A novel function for Sam68: Enhancement of HIV-1 RNA 3′ end processing

Human Immunodeficiency Virus Type 1 (HIV-1) Induces the Cytoplasmic Retention of Heterogeneous Nuclear Ribonucleoprotein A1 by Disrupting Nuclear Import

Splicing regulation: From a parts list of regulatory elements to an integrated splicing code

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