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  • 1.
    Lan, Susan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    The Multifunctional Nature of the Adenovirus L4-22K Protein2016Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The adenovirus major late transcription unit (MLTU) encodes for most of the mRNAs that are translated into the structural proteins of the virus capsid. Transcription from the MLTU is directed by the major late promoter (MLP), which is highly activated during the late phase of infection. This thesis discusses how the adenovirus-encoded L4-22K protein regulates the MLP at both the level of transcription and pre-mRNA splicing. The study shed new light on the complex regulation of the early to late shift of adenoviral gene expression.

    Here we show that the L4-22K protein has opposing effects on MLP transcription, functioning both as an activator and a repressor protein. The stimulatory effect mainly depends on the direct interaction of the L4-22K protein with the downstream element (DE element) located approximately 100 nucleotides downstream of the transcription initiation site. In addition to the DE element we also show that the promoter-proximal upstream element (UPE) acts as an L4-22K responsive enhancer element in the MLP. Preliminary data suggests that the activation of MLP transcription via DE and UPE differs mechanistically. The transactivation domain of the L4-22K protein is localized to the conserved carboxy-terminus of the protein.

    Our results also defined a novel low affinity L4-22K binding site, the R1 region, which functions as a repressor element in MLP transcription. At high concentrations L4-22K binds to R1 and recruits the cellular transcription factor Sp1 to a DNA segment covering the major late first leader 5´ splice site that is embedded in the R1 region. Sp1 binding to R1 results in a suppression of L4-22K-mediated activation of MLP transcription. This self-limiting effect on MLP transcription might have a function to fine-tune the MLTU gene expression.

    Interestingly, the L4-22K protein binds with the same sequence specificity to both the R1 double-stranded DNA and R1 single-stranded RNA (ssRNA). L4-22K binds to the R1 ssRNA with the same polarity as the MLTU nascent RNA. This binding results in the recruitment of U1 snRNA to the major late first leader 5´ splice site. This enhanced U1 snRNA recruitment leads to a suppression of MLP transcription and simultaneously an increase of major late first intron splicing.

    List of papers
    1. Adenovirus L4-22K stimulates major late transcription by a mechanism requiring the intragenic late-specific transcription factor-binding site
    Open this publication in new window or tab >>Adenovirus L4-22K stimulates major late transcription by a mechanism requiring the intragenic late-specific transcription factor-binding site
    2010 (English)In: Virus Research, ISSN 0168-1702, E-ISSN 1872-7492, Vol. 151, no 2, p. 220-228Article in journal (Refereed) Published
    Abstract [en]

    The adenovirus major late promoter (MLP) generates a primary transcript that undergoes a complex pattern of regulated alternative RNA splicing and polyadenylation events. The late-specific activation of the MLP requires binding of two infected-cell specific transcription factor complexes, DEF-A and DEF-B, to the so-called DE sequence located downstream of the MLP start site. Previous studies have shown that DEF-B is a homodimer of the viral IVa2 protein and suggested that DEF-A is a heterodimer of IVa2 and an unknown protein. Here we have searched for a possible DEF-A candidate protein. The adenovirus L4-33K protein functions as a virus-encoded alternative RNA splicing factor, stimulating cytoplasmic accumulation of most late viral mRNAs. Interestingly, the L4 region also encodes for a second related protein, L4-22K, which share the 105 amino-terminal amino acids with L4-33K. Here we show that L4-22K both in vivo and in vitro stimulates transcription from the MLP in a DE sequence dependent manner. We also show that the viral pIX promoter is a natural target, activated by L4-22K. Interestingly, the position of the L4-22K DNA binding site in a promoter does not appear to be critical for function. Thus, tethering L4-22K, as a BPV E2 DNA binding domain fusion protein either to a position upstream or downstream of the MLP start site, or upstream of a minimal E1B promoter, resulted in an activation of transcription. Collectively, our results are compatible with the hypothesis that L4-22K may be the elusive component of DEF-A that partakes in activation of the MLP.

    Keywords
    Adenovirus, L4-22K, MLP, transcription, DE elements
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:uu:diva-101310 (URN)10.1016/j.virusres.2010.05.013 (DOI)000280210000015 ()20621673 (PubMedID)
    Available from: 2009-04-22 Created: 2009-04-22 Last updated: 2017-12-13Bibliographically approved
    2. A suppressive effect of Sp1 recruitment to the first leader 5' splice site region on L4-22K-mediated activation of the adenovirus major late promoter
    Open this publication in new window or tab >>A suppressive effect of Sp1 recruitment to the first leader 5' splice site region on L4-22K-mediated activation of the adenovirus major late promoter
    2015 (English)In: Virus Research, ISSN 0168-1702, E-ISSN 1872-7492, Vol. 210, p. 133-140Article in journal (Refereed) Published
    Abstract [en]

    Transcription from the adenovirus major late promoter (MLP) requires binding of late phase-specific factors to the so-called DE element located approximately 100 base pairs downstream of the MLP transcriptional start site. The adenovirus L4-22K protein binds to the DE element and stimulates transcription from the MLP via a DE sequence-dependent mechanism. Here we use a transient expression approach to show that L4-22K binds to an additional site downstream of the MLP start site, the so-called R1 region, which includes the major late first leader 5' splice site. Binding of L4-22K to R1 has a suppressive effect on MLP transcription. L4-22K binds to the distal part of R1 and stimulates the recruitment of Sp1 and other cellular factors to a site overlapping the first leader 5' splice site. Binding of Sp1 to the 5' splice site region had an inhibitory effect on L4-22K-activated MLP transcription.

    National Category
    Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
    Identifiers
    urn:nbn:se:uu:diva-275814 (URN)10.1016/j.virusres.2015.07.026 (DOI)000365455500020 ()26247419 (PubMedID)
    Funder
    Swedish Cancer Society, 12 0504Swedish Research Council, K2012-99X-21959-01-3 2006-5038-36531-16
    Available from: 2016-02-07 Created: 2016-02-07 Last updated: 2017-11-30Bibliographically approved
    3. The adenovirus L4-22K protein regulates major late transcription and RNA splicing through a sequence-specific binding to single-stranded RNA
    Open this publication in new window or tab >>The adenovirus L4-22K protein regulates major late transcription and RNA splicing through a sequence-specific binding to single-stranded RNA
    (English)Manuscript (preprint) (Other academic)
    National Category
    Microbiology in the medical area
    Identifiers
    urn:nbn:se:uu:diva-278134 (URN)
    Available from: 2016-02-23 Created: 2016-02-23 Last updated: 2018-01-10
    4. Adenovirus L4-22K protein responsive elements in the adenovirus major late promoter
    Open this publication in new window or tab >>Adenovirus L4-22K protein responsive elements in the adenovirus major late promoter
    (English)Manuscript (preprint) (Other academic)
    National Category
    Microbiology in the medical area
    Identifiers
    urn:nbn:se:uu:diva-278133 (URN)
    Available from: 2016-02-23 Created: 2016-02-23 Last updated: 2018-01-10
  • 2.
    Lan, Susan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Kamel, Wael
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Punga, Tanel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Akusjärvi, Göran
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    The adenovirus L4-22K protein regulates transcription and RNA splicing via a sequence-specific single-stranded RNA binding2017In: Nucleic Acids Research, ISSN 0305-1048, E-ISSN 1362-4962, Vol. 45, no 4, p. 1731-1742Article in journal (Refereed)
    Abstract [en]

    The adenovirus L4-22K protein both activates and suppresses transcription from the adenovirus major late promoter (MLP) by binding to DNA elements located downstream of the MLP transcriptional start site: the so-called DE element ( positive) and the R1 region ( negative). Here we show that L4-22K preferentially binds to the RNA form of the R1 region, both to the double-stranded RNA and the single-stranded RNA of the same polarity as the nascent MLP transcript. Further, L4-22K binds to a 5'-CAAA-3' motif in the single-stranded RNA, which is identical to the sequence motif characterized for L4-22K DNA binding. L4-22K binding to single-stranded RNA results in an enhancement of U1 snRNA recruitment to the major late first leader 5' splice site. This increase in U1 snRNA binding results in a suppression of MLP transcription and a concurrent stimulation of major late first intron splicing.

  • 3.
    Lan, Susan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Östberg, Sara
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Punga, Tanel
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Akusjärvi, Göran
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    A suppressive effect of Sp1 recruitment to the first leader 5' splice site region on L4-22K-mediated activation of the adenovirus major late promoter2015In: Virus Research, ISSN 0168-1702, E-ISSN 1872-7492, Vol. 210, p. 133-140Article in journal (Refereed)
    Abstract [en]

    Transcription from the adenovirus major late promoter (MLP) requires binding of late phase-specific factors to the so-called DE element located approximately 100 base pairs downstream of the MLP transcriptional start site. The adenovirus L4-22K protein binds to the DE element and stimulates transcription from the MLP via a DE sequence-dependent mechanism. Here we use a transient expression approach to show that L4-22K binds to an additional site downstream of the MLP start site, the so-called R1 region, which includes the major late first leader 5' splice site. Binding of L4-22K to R1 has a suppressive effect on MLP transcription. L4-22K binds to the distal part of R1 and stimulates the recruitment of Sp1 and other cellular factors to a site overlapping the first leader 5' splice site. Binding of Sp1 to the 5' splice site region had an inhibitory effect on L4-22K-activated MLP transcription.

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