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Martin C Stoltzfus - One of the best experts on this subject based on the ideXlab platform.
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an Exonic Splicing Silencer downstream of the 3 splice site a2 is required for efficient human immunodeficiency virus type 1 replication
Journal of Virology, 2005Co-Authors: Joshua M Madsen, Martin C StoltzfusAbstract:Alternative Splicing of the human immunodeficiency virus type 1 (HIV-1) genomic mRNA produces more than 40 unique viral mRNA species, of which more than half remain incompletely spliced within an HIV-1-infected cell. Regulation of Splicing at HIV-1 3' splice sites (3'ss) requires suboptimal polypyrimidine tracts, and positive or negative regulation of Splicing occurs through binding of cellular factors to cis-acting Splicing regulatory elements. We have previously shown that Splicing at HIV-1 3'ss A2, which produces vpr mRNA and promotes inclusion of HIV-1 exon 3, is repressed by the hnRNP A/B-dependent Exonic Splicing Silencer ESSV. Here we show that ESSV activity downstream of 3'ss A2 is localized to a 16-nucleotide element within HIV-1 exon 3. HIV-1 replication was reduced by 95% when ESSV was inactivated by mutagenesis. Reduced replication was concomitant with increased inclusion of exon 3 within spliced viral mRNA and decreased accumulation of unspliced viral mRNA, resulting in decreased cell-associated p55 Gag. Prolonged culture of ESSV mutant viruses resulted in two independent second-site reversions disrupting the splice sites that define exon 3, 3'ss A2 and 5' splice site D3. Either of these changes restored both HIV-1 replication and regulated viral Splicing. Therefore, inhibition of HIV-1 3'ss A2 Splicing is necessary for HIV-1 replication.
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human immunodeficiency virus type 1 hnrnp a b dependent Exonic Splicing Silencer essv antagonizes binding of u2af65 to viral polypyrimidine tracts
Molecular and Cellular Biology, 2003Co-Authors: Jeffrey K Domsic, Akila Mayeda, Adrian R Krainer, Yibin Wang, Martin C StoltzfusAbstract:Human immunodeficiency virus type 1 (HIV-1) Exonic Splicing Silencers (ESSs) inhibit production of certain spliced viral RNAs by repressing alternative Splicing of the viral precursor RNA. Several HIV-1 ESSs interfere with spliceosome assembly by binding cellular hnRNP A/B proteins. Here, we have further characterized the mechanism of Splicing repression using a representative HIV-1 hnRNP A/B-dependent ESS, ESSV, which regulates Splicing at the vpr 3' splice site. We show that hnRNP A/B proteins bound to ESSV are necessary to inhibit E complex assembly by competing with the binding of U2AF65 to the polypyrimidine tracts of repressed 3' splice sites. We further show evidence suggesting that U1 snRNP binds the 5' splice site despite an almost complete block of Splicing by ESSV. Possible Splicing-independent functions of U1 snRNP-5' splice site interactions during virus replication are discussed.
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rna Splicing at human immunodeficiency virus type 1 3 splice site a2 is regulated by binding of hnrnp a b proteins to an Exonic Splicing Silencer element
Journal of Virology, 2001Co-Authors: Patricia S Bilodeau, Jeffrey K Domsic, Akila Mayeda, Adrian R Krainer, Martin C StoltzfusAbstract:Both simple and complex retroviruses require Splicing of a single primary RNA transcript in order to generate mRNA for the viral envelope protein (Env). Complex retroviruses, such as human immunodeficiency virus type 1 (HIV-1), require the production of additional mRNAs for regulatory and accessory proteins. For HIV-1 these include mRNAs for Tat, Rev, Vif, Vpr, and Nef (6, 19, 35, 37, 39). The Rev protein binds to RNAs containing the Rev-responsive element in the env gene sequence. This interaction facilitates nuclear export of unspliced and partially spliced RNAs required for translation and for packaging into progeny virions (16, 17, 20, 21, 30; for a recent review, see reference 12). Early in infection of cells with HIV-1 and prior to the accumulation of Rev, multiply spliced mRNAs predominate in the cytoplasm. Later in infection, the production of Rev allows the cytoplasmic accumulation of unspliced and partially spliced RNAs (24, 25) In order to generate mRNAs required for the synthesis of viral proteins, HIV-1 primary RNA transcripts undergo a complex Splicing process (Fig. (Fig.1).1). The viral RNA contains both constitutive and alternative 5′ and 3′ splice sites. All spliced mRNAs contain 5′-terminal noncoding exon 1, which is flanked by consensus 5′ splice site D1. Selection of the alternative 3′ splice sites near the middle of the genome determines which proteins are encoded by the mRNAs. Two size classes of spliced RNAs are produced, depending on the removal of the intron spanning D4 to A7 (∼1.8 kb for the small size class and ∼4 kb for the intermediate size class). For instance, Splicing at A3 coupled with Splicing at D4 to A7 generates ∼1.8-kb Tat mRNA. Similarly, Splicing at A4a, A4b, or A4c coupled with Splicing at D4 to A7 generates ∼1.8-kb Rev mRNA; Splicing at A5 coupled with Splicing at D4 to A7 generates ∼1.8-kb Nef mRNA. Splicing at A3 generates an ∼4-kb mRNA encoding a single-exon form of Tat. Splicing of mRNAs at A4a, A4b, A4c, and A5 generates ∼4-kb mRNAs encoding Env. Splicing at A1 and A2 generates ∼4-kb mRNAs encoding Vif and Vpr, respectively. As a further complexity, some mRNAs of both size classes include one or both of two alternative noncoding exons (Fig. (Fig.1B):1B): exon 2, which is flanked by A1 and D2, and exon 3, which is flanked by A2 and D3 (18, 35, 39). Finally, some virus strains contain within the env gene cryptic splice sites (D5 and A6) whose usage results in the synthesis of an mRNA encoding a hybrid protein, Tev (8, 38). FIG. 1 (A) Structure of the HIV-1 NL4-3 genome. Boxes indicate open reading frames. Hash marks represent endpoints of gag-pol deletion in pΔPSP. ESS sequences are shown by shaded boxes. Oligonucleotide primers used are indicated by arrows designating ... Different spliced HIV-1 mRNAs are generated with very different efficiencies. For example, ∼1.8-kb mRNAs encoding Tat are present at low levels compared to mRNAs encoding Rev or Nef. Similarly, ∼4-kb mRNAs encoding single-exon Tat are present at low levels compared to mRNAs encoding Env (35). It was previously shown that splice site A3 is repressed by ESS2, an Exonic Splicing Silencer (ESS) within the first tat coding exon (exon 4 in Fig. Fig.1).1). Mutations within the ESS2 element result in a selective increase in Splicing at A3 (3, 4). A second ESS (ESS3) was identified within the second tat/rev coding exon downstream of A7 (exon 7 in Fig. Fig.1).1). In this case, an adjacent upstream Exonic Splicing enhancer is juxtaposed to the ESS (4, 43). Both ESS2 and ESS3 appear to bind to a common cellular factor or factors that act to repress Splicing (42). It has been reported that ESS2 selectively binds to members of the A/B hnRNP family (hnRNPs A1, A1B, A2, and B1) (11, 14). The addition of hnRNP A1 and other members of the hnRNP A/B protein family restores specific Splicing repression in HeLa cell nuclear extracts depleted of ESS2-binding proteins (11). A third potential ESS is present in the env gene, where it may prevent the activation of cryptic exon 6D, which is bordered by splice sites A6 and D5 (46). The levels of Vpr mRNA singly spliced at 3′ splice site A2 also have been shown to be low in cells infected with HIV-1, indicating that Splicing at A2 is inefficient. Furthermore, noncoding exon 3 (Fig. (Fig.1)1) is skipped in the majority of the mRNAs (35). This exon skipping also suggests that splice site A2 or D3 or both of these splice sites are used inefficiently. It has been shown that the branch point used for Splicing at splice site A2 is a G rather than the consensus A that is used for most 3′ splice sites. However, replacing the nonconsensus wild-type branch-point sequence with a consensus sequence did not significantly affect Splicing efficiency in an in vitro Splicing system (13). In this report, we describe additional elements downstream of 3′ splice site A2 that act to repress Splicing at this splice site.
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rna Splicing at human immunodeficiency virus type 1 3 splice site a2 is regulated by binding of hnrnp a b proteins to an Exonic Splicing Silencer element
Journal of Virology, 2001Co-Authors: Patricia S Bilodeau, Jeffrey K Domsic, Akila Mayeda, Adrian R Krainer, Martin C StoltzfusAbstract:The synthesis of human immunodeficiency virus type 1 (HIV-1) mRNAs is a complex process by which more than 30 different mRNA species are produced by alternative Splicing of a single primary RNA transcript. HIV-1 splice sites are used with significantly different efficiencies, resulting in different levels of mRNA species in infected cells. Splicing of Tat mRNA, which is present at relatively low levels in infected cells, is repressed by the presence of Exonic Splicing Silencers (ESS) within the two tat coding exons (ESS2 and ESS3). These ESS elements contain the consensus sequence PyUAG. Here we show that the efficiency of Splicing at 3' splice site A2, which is used to generate Vpr mRNA, is also regulated by the presence of an ESS (ESSV), which has sequence homology to ESS2 and ESS3. Mutagenesis of the three PyUAG motifs within ESSV increases Splicing at splice site A2, resulting in increased Vpr mRNA levels and reduced skipping of the noncoding exon flanked by A2 and D3. The increase in Vpr mRNA levels and the reduced skipping also occur when splice site D3 is mutated toward the consensus sequence. By in vitro Splicing assays, we show that ESSV represses Splicing when placed downstream of a heterologous splice site. A1, A1(B), A2, and B1 hnRNPs preferentially bind to ESSV RNA compared to ESSV mutant RNA. Each of these proteins, when added back to HeLa cell nuclear extracts depleted of ESSV-binding factors, is able to restore Splicing repression. The results suggest that coordinate repression of HIV-1 RNA Splicing is mediated by members of the hnRNP A/B protein family.
Adrian R Krainer - One of the best experts on this subject based on the ideXlab platform.
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An Exonic Splicing enhancer is required for Splicing of IgM M1-M2 with ΔNΔRS.
2013Co-Authors: Stephanie D. Shaw, Sutapa Chakrabarti, Gourisankar Ghosh, Adrian R KrainerAbstract:In vitro Splicing of IgM M1-M2 and derivative pre-mRNAs with mutations in the polypyrimidine tract, Exonic Splicing enhancer, and/or Exonic Splicing Silencer: IgM M1-M2 (lanes 1-3), IgMΔE (lanes 4–6), IgMPy↑ (lanes 7–9), IgMPy↑ΔE (lanes 10–12), IgMPTB (lanes 13–15), IgMΔEPTB (lanes 16–18), IgMPy↑PTB (lanes 19–21), and IgMPy↑ΔE PTB (lanes 22–24, mRNA position indicated by asterisk); in S100 alone (lanes 1, 4, 7, 10, 13, 16, 19, and 22), and S100 complemented with 16 pmol of SF2/ASF (lanes 2, 5, 8, 11, 14, 17, 20, and 23), or ΔNΔRS (lanes 3, 6, 9, 12, 15, 18, 21, and 24). The Splicing efficiency is indicated below each lane.
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determinants of exon 7 Splicing in the spinal muscular atrophy genes smn1 and smn2
American Journal of Human Genetics, 2006Co-Authors: Luca Cartegni, John A Calarco, Elisa De Stanchina, Michelle L Hastings, Adrian R KrainerAbstract:Spinal muscular atrophy is a neurodegenerative disorder caused by the deletion or mutation of the survival-of-motor-neuron gene, SMN1. An SMN1 paralog, SMN2, differs by a C→T transition in exon 7 that causes substantial skipping of this exon, such that SMN2 expresses only low levels of functional protein. A better understanding of SMN Splicing mechanisms should facilitate the development of drugs that increase survival motor neuron (SMN) protein levels by improving SMN2 exon 7 inclusion. In addition, Exonic mutations that cause defective Splicing give rise to many genetic diseases, and the SMN1/2 system is a useful paradigm for understanding exon-identity determinants and alternative-Splicing mechanisms. Skipping of SMN2 exon 7 was previously attributed either to the loss of an SF2/ASF–dependent Exonic Splicing enhancer or to the creation of an hnRNP A/B–dependent Exonic Splicing Silencer, as a result of the C→T transition. We report the extensive testing of the enhancer-loss and Silencer-gain models by mutagenesis, RNA interference, overexpression, RNA Splicing, and RNA-protein interaction experiments. Our results support the enhancer-loss model but also demonstrate that hnRNP A/B proteins antagonize SF2/ASF–dependent ESE activity and promote exon 7 skipping by a mechanism that is independent of the C→T transition and is, therefore, common to both SMN1 and SMN2. Our findings explain the basis of defective SMN2 Splicing, illustrate the fine balance between positive and negative determinants of exon identity and alternative Splicing, and underscore the importance of antagonistic Splicing factors and Exonic elements in a disease context.
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human immunodeficiency virus type 1 hnrnp a b dependent Exonic Splicing Silencer essv antagonizes binding of u2af65 to viral polypyrimidine tracts
Molecular and Cellular Biology, 2003Co-Authors: Jeffrey K Domsic, Akila Mayeda, Adrian R Krainer, Yibin Wang, Martin C StoltzfusAbstract:Human immunodeficiency virus type 1 (HIV-1) Exonic Splicing Silencers (ESSs) inhibit production of certain spliced viral RNAs by repressing alternative Splicing of the viral precursor RNA. Several HIV-1 ESSs interfere with spliceosome assembly by binding cellular hnRNP A/B proteins. Here, we have further characterized the mechanism of Splicing repression using a representative HIV-1 hnRNP A/B-dependent ESS, ESSV, which regulates Splicing at the vpr 3' splice site. We show that hnRNP A/B proteins bound to ESSV are necessary to inhibit E complex assembly by competing with the binding of U2AF65 to the polypyrimidine tracts of repressed 3' splice sites. We further show evidence suggesting that U1 snRNP binds the 5' splice site despite an almost complete block of Splicing by ESSV. Possible Splicing-independent functions of U1 snRNP-5' splice site interactions during virus replication are discussed.
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exon identity established through differential antagonism between Exonic Splicing Silencer bound hnrnp a1 and enhancer bound sr proteins
Molecular Cell, 2001Co-Authors: Akila Mayeda, Jun Zhu, Adrian R KrainerAbstract:SR proteins recognize Exonic Splicing enhancer (ESE) elements and promote exon use, whereas certain hnRNP proteins bind to Exonic Splicing Silencer (ESS) elements and block exon recognition. We investigated how ESS3 in HIV-1 tat exon 3 blocks Splicing promoted by one SR protein (SC35) but not another (SF2/ASF). hnRNP A1 mediates silencing by binding initially to a required high-affinity site in ESS3, which then promotes further hnRNP A1 association with the upstream region of the exon. Both SC35 and SF2/ASF recognize upstream ESE motifs, but only SF2/ASF prevents secondary hnRNP A1 binding, presumably by blocking its cooperative propagation along the exon. The differential antagonism between a negative and two positive regulators exemplifies how inclusion of an alternative exon can be modulated.
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rna Splicing at human immunodeficiency virus type 1 3 splice site a2 is regulated by binding of hnrnp a b proteins to an Exonic Splicing Silencer element
Journal of Virology, 2001Co-Authors: Patricia S Bilodeau, Jeffrey K Domsic, Akila Mayeda, Adrian R Krainer, Martin C StoltzfusAbstract:Both simple and complex retroviruses require Splicing of a single primary RNA transcript in order to generate mRNA for the viral envelope protein (Env). Complex retroviruses, such as human immunodeficiency virus type 1 (HIV-1), require the production of additional mRNAs for regulatory and accessory proteins. For HIV-1 these include mRNAs for Tat, Rev, Vif, Vpr, and Nef (6, 19, 35, 37, 39). The Rev protein binds to RNAs containing the Rev-responsive element in the env gene sequence. This interaction facilitates nuclear export of unspliced and partially spliced RNAs required for translation and for packaging into progeny virions (16, 17, 20, 21, 30; for a recent review, see reference 12). Early in infection of cells with HIV-1 and prior to the accumulation of Rev, multiply spliced mRNAs predominate in the cytoplasm. Later in infection, the production of Rev allows the cytoplasmic accumulation of unspliced and partially spliced RNAs (24, 25) In order to generate mRNAs required for the synthesis of viral proteins, HIV-1 primary RNA transcripts undergo a complex Splicing process (Fig. (Fig.1).1). The viral RNA contains both constitutive and alternative 5′ and 3′ splice sites. All spliced mRNAs contain 5′-terminal noncoding exon 1, which is flanked by consensus 5′ splice site D1. Selection of the alternative 3′ splice sites near the middle of the genome determines which proteins are encoded by the mRNAs. Two size classes of spliced RNAs are produced, depending on the removal of the intron spanning D4 to A7 (∼1.8 kb for the small size class and ∼4 kb for the intermediate size class). For instance, Splicing at A3 coupled with Splicing at D4 to A7 generates ∼1.8-kb Tat mRNA. Similarly, Splicing at A4a, A4b, or A4c coupled with Splicing at D4 to A7 generates ∼1.8-kb Rev mRNA; Splicing at A5 coupled with Splicing at D4 to A7 generates ∼1.8-kb Nef mRNA. Splicing at A3 generates an ∼4-kb mRNA encoding a single-exon form of Tat. Splicing of mRNAs at A4a, A4b, A4c, and A5 generates ∼4-kb mRNAs encoding Env. Splicing at A1 and A2 generates ∼4-kb mRNAs encoding Vif and Vpr, respectively. As a further complexity, some mRNAs of both size classes include one or both of two alternative noncoding exons (Fig. (Fig.1B):1B): exon 2, which is flanked by A1 and D2, and exon 3, which is flanked by A2 and D3 (18, 35, 39). Finally, some virus strains contain within the env gene cryptic splice sites (D5 and A6) whose usage results in the synthesis of an mRNA encoding a hybrid protein, Tev (8, 38). FIG. 1 (A) Structure of the HIV-1 NL4-3 genome. Boxes indicate open reading frames. Hash marks represent endpoints of gag-pol deletion in pΔPSP. ESS sequences are shown by shaded boxes. Oligonucleotide primers used are indicated by arrows designating ... Different spliced HIV-1 mRNAs are generated with very different efficiencies. For example, ∼1.8-kb mRNAs encoding Tat are present at low levels compared to mRNAs encoding Rev or Nef. Similarly, ∼4-kb mRNAs encoding single-exon Tat are present at low levels compared to mRNAs encoding Env (35). It was previously shown that splice site A3 is repressed by ESS2, an Exonic Splicing Silencer (ESS) within the first tat coding exon (exon 4 in Fig. Fig.1).1). Mutations within the ESS2 element result in a selective increase in Splicing at A3 (3, 4). A second ESS (ESS3) was identified within the second tat/rev coding exon downstream of A7 (exon 7 in Fig. Fig.1).1). In this case, an adjacent upstream Exonic Splicing enhancer is juxtaposed to the ESS (4, 43). Both ESS2 and ESS3 appear to bind to a common cellular factor or factors that act to repress Splicing (42). It has been reported that ESS2 selectively binds to members of the A/B hnRNP family (hnRNPs A1, A1B, A2, and B1) (11, 14). The addition of hnRNP A1 and other members of the hnRNP A/B protein family restores specific Splicing repression in HeLa cell nuclear extracts depleted of ESS2-binding proteins (11). A third potential ESS is present in the env gene, where it may prevent the activation of cryptic exon 6D, which is bordered by splice sites A6 and D5 (46). The levels of Vpr mRNA singly spliced at 3′ splice site A2 also have been shown to be low in cells infected with HIV-1, indicating that Splicing at A2 is inefficient. Furthermore, noncoding exon 3 (Fig. (Fig.1)1) is skipped in the majority of the mRNAs (35). This exon skipping also suggests that splice site A2 or D3 or both of these splice sites are used inefficiently. It has been shown that the branch point used for Splicing at splice site A2 is a G rather than the consensus A that is used for most 3′ splice sites. However, replacing the nonconsensus wild-type branch-point sequence with a consensus sequence did not significantly affect Splicing efficiency in an in vitro Splicing system (13). In this report, we describe additional elements downstream of 3′ splice site A2 that act to repress Splicing at this splice site.
Akila Mayeda - One of the best experts on this subject based on the ideXlab platform.
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human immunodeficiency virus type 1 hnrnp a b dependent Exonic Splicing Silencer essv antagonizes binding of u2af65 to viral polypyrimidine tracts
Molecular and Cellular Biology, 2003Co-Authors: Jeffrey K Domsic, Akila Mayeda, Adrian R Krainer, Yibin Wang, Martin C StoltzfusAbstract:Human immunodeficiency virus type 1 (HIV-1) Exonic Splicing Silencers (ESSs) inhibit production of certain spliced viral RNAs by repressing alternative Splicing of the viral precursor RNA. Several HIV-1 ESSs interfere with spliceosome assembly by binding cellular hnRNP A/B proteins. Here, we have further characterized the mechanism of Splicing repression using a representative HIV-1 hnRNP A/B-dependent ESS, ESSV, which regulates Splicing at the vpr 3' splice site. We show that hnRNP A/B proteins bound to ESSV are necessary to inhibit E complex assembly by competing with the binding of U2AF65 to the polypyrimidine tracts of repressed 3' splice sites. We further show evidence suggesting that U1 snRNP binds the 5' splice site despite an almost complete block of Splicing by ESSV. Possible Splicing-independent functions of U1 snRNP-5' splice site interactions during virus replication are discussed.
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exon identity established through differential antagonism between Exonic Splicing Silencer bound hnrnp a1 and enhancer bound sr proteins
Molecular Cell, 2001Co-Authors: Akila Mayeda, Jun Zhu, Adrian R KrainerAbstract:SR proteins recognize Exonic Splicing enhancer (ESE) elements and promote exon use, whereas certain hnRNP proteins bind to Exonic Splicing Silencer (ESS) elements and block exon recognition. We investigated how ESS3 in HIV-1 tat exon 3 blocks Splicing promoted by one SR protein (SC35) but not another (SF2/ASF). hnRNP A1 mediates silencing by binding initially to a required high-affinity site in ESS3, which then promotes further hnRNP A1 association with the upstream region of the exon. Both SC35 and SF2/ASF recognize upstream ESE motifs, but only SF2/ASF prevents secondary hnRNP A1 binding, presumably by blocking its cooperative propagation along the exon. The differential antagonism between a negative and two positive regulators exemplifies how inclusion of an alternative exon can be modulated.
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rna Splicing at human immunodeficiency virus type 1 3 splice site a2 is regulated by binding of hnrnp a b proteins to an Exonic Splicing Silencer element
Journal of Virology, 2001Co-Authors: Patricia S Bilodeau, Jeffrey K Domsic, Akila Mayeda, Adrian R Krainer, Martin C StoltzfusAbstract:Both simple and complex retroviruses require Splicing of a single primary RNA transcript in order to generate mRNA for the viral envelope protein (Env). Complex retroviruses, such as human immunodeficiency virus type 1 (HIV-1), require the production of additional mRNAs for regulatory and accessory proteins. For HIV-1 these include mRNAs for Tat, Rev, Vif, Vpr, and Nef (6, 19, 35, 37, 39). The Rev protein binds to RNAs containing the Rev-responsive element in the env gene sequence. This interaction facilitates nuclear export of unspliced and partially spliced RNAs required for translation and for packaging into progeny virions (16, 17, 20, 21, 30; for a recent review, see reference 12). Early in infection of cells with HIV-1 and prior to the accumulation of Rev, multiply spliced mRNAs predominate in the cytoplasm. Later in infection, the production of Rev allows the cytoplasmic accumulation of unspliced and partially spliced RNAs (24, 25) In order to generate mRNAs required for the synthesis of viral proteins, HIV-1 primary RNA transcripts undergo a complex Splicing process (Fig. (Fig.1).1). The viral RNA contains both constitutive and alternative 5′ and 3′ splice sites. All spliced mRNAs contain 5′-terminal noncoding exon 1, which is flanked by consensus 5′ splice site D1. Selection of the alternative 3′ splice sites near the middle of the genome determines which proteins are encoded by the mRNAs. Two size classes of spliced RNAs are produced, depending on the removal of the intron spanning D4 to A7 (∼1.8 kb for the small size class and ∼4 kb for the intermediate size class). For instance, Splicing at A3 coupled with Splicing at D4 to A7 generates ∼1.8-kb Tat mRNA. Similarly, Splicing at A4a, A4b, or A4c coupled with Splicing at D4 to A7 generates ∼1.8-kb Rev mRNA; Splicing at A5 coupled with Splicing at D4 to A7 generates ∼1.8-kb Nef mRNA. Splicing at A3 generates an ∼4-kb mRNA encoding a single-exon form of Tat. Splicing of mRNAs at A4a, A4b, A4c, and A5 generates ∼4-kb mRNAs encoding Env. Splicing at A1 and A2 generates ∼4-kb mRNAs encoding Vif and Vpr, respectively. As a further complexity, some mRNAs of both size classes include one or both of two alternative noncoding exons (Fig. (Fig.1B):1B): exon 2, which is flanked by A1 and D2, and exon 3, which is flanked by A2 and D3 (18, 35, 39). Finally, some virus strains contain within the env gene cryptic splice sites (D5 and A6) whose usage results in the synthesis of an mRNA encoding a hybrid protein, Tev (8, 38). FIG. 1 (A) Structure of the HIV-1 NL4-3 genome. Boxes indicate open reading frames. Hash marks represent endpoints of gag-pol deletion in pΔPSP. ESS sequences are shown by shaded boxes. Oligonucleotide primers used are indicated by arrows designating ... Different spliced HIV-1 mRNAs are generated with very different efficiencies. For example, ∼1.8-kb mRNAs encoding Tat are present at low levels compared to mRNAs encoding Rev or Nef. Similarly, ∼4-kb mRNAs encoding single-exon Tat are present at low levels compared to mRNAs encoding Env (35). It was previously shown that splice site A3 is repressed by ESS2, an Exonic Splicing Silencer (ESS) within the first tat coding exon (exon 4 in Fig. Fig.1).1). Mutations within the ESS2 element result in a selective increase in Splicing at A3 (3, 4). A second ESS (ESS3) was identified within the second tat/rev coding exon downstream of A7 (exon 7 in Fig. Fig.1).1). In this case, an adjacent upstream Exonic Splicing enhancer is juxtaposed to the ESS (4, 43). Both ESS2 and ESS3 appear to bind to a common cellular factor or factors that act to repress Splicing (42). It has been reported that ESS2 selectively binds to members of the A/B hnRNP family (hnRNPs A1, A1B, A2, and B1) (11, 14). The addition of hnRNP A1 and other members of the hnRNP A/B protein family restores specific Splicing repression in HeLa cell nuclear extracts depleted of ESS2-binding proteins (11). A third potential ESS is present in the env gene, where it may prevent the activation of cryptic exon 6D, which is bordered by splice sites A6 and D5 (46). The levels of Vpr mRNA singly spliced at 3′ splice site A2 also have been shown to be low in cells infected with HIV-1, indicating that Splicing at A2 is inefficient. Furthermore, noncoding exon 3 (Fig. (Fig.1)1) is skipped in the majority of the mRNAs (35). This exon skipping also suggests that splice site A2 or D3 or both of these splice sites are used inefficiently. It has been shown that the branch point used for Splicing at splice site A2 is a G rather than the consensus A that is used for most 3′ splice sites. However, replacing the nonconsensus wild-type branch-point sequence with a consensus sequence did not significantly affect Splicing efficiency in an in vitro Splicing system (13). In this report, we describe additional elements downstream of 3′ splice site A2 that act to repress Splicing at this splice site.
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rna Splicing at human immunodeficiency virus type 1 3 splice site a2 is regulated by binding of hnrnp a b proteins to an Exonic Splicing Silencer element
Journal of Virology, 2001Co-Authors: Patricia S Bilodeau, Jeffrey K Domsic, Akila Mayeda, Adrian R Krainer, Martin C StoltzfusAbstract:The synthesis of human immunodeficiency virus type 1 (HIV-1) mRNAs is a complex process by which more than 30 different mRNA species are produced by alternative Splicing of a single primary RNA transcript. HIV-1 splice sites are used with significantly different efficiencies, resulting in different levels of mRNA species in infected cells. Splicing of Tat mRNA, which is present at relatively low levels in infected cells, is repressed by the presence of Exonic Splicing Silencers (ESS) within the two tat coding exons (ESS2 and ESS3). These ESS elements contain the consensus sequence PyUAG. Here we show that the efficiency of Splicing at 3' splice site A2, which is used to generate Vpr mRNA, is also regulated by the presence of an ESS (ESSV), which has sequence homology to ESS2 and ESS3. Mutagenesis of the three PyUAG motifs within ESSV increases Splicing at splice site A2, resulting in increased Vpr mRNA levels and reduced skipping of the noncoding exon flanked by A2 and D3. The increase in Vpr mRNA levels and the reduced skipping also occur when splice site D3 is mutated toward the consensus sequence. By in vitro Splicing assays, we show that ESSV represses Splicing when placed downstream of a heterologous splice site. A1, A1(B), A2, and B1 hnRNPs preferentially bind to ESSV RNA compared to ESSV mutant RNA. Each of these proteins, when added back to HeLa cell nuclear extracts depleted of ESSV-binding factors, is able to restore Splicing repression. The results suggest that coordinate repression of HIV-1 RNA Splicing is mediated by members of the hnRNP A/B protein family.
Jeffrey K Domsic - One of the best experts on this subject based on the ideXlab platform.
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human immunodeficiency virus type 1 hnrnp a b dependent Exonic Splicing Silencer essv antagonizes binding of u2af65 to viral polypyrimidine tracts
Molecular and Cellular Biology, 2003Co-Authors: Jeffrey K Domsic, Akila Mayeda, Adrian R Krainer, Yibin Wang, Martin C StoltzfusAbstract:Human immunodeficiency virus type 1 (HIV-1) Exonic Splicing Silencers (ESSs) inhibit production of certain spliced viral RNAs by repressing alternative Splicing of the viral precursor RNA. Several HIV-1 ESSs interfere with spliceosome assembly by binding cellular hnRNP A/B proteins. Here, we have further characterized the mechanism of Splicing repression using a representative HIV-1 hnRNP A/B-dependent ESS, ESSV, which regulates Splicing at the vpr 3' splice site. We show that hnRNP A/B proteins bound to ESSV are necessary to inhibit E complex assembly by competing with the binding of U2AF65 to the polypyrimidine tracts of repressed 3' splice sites. We further show evidence suggesting that U1 snRNP binds the 5' splice site despite an almost complete block of Splicing by ESSV. Possible Splicing-independent functions of U1 snRNP-5' splice site interactions during virus replication are discussed.
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rna Splicing at human immunodeficiency virus type 1 3 splice site a2 is regulated by binding of hnrnp a b proteins to an Exonic Splicing Silencer element
Journal of Virology, 2001Co-Authors: Patricia S Bilodeau, Jeffrey K Domsic, Akila Mayeda, Adrian R Krainer, Martin C StoltzfusAbstract:Both simple and complex retroviruses require Splicing of a single primary RNA transcript in order to generate mRNA for the viral envelope protein (Env). Complex retroviruses, such as human immunodeficiency virus type 1 (HIV-1), require the production of additional mRNAs for regulatory and accessory proteins. For HIV-1 these include mRNAs for Tat, Rev, Vif, Vpr, and Nef (6, 19, 35, 37, 39). The Rev protein binds to RNAs containing the Rev-responsive element in the env gene sequence. This interaction facilitates nuclear export of unspliced and partially spliced RNAs required for translation and for packaging into progeny virions (16, 17, 20, 21, 30; for a recent review, see reference 12). Early in infection of cells with HIV-1 and prior to the accumulation of Rev, multiply spliced mRNAs predominate in the cytoplasm. Later in infection, the production of Rev allows the cytoplasmic accumulation of unspliced and partially spliced RNAs (24, 25) In order to generate mRNAs required for the synthesis of viral proteins, HIV-1 primary RNA transcripts undergo a complex Splicing process (Fig. (Fig.1).1). The viral RNA contains both constitutive and alternative 5′ and 3′ splice sites. All spliced mRNAs contain 5′-terminal noncoding exon 1, which is flanked by consensus 5′ splice site D1. Selection of the alternative 3′ splice sites near the middle of the genome determines which proteins are encoded by the mRNAs. Two size classes of spliced RNAs are produced, depending on the removal of the intron spanning D4 to A7 (∼1.8 kb for the small size class and ∼4 kb for the intermediate size class). For instance, Splicing at A3 coupled with Splicing at D4 to A7 generates ∼1.8-kb Tat mRNA. Similarly, Splicing at A4a, A4b, or A4c coupled with Splicing at D4 to A7 generates ∼1.8-kb Rev mRNA; Splicing at A5 coupled with Splicing at D4 to A7 generates ∼1.8-kb Nef mRNA. Splicing at A3 generates an ∼4-kb mRNA encoding a single-exon form of Tat. Splicing of mRNAs at A4a, A4b, A4c, and A5 generates ∼4-kb mRNAs encoding Env. Splicing at A1 and A2 generates ∼4-kb mRNAs encoding Vif and Vpr, respectively. As a further complexity, some mRNAs of both size classes include one or both of two alternative noncoding exons (Fig. (Fig.1B):1B): exon 2, which is flanked by A1 and D2, and exon 3, which is flanked by A2 and D3 (18, 35, 39). Finally, some virus strains contain within the env gene cryptic splice sites (D5 and A6) whose usage results in the synthesis of an mRNA encoding a hybrid protein, Tev (8, 38). FIG. 1 (A) Structure of the HIV-1 NL4-3 genome. Boxes indicate open reading frames. Hash marks represent endpoints of gag-pol deletion in pΔPSP. ESS sequences are shown by shaded boxes. Oligonucleotide primers used are indicated by arrows designating ... Different spliced HIV-1 mRNAs are generated with very different efficiencies. For example, ∼1.8-kb mRNAs encoding Tat are present at low levels compared to mRNAs encoding Rev or Nef. Similarly, ∼4-kb mRNAs encoding single-exon Tat are present at low levels compared to mRNAs encoding Env (35). It was previously shown that splice site A3 is repressed by ESS2, an Exonic Splicing Silencer (ESS) within the first tat coding exon (exon 4 in Fig. Fig.1).1). Mutations within the ESS2 element result in a selective increase in Splicing at A3 (3, 4). A second ESS (ESS3) was identified within the second tat/rev coding exon downstream of A7 (exon 7 in Fig. Fig.1).1). In this case, an adjacent upstream Exonic Splicing enhancer is juxtaposed to the ESS (4, 43). Both ESS2 and ESS3 appear to bind to a common cellular factor or factors that act to repress Splicing (42). It has been reported that ESS2 selectively binds to members of the A/B hnRNP family (hnRNPs A1, A1B, A2, and B1) (11, 14). The addition of hnRNP A1 and other members of the hnRNP A/B protein family restores specific Splicing repression in HeLa cell nuclear extracts depleted of ESS2-binding proteins (11). A third potential ESS is present in the env gene, where it may prevent the activation of cryptic exon 6D, which is bordered by splice sites A6 and D5 (46). The levels of Vpr mRNA singly spliced at 3′ splice site A2 also have been shown to be low in cells infected with HIV-1, indicating that Splicing at A2 is inefficient. Furthermore, noncoding exon 3 (Fig. (Fig.1)1) is skipped in the majority of the mRNAs (35). This exon skipping also suggests that splice site A2 or D3 or both of these splice sites are used inefficiently. It has been shown that the branch point used for Splicing at splice site A2 is a G rather than the consensus A that is used for most 3′ splice sites. However, replacing the nonconsensus wild-type branch-point sequence with a consensus sequence did not significantly affect Splicing efficiency in an in vitro Splicing system (13). In this report, we describe additional elements downstream of 3′ splice site A2 that act to repress Splicing at this splice site.
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rna Splicing at human immunodeficiency virus type 1 3 splice site a2 is regulated by binding of hnrnp a b proteins to an Exonic Splicing Silencer element
Journal of Virology, 2001Co-Authors: Patricia S Bilodeau, Jeffrey K Domsic, Akila Mayeda, Adrian R Krainer, Martin C StoltzfusAbstract:The synthesis of human immunodeficiency virus type 1 (HIV-1) mRNAs is a complex process by which more than 30 different mRNA species are produced by alternative Splicing of a single primary RNA transcript. HIV-1 splice sites are used with significantly different efficiencies, resulting in different levels of mRNA species in infected cells. Splicing of Tat mRNA, which is present at relatively low levels in infected cells, is repressed by the presence of Exonic Splicing Silencers (ESS) within the two tat coding exons (ESS2 and ESS3). These ESS elements contain the consensus sequence PyUAG. Here we show that the efficiency of Splicing at 3' splice site A2, which is used to generate Vpr mRNA, is also regulated by the presence of an ESS (ESSV), which has sequence homology to ESS2 and ESS3. Mutagenesis of the three PyUAG motifs within ESSV increases Splicing at splice site A2, resulting in increased Vpr mRNA levels and reduced skipping of the noncoding exon flanked by A2 and D3. The increase in Vpr mRNA levels and the reduced skipping also occur when splice site D3 is mutated toward the consensus sequence. By in vitro Splicing assays, we show that ESSV represses Splicing when placed downstream of a heterologous splice site. A1, A1(B), A2, and B1 hnRNPs preferentially bind to ESSV RNA compared to ESSV mutant RNA. Each of these proteins, when added back to HeLa cell nuclear extracts depleted of ESSV-binding factors, is able to restore Splicing repression. The results suggest that coordinate repression of HIV-1 RNA Splicing is mediated by members of the hnRNP A/B protein family.
Zhiming Zheng - One of the best experts on this subject based on the ideXlab platform.
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serine arginine rich Splicing factor 3 and heterogeneous nuclear ribonucleoprotein a1 regulate alternative rna Splicing and gene expression of human papillomavirus 18 through two functionally distinguishable cis elements
Journal of Virology, 2016Co-Authors: Masahiko Ajiro, Shuang Tang, John Doorbar, Zhiming ZhengAbstract:UNLABELLED: Human papillomavirus 18 (HPV18) is the second most common oncogenic HPV type associated with cervical, anogenital, and oropharyngeal cancers. Like other oncogenic HPVs, HPV18 encodes two major (one early and one late) polycistronic pre-mRNAs that are regulated by alternative RNA Splicing to produce a repertoire of viral transcripts for the expression of individual viral genes. However, RNA cis-regulatory elements and trans-acting factors contributing to HPV18 alternative RNA Splicing remain unknown. In this study, an Exonic Splicing enhancer (ESE) in the nucleotide (nt) 3520 to 3550 region in the HPV18 genome was identified and characterized for promotion of HPV18 929^3434 Splicing and E1^E4 production through interaction with SRSF3, a host oncogenic Splicing factor differentially expressed in epithelial cells and keratinocytes. Introduction of point mutations in the SRSF3-binding site or knockdown of SRSF3 expression in cells reduces 929^3434 Splicing and E1^E4 production but activates other, minor 929^3465 and 929^3506 Splicing. Knockdown of SRSF3 expression also enhances the expression of E2 and L1 mRNAs. An Exonic Splicing Silencer (ESS) in the HPV18 nt 612 to 639 region was identified as being inhibitory to the 233^416 Splicing of HPV18 E6E7 pre-mRNAs via binding to hnRNP A1, a well-characterized, abundantly and ubiquitously expressed RNA-binding protein. Introduction of point mutations into the hnRNP A1-binding site or knockdown of hnRNP A1 expression promoted 233^416 Splicing and reduced E6 expression. These data provide the first evidence that the alternative RNA Splicing of HPV18 pre-mRNAs is subject to regulation by viral RNA cis elements and host trans-acting Splicing factors. IMPORTANCE: Expression of HPV18 genes is regulated by alternative RNA Splicing of viral polycistronic pre-mRNAs to produce a repertoire of viral early and late transcripts. RNA cis elements and trans-acting factors contributing to HPV18 alternative RNA Splicing have been discovered in this study for the first time. The identified ESS at the E7 open reading frame (ORF) prevents HPV18 233^416 Splicing in the E6 ORF through interaction with a host Splicing factor, hnRNP A1, and regulates E6 and E7 expression of the early E6E7 polycistronic pre-mRNA. The identified ESE at the E1^E4 ORF promotes HPV18 929^3434 Splicing of both viral early and late pre-mRNAs and E1^E4 production through interaction with SRSF3. This study provides important observations on how alternative RNA Splicing of HPV18 pre-mRNAs is subject to regulation by viral RNA cis elements and host Splicing factors and offers potential therapeutic targets to overcome HPV-related cancer.
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serine arginine rich Splicing factor 3 and heterogeneous nuclear ribonucleoprotein a1 regulate alternative rna Splicing and gene expression of human papillomavirus 18 through two functionally distinguishable cis elements
Journal of Virology, 2016Co-Authors: Masahiko Ajiro, Shuang Tang, John Doorbar, Zhiming ZhengAbstract:UNLABELLED: Human papillomavirus 18 (HPV18) is the second most common oncogenic HPV type associated with cervical, anogenital, and oropharyngeal cancers. Like other oncogenic HPVs, HPV18 encodes two major (one early and one late) polycistronic pre-mRNAs that are regulated by alternative RNA Splicing to produce a repertoire of viral transcripts for the expression of individual viral genes. However, RNA cis-regulatory elements and trans-acting factors contributing to HPV18 alternative RNA Splicing remain unknown. In this study, an Exonic Splicing enhancer (ESE) in the nucleotide (nt) 3520 to 3550 region in the HPV18 genome was identified and characterized for promotion of HPV18 929^3434 Splicing and E1^E4 production through interaction with SRSF3, a host oncogenic Splicing factor differentially expressed in epithelial cells and keratinocytes. Introduction of point mutations in the SRSF3-binding site or knockdown of SRSF3 expression in cells reduces 929^3434 Splicing and E1^E4 production but activates other, minor 929^3465 and 929^3506 Splicing. Knockdown of SRSF3 expression also enhances the expression of E2 and L1 mRNAs. An Exonic Splicing Silencer (ESS) in the HPV18 nt 612 to 639 region was identified as being inhibitory to the 233^416 Splicing of HPV18 E6E7 pre-mRNAs via binding to hnRNP A1, a well-characterized, abundantly and ubiquitously expressed RNA-binding protein. Introduction of point mutations into the hnRNP A1-binding site or knockdown of hnRNP A1 expression promoted 233^416 Splicing and reduced E6 expression. These data provide the first evidence that the alternative RNA Splicing of HPV18 pre-mRNAs is subject to regulation by viral RNA cis elements and host trans-acting Splicing factors. IMPORTANCE: Expression of HPV18 genes is regulated by alternative RNA Splicing of viral polycistronic pre-mRNAs to produce a repertoire of viral early and late transcripts. RNA cis elements and trans-acting factors contributing to HPV18 alternative RNA Splicing have been discovered in this study for the first time. The identified ESS at the E7 open reading frame (ORF) prevents HPV18 233^416 Splicing in the E6 ORF through interaction with a host Splicing factor, hnRNP A1, and regulates E6 and E7 expression of the early E6E7 polycistronic pre-mRNA. The identified ESE at the E1^E4 ORF promotes HPV18 929^3434 Splicing of both viral early and late pre-mRNAs and E1^E4 production through interaction with SRSF3. This study provides important observations on how alternative RNA Splicing of HPV18 pre-mRNAs is subject to regulation by viral RNA cis elements and host Splicing factors and offers potential therapeutic targets to overcome HPV-related cancer.