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Giuseppe Biamonti - One of the best experts on this subject based on the ideXlab platform.

  • RNA recognition motif 2 directs the recruitment of SF2/ASF to nuclear stress bodies.
    Nucleic acids research, 2004
    Co-Authors: Ilaria Chiodi, Margherita Corioni, Manuela Giordano, Rut Valgardsdottir, Claudia Ghigna, Fabio Cobianchi, Silvano Riva, Giuseppe Biamonti
    Abstract:

    Heat shock induces the transcriptional activation of large heterochromatic regions of the human genome composed of arrays of satellite III DNA repeats. A number of RNA-processing factors, among them splicing factor SF2/ASF, associate with these transcription factors giving rise to nuclear stress bodies (nSBs). Here, we show that the recruitment of SF2/ASF to these structures is mediated by its second RNA recognition motif. Amino acid substitutions in the first α-helix of this Domain, but not in the β-strand regions, abrogate the association with nSBs. The same mutations drastically affect the in vivo activity of SF2/ASF in the alternative splicing of adenoviral E1A transcripts. Sequence analysis identifies four putative high-affinity binding sites for SF2/ASF in the transcribed strand of the satellite III DNA. We have verified by gel mobility shift assays that the second RNA-binding Domain of SF2/ASF binds at least one of these sites. Our analysis suggests that the recruitment of SF2/ASF to nSBs is mediated by a direct interaction with satellite III transcripts and points to the second RNA-binding Domain of the protein as the major determinant of this interaction.

  • rna recognition motif 2 directs the recruitment of sf2 asf to nuclear stress bodies
    Nucleic Acids Research, 2004
    Co-Authors: Ilaria Chiodi, Margherita Corioni, Manuela Giordano, Rut Valgardsdottir, Claudia Ghigna, Fabio Cobianchi, Silvano Riva, Giuseppe Biamonti
    Abstract:

    Heat shock induces the transcriptional activation of large heterochromatic regions of the human genome composed of arrays of satellite III DNA repeats. A number of RNA-processing factors, among them splicing factor SF2/ASF, associate with these transcription factors giving rise to nuclear stress bodies (nSBs). Here, we show that the recruitment of SF2/ASF to these structures is mediated by its second RNA recognition motif. Amino acid substitutions in the first α-helix of this Domain, but not in the β-strand regions, abrogate the association with nSBs. The same mutations drastically affect the in vivo activity of SF2/ASF in the alternative splicing of adenoviral E1A transcripts. Sequence analysis identifies four putative high-affinity binding sites for SF2/ASF in the transcribed strand of the satellite III DNA. We have verified by gel mobility shift assays that the second RNA-binding Domain of SF2/ASF binds at least one of these sites. Our analysis suggests that the recruitment of SF2/ASF to nSBs is mediated by a direct interaction with satellite III transcripts and points to the second RNA-binding Domain of the protein as the major determinant of this interaction.

Mark Bycroft - One of the best experts on this subject based on the ideXlab platform.

  • RNA recognition by a Staufen double‐stranded RNA‐binding Domain
    The EMBO journal, 2000
    Co-Authors: Andres Ramos, Stefan Grünert, Jan Adams, David Micklem, Mark R. Proctor, Stefan M.v. Freund, Mark Bycroft, Daniel St Johnston, Gabriele Varani
    Abstract:

    The double-stranded RNA-binding Domain (dsRBD) is a common RNA-binding motif found in many proteins involved in RNA maturation and localization. To determine how this Domain recognizes RNA, we have studied the third dsRBD from Drosophila Staufen. The Domain binds optimally to RNA stem-loops containing 12 uninterrupted base pairs, and we have identified the amino acids required for this interaction. By mutating these residues in a staufen transgene, we show that the RNA-binding activity of dsRBD3 is required in vivo for Staufen-dependent localization of bicoid and oskar mRNAs. Using high-resolution NMR, we have determined the structure of the complex between dsRBD3 and an RNA stem-loop. The dsRBD recognizes the shape of A-form dsRNA through interactions between conserved residues within loop 2 and the minor groove, and between loop 4 and the phosphodiester backbone across the adjacent major groove. In addition, helix alpha1 interacts with the single-stranded loop that caps the RNA helix. Interactions between helix alpha1 and single-stranded RNA may be important determinants of the specificity of dsRBD proteins.

  • The solution structure of the S1 RNA binding Domain: a member of an ancient nucleic acid-binding fold.
    Cell, 1997
    Co-Authors: Mark Bycroft, Mark R. Proctor, Stefan M.v. Freund, Tim Hubbard, Alexey G. Murzin
    Abstract:

    Abstract The S1 Domain, originally identified in ribosomal protein S1, is found in a large number of RNA-associated proteins. The structure of the S1 RNA-binding Domain from the E. coli polynucleotide phosphorylase has been determined using NMR methods and consists of a five-stranded antiparallel β barrel. Conserved residues on one face of the barrel and adjacent loops form the putative RNA-binding site. The structure of the S1 Domain is very similar to that of cold shock protein, suggesting that they are both derived from an ancient nucleic acid–binding protein. Enhanced sequence searches reveal hitherto unidentified S1 Domains in RNase E, RNase II, NusA, EMB-5, and other proteins.

  • Assignment of the backbone 1H,15N,13C NMR resonances and secondary structure of a double-stranded RNA binding Domain from the Drosophila protein staufen
    FEBS letters, 1995
    Co-Authors: Mark Bycroft, Mark R. Proctor, Stefan M.v. Freund, Daniel St Johnston
    Abstract:

    Abstract NMR spectroscopy has been used to determine the secondary structure of one of the double-stranded RNA binding Domains from the Drosophila protein staufen. The Domain has an αβββα arrangement of secondary structure, with the β strands forming an antiparallel β sheet. The secondary structure differs from that found in the RNP RNA binding Domain.

Denis Soubieux - One of the best experts on this subject based on the ideXlab platform.

  • Major contribution of the RNA-binding Domain of NS1 in the pathogenicity and replication potential of an avian H7N1 influenza virus in chickens
    Virology Journal, 2018
    Co-Authors: Sascha Trapp, Denis Soubieux, Alexandra Lidove, Evelyne Esnault, Adrien Lion, Vanaïque Guillory, Alan Wacquiez, Emmanuel Kut, Pascale Quéré, Thibaut Larcher
    Abstract:

    Background: Non-structural protein NS1 of influenza A viruses harbours several determinants of pathogenicity and host-range. However it is still unclear to what extent each of its two structured Domains (i.e. RNA-binding Domain, RBD, and effector Domain, ED) contribute to its various activities. Methods: To evaluate the respective contributions of the two Domains, we genetically engineered two variants of an H7N1 low pathogenicity avian influenza virus harbouring amino-acid substitutions that impair the functionality of either Domain. The RBD- and ED-mutant viruses were compared to their wt- counterpart in vivo and in vitro, notably in chicken infection and avian cell culture models. Results: The double substitution R38A-K41A in the RBD dramatically reduced the pathogenicity and replication potential of the virus, whereas the substitution A149V that was considered to abrogate the IFN-antagonistic activity of the effector Domain entailed much less effects. While all three viruses initiated the viral life cycle in avian cells, replication of the R38A-K41A virus was severely impaired. This defect was associated with a delayed synthesis of nucleoprotein NP and a reduced accumulation of NS1, which was found to reach a concentration of about 30 micromol.L− 1 in wt-infected cells at 8 h post-infection. When overexpressed in avian lung epithelial cells, both the wt-NS1 and 3841AA-NS1, but not the A149V-NS1, reduced the poly(I:C)-induced activation of the IFN-sensitive chicken Mx promoter. Unexpectedly, the R38A-K41A substitution in the recombinant RBD did not alter its in vitro affinity for a model dsRNA. When overexpressed in avian cells, both the wt- and A149V-NS1s, as well as the individually expressed wt-RBD to a lesser extent, enhanced the activity of the reconstituted viral RNA-polymerase in a minireplicon assay. Conclusions: Collectively, our data emphasized the critical importance and essential role of the RNA-binding Domain in essential steps of the virus replication cycle, notably expression and translation of viral mRNAs. Keywords: Influenza A, NS1, Viral replication, Chicken

  • Influenza virus non-structural protein NS1 cooperatively binds virus-specific (+)-strand RNA sequences
    2013
    Co-Authors: Daniel Marc, Denis Soubieux
    Abstract:

    Non-structural protein NS1 of influenza viruses plays a major role in countering the interferon response of the host, and is involved in the metabolism of viral and cellular RNAs. Its multiple activities all require a functional RNA-binding Domain. NS1 is generally thought to bind non-specifically to several viral and cellular RNAs, notably to double-stranded RNAs (dsRNAs). We asked whether NS1 could exhibit some sequence-specificity towards its RNA ligands, and performed an in vitro selection (SELEX) to isolate NS1-specific aptamers. We identified two virus-specific sequences that are characteristic of the viral RNAs of positive polarity. The first motif, AGCAAAAG, is strictly conserved at the 5’-end of all (+)-strand RNAs of influenzaviruses A. The second motif, UGAUUGAAG, is highly conserved in NS1-mRNA, 15 nucleotides downstream of NS1’s stop codon. In addition, most of NS1-aptamers had one or two symmetrically positioned copies of the 5’-GUAAC / 3’-CUUAG double-stranded motif, which closely resembles the canonical 5’-splice site. We characterized the interaction of NS1 with its RNA-aptamers and showed that NS1’s RNA-binding Domain specifically recognizes both the sequence and structure of the virus-specific RNA-sequences. Cooperative binding of NS1’s RNA-binding Domain leads to its oligomerization on the bound RNA. This strong and intimate interaction suggests that NS1 activity towards viral RNAs is much more specific than previously thought.

  • The RNA-binding Domain of influenzavirus non-structural protein-1 cooperatively binds to virus-specific RNA sequences in a structure-dependent manner
    Nucleic Acids Research, 2013
    Co-Authors: Daniel Marc, Sosthène Barbachou, Denis Soubieux
    Abstract:

    Influenzavirus non-structural protein NS1 is involved in several steps of the virus replication cycle. It counteracts the interferon response, and also exhibits other activities towards viral and cellular RNAs.NS1 is known to bind non-specifically to dsRNAs as well as to viral and cellular RNAs. We set out to search whether NS1 could preferentially bind sequence-specific RNA patterns, and performed an in vitro selection (SELEX) to isolate NS1-specific aptamers from a pool of 80-nucleotide(nt)-long RNAs.Among the 63 aptamers characterized, two families were found to harbour a sequence that is strictly conserved at the 5’terminus of all positive-strand RNAs of influenzaviruses A. We found a second virus-specific motif, a 9 nucleotide-sequence located 15 nucleotides downstream from NS1’s stop codon. In addition, a majority of aptamers had one or two symmetrically positioned copies of the 5’-GUAAC / 3’-CUUAG double-stranded motif, which closely resembles the canonical 5’-splice site.Through an in depth analysis of the interaction combining fluorimetry and gel-shift assays, we showed that NS1’s RNA binding Domain (RBD) specifically recognizes sequence patterns in a structuredependent manner, resulting in an intimate interaction with high affinity (low nanomolar to subnanomolar KD values) that leads to oligomerization of the RNA binding Domain on its RNA ligands.

Ilaria Chiodi - One of the best experts on this subject based on the ideXlab platform.

  • RNA recognition motif 2 directs the recruitment of SF2/ASF to nuclear stress bodies.
    Nucleic acids research, 2004
    Co-Authors: Ilaria Chiodi, Margherita Corioni, Manuela Giordano, Rut Valgardsdottir, Claudia Ghigna, Fabio Cobianchi, Silvano Riva, Giuseppe Biamonti
    Abstract:

    Heat shock induces the transcriptional activation of large heterochromatic regions of the human genome composed of arrays of satellite III DNA repeats. A number of RNA-processing factors, among them splicing factor SF2/ASF, associate with these transcription factors giving rise to nuclear stress bodies (nSBs). Here, we show that the recruitment of SF2/ASF to these structures is mediated by its second RNA recognition motif. Amino acid substitutions in the first α-helix of this Domain, but not in the β-strand regions, abrogate the association with nSBs. The same mutations drastically affect the in vivo activity of SF2/ASF in the alternative splicing of adenoviral E1A transcripts. Sequence analysis identifies four putative high-affinity binding sites for SF2/ASF in the transcribed strand of the satellite III DNA. We have verified by gel mobility shift assays that the second RNA-binding Domain of SF2/ASF binds at least one of these sites. Our analysis suggests that the recruitment of SF2/ASF to nSBs is mediated by a direct interaction with satellite III transcripts and points to the second RNA-binding Domain of the protein as the major determinant of this interaction.

  • rna recognition motif 2 directs the recruitment of sf2 asf to nuclear stress bodies
    Nucleic Acids Research, 2004
    Co-Authors: Ilaria Chiodi, Margherita Corioni, Manuela Giordano, Rut Valgardsdottir, Claudia Ghigna, Fabio Cobianchi, Silvano Riva, Giuseppe Biamonti
    Abstract:

    Heat shock induces the transcriptional activation of large heterochromatic regions of the human genome composed of arrays of satellite III DNA repeats. A number of RNA-processing factors, among them splicing factor SF2/ASF, associate with these transcription factors giving rise to nuclear stress bodies (nSBs). Here, we show that the recruitment of SF2/ASF to these structures is mediated by its second RNA recognition motif. Amino acid substitutions in the first α-helix of this Domain, but not in the β-strand regions, abrogate the association with nSBs. The same mutations drastically affect the in vivo activity of SF2/ASF in the alternative splicing of adenoviral E1A transcripts. Sequence analysis identifies four putative high-affinity binding sites for SF2/ASF in the transcribed strand of the satellite III DNA. We have verified by gel mobility shift assays that the second RNA-binding Domain of SF2/ASF binds at least one of these sites. Our analysis suggests that the recruitment of SF2/ASF to nSBs is mediated by a direct interaction with satellite III transcripts and points to the second RNA-binding Domain of the protein as the major determinant of this interaction.

Daniel St Johnston - One of the best experts on this subject based on the ideXlab platform.

  • RNA recognition by a Staufen double‐stranded RNA‐binding Domain
    The EMBO journal, 2000
    Co-Authors: Andres Ramos, Stefan Grünert, Jan Adams, David Micklem, Mark R. Proctor, Stefan M.v. Freund, Mark Bycroft, Daniel St Johnston, Gabriele Varani
    Abstract:

    The double-stranded RNA-binding Domain (dsRBD) is a common RNA-binding motif found in many proteins involved in RNA maturation and localization. To determine how this Domain recognizes RNA, we have studied the third dsRBD from Drosophila Staufen. The Domain binds optimally to RNA stem-loops containing 12 uninterrupted base pairs, and we have identified the amino acids required for this interaction. By mutating these residues in a staufen transgene, we show that the RNA-binding activity of dsRBD3 is required in vivo for Staufen-dependent localization of bicoid and oskar mRNAs. Using high-resolution NMR, we have determined the structure of the complex between dsRBD3 and an RNA stem-loop. The dsRBD recognizes the shape of A-form dsRNA through interactions between conserved residues within loop 2 and the minor groove, and between loop 4 and the phosphodiester backbone across the adjacent major groove. In addition, helix alpha1 interacts with the single-stranded loop that caps the RNA helix. Interactions between helix alpha1 and single-stranded RNA may be important determinants of the specificity of dsRBD proteins.

  • Assignment of the backbone 1H,15N,13C NMR resonances and secondary structure of a double-stranded RNA binding Domain from the Drosophila protein staufen
    FEBS letters, 1995
    Co-Authors: Mark Bycroft, Mark R. Proctor, Stefan M.v. Freund, Daniel St Johnston
    Abstract:

    Abstract NMR spectroscopy has been used to determine the secondary structure of one of the double-stranded RNA binding Domains from the Drosophila protein staufen. The Domain has an αβββα arrangement of secondary structure, with the β strands forming an antiparallel β sheet. The secondary structure differs from that found in the RNP RNA binding Domain.