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  • Structural divergence of the group I intron binding surface in fungal mitochondrial tyrosyl-tRNA synthetases that function in RNA splicing
    The Journal of biological chemistry, 2016
    Co-Authors: Lilian T. Lamech, M.m. Saoji, Paul J. Paukstelis, Alan M. Lambowitz
    Abstract:

    The mitochondrial tyrosyl-tRNA synthetases (mtTyrRSs) of Pezizomycotina fungi, a subphylum that includes many pathogenic species, are bifunctional proteins that both charge mitochondrial tRNA(Tyr) and act as splicing cofactors for autocatalytic group I introns. Previous studies showed that one of these proteins, Neurospora crassa CYT-18, binds group I introns by using both its N-terminal catalytic and C-terminal anticodon binding domains and that the catalytic domain uses a newly evolved group I intron binding surface that includes an N-terminal extension and two small insertions (insertions 1 and 2) with distinctive features not found in non-splicing mtTyrRSs. To explore how this RNA binding surface diverged to accommodate different group I introns in other Pezizomycotina fungi, we determined x-ray crystal structures of C-terminally truncated Aspergillus nidulans and Coccidioides posadasii mtTyrRSs. Comparisons with previous N. crassa CYT-18 structures and a structural model of the Aspergillus fumigatus mtTyrRS showed that the overall topology of the group I intron binding surface is conserved but with variations in key intron binding regions, particularly the Pezizomycotina-specific insertions. These insertions, which arose by expansion of flexible termini or internal loops, show greater variation in structure and amino acids potentially involved in group I intron binding than do neighboring protein core regions, which also function in intron binding but may be more constrained to preserve mtTyrRS activity. Our results suggest a structural basis for the intron specificity of different Pezizomycotina mtTyrRSs, highlight flexible terminal and loop regions as major sites for enzyme diversification, and identify targets for therapeutic intervention by disrupting an essential RNA-protein interaction in pathogenic fungi.

  • nmr structure of the c terminal domain of a tyrosyl trna synthetase that functions in group i intron splicing
    Biochemistry, 2011
    Co-Authors: Paul J. Paukstelis, Alan M. Lambowitz, Nandini S Chari, David W Hoffman
    Abstract:

    The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (mt TyrRS; CYT-18 protein) and those of other fungi of the subphylum Pezizomycotina are bifunctional proteins that both aminoacylate mt tRNATyr and promote the splicing of mt group I introns (1, 2). Previous studies showed that CYT-18 recognizes conserved structural features of the group I intron catalytic core and promotes splicing by stabilizing the catalytically active RNA structure (3–5). The group I intron catalytic core consists of two extended helical domains: P4–P6 consisting of stacked helices P5, P4, P6, and P6a/b, and P3–P9 consisting of helices P9, P7, P3, and P8 (6). The two domains interact via a series of tertiary contacts, with the P3–P9 domain wrapping around the P4–P6 domain forming a cleft that contains the intron RNA’s active site. This active site binds the splice sites and guanosine cofactor and uses specifically bound Mg2+ ions to catalyze splicing via guanosine-initiated transesterification reactions. Biochemical and genetic experiments suggested that CYT-18 binds first to the P4–P6 domain to promote its assembly and then makes additional contacts with the P3–P9 domain that stabilize the active RNA structure relative to alternative non-native structures (3–5, 7). The structural stabilization of the group I intron core afforded by CYT-18 compensates for RNA structural defects that impair self-splicing (3). Bacterial TyrRSs are comprised of an N-terminal nucleotide-binding fold or catalytic domain, an intermediate α-helical domain, and a C-terminal tRNA-binding domain. The latter has a fold similar to that of ribosomal protein S4 and is attached to the remainder of the protein via a flexible linker (8, 9). The functional TyrRS is a homodimer, which binds tRNATyr asymmetrically across the two subunits (10, 11). The catalytic domain of one subunit (subunit A) binds the tRNA’s acceptor stem and catalyzes aminoacylation, while the intermediate α-helical and C-terminal domains of the other subunit (subunit B) bind the tRNA’s anticodon and variable arms. Although TyrRS homodimers contain two active sites, only a single tRNATyr is bound and charged, a phenomenon known as “half-sites reactivity” (12). Mt TyrRSs are structurally homologous to the bacterial enzymes, and CYT-18 uses both its N-terminal catalytic and C-terminal tRNA-binding domains to bind group I intron RNAs (13–15). However, group I intron splicing activity has been found only for the mt TyrRSs of Pezizomycotina, filamentous fungi that includes the model organisms Neurospora crassa, Aspergillus nidulans, and Podospora anserina, as well as important human pathogens, such as Histoplasma capsulatum, Coccidioides posadasii, and Aspergillus fumigatus (2). The acquisition of group I intron splicing activity by the mt TyrRSs of these fungi can be traced to a series of structural adaptations in different regions of the protein, including a number of small “insertions”, which occurred during or after the divergence of Pezizomycotina from yeast (2, 16). As illustrated in Figure 1 for several representative examples, these Pezizomycotina-specific insertions include an α-helical N-terminal extension (H0), two small insertions in the catalytic domain (Ins1 and Ins2), three additional insertions in the C-terminal domain (Ins3, 4, and 5), and a variable length C-terminal extension (CTE). Studies with CYT-18 showed that the N-terminal domain insertions H0, Ins1 and Ins2 are required for group I intron splicing but not TyrRS activity and form part of a new group I intron-binding site distinct from that which binds tRNATyr (14, 16, 17). A co-crystal structure of a splicing active C-terminally truncated CYT-18 protein (CYT-18/Δ424–669) with a group I intron RNA (the bacteriophage Twort orf142-I2 ribozyme) revealed key features of the RNA-protein interface and showed that H0, Ins1, and Ins2 bind directly to the group I intron catalytic core in position to stabilize key tertiary interactions (18). Figure 1 Comparison of splicing-active Pezizomycotina mt TyrRSs with non-splicing bacterial and mt TyrRSs. The Pezizomycotina mt TyrRSs are distinguished by a series of insertions, including an α-helical N-terminal extension H0, Ins1 and Ins2 in the catalytic ... Thus far, there has been relatively little information about how CYT-18’s C-terminal domain contributes to group I intron splicing or about the structure and function of the C-terminal domain insertions. Although a C-terminally truncated CYT-18 protein consisting of the N-terminal catalytic and intermediate α-helical domains can splice many group I introns, the C-terminal domain contributes to group I intron binding and is essential for splicing some introns, e.g., the N. crassa mt large ribosomal subunit (Nc mt LSU) intron (14). Genetic studies showed that CYT-18’s C-terminal domain is needed to compensate for certain structural mutations that impair self-splicing of group I intron RNAs, including mutations that weaken two key long-range tertiary interactions: L9-P5, which helps establish the correct relative orientation of the two catalytic core domains, and L2-P8, which helps position the P1 helix containing the 5’-splice site at the RNA’s active site (15). Additionally, small deletions within the catalytic domain’s Ins2, which binds near the L9-P5 tetraloop-tetraloop receptor interaction, have more severe effects in the C-terminally truncated CYT-18/Δ424–669 protein than in the full-length protein, suggesting that C-terminal domain binding can compensate for loss of some N-terminal domain interactions (17). Together, these findings suggest that CYT-18’s N- and C-terminal domains both contribute to group I intron binding, with greater or lesser dependence on the C-terminal domain reflecting different structural defects that must be compensated for in each intron. Site-directed hydroxyl-radical cleavage experiments showed that CYT-18’s C-terminal domain binds near P6–P6a, P3–P8 and P5 in the Nc ND1 intron and near P6–P6a, P2, P4 and P5 in the Nc mt LSU intron (19). While CYT-18’s C-terminal domain contributes to the splicing of a number of group I introns, it inhibits the second step of splicing in some heterologous group I introns, suggesting that it co-evolved to function optimally with N. crassa mt group I introns (20). The C-terminal domain of CYT-18, like those of most bacterial TyrRSs, has been intractable to X-ray crystallography in the full-length protein, presumably due to its attachment via a flexible linker that impedes crystallization in a specific orientation (8). Here, we used heteronuclear multidimensional NMR to determine the solution structure of the closely related but smaller C-terminal domain of the splicing-active Aspergillus nidulans (An) mt TyrRS. The structure confirmed the S4-like fold, but with Pezizomycotina-specific features, including the C-terminal domain insertions, which potentially contribute to novel functions. Modeling indicated that flexible attachment of the C-terminal domain is critical for its ability to interact with mt tRNATyr and group I intron RNAs on opposite sides of the catalytic domain. Surprisingly, however, NMR experiments showed that the C-terminal domains of the full-length An mt TyrRS and GeoBacillus stearothermophilus (Gs) TyrRSs do not tumble independently, implying that their attachment to the remainder of the protein is less flexible than was believed previously.

  • NMR structure of the C-terminal domain of a tyrosyl-tRNA synthetase that functions in group I intron splicing †
    Biochemistry, 2011
    Co-Authors: Paul J. Paukstelis, Alan M. Lambowitz, Nandini S Chari, David W Hoffman
    Abstract:

    The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (mt TyrRS; CYT-18 protein) and those of other fungi of the subphylum Pezizomycotina are bifunctional proteins that both aminoacylate mt tRNATyr and promote the splicing of mt group I introns (1, 2). Previous studies showed that CYT-18 recognizes conserved structural features of the group I intron catalytic core and promotes splicing by stabilizing the catalytically active RNA structure (3–5). The group I intron catalytic core consists of two extended helical domains: P4–P6 consisting of stacked helices P5, P4, P6, and P6a/b, and P3–P9 consisting of helices P9, P7, P3, and P8 (6). The two domains interact via a series of tertiary contacts, with the P3–P9 domain wrapping around the P4–P6 domain forming a cleft that contains the intron RNA’s active site. This active site binds the splice sites and guanosine cofactor and uses specifically bound Mg2+ ions to catalyze splicing via guanosine-initiated transesterification reactions. Biochemical and genetic experiments suggested that CYT-18 binds first to the P4–P6 domain to promote its assembly and then makes additional contacts with the P3–P9 domain that stabilize the active RNA structure relative to alternative non-native structures (3–5, 7). The structural stabilization of the group I intron core afforded by CYT-18 compensates for RNA structural defects that impair self-splicing (3). Bacterial TyrRSs are comprised of an N-terminal nucleotide-binding fold or catalytic domain, an intermediate α-helical domain, and a C-terminal tRNA-binding domain. The latter has a fold similar to that of ribosomal protein S4 and is attached to the remainder of the protein via a flexible linker (8, 9). The functional TyrRS is a homodimer, which binds tRNATyr asymmetrically across the two subunits (10, 11). The catalytic domain of one subunit (subunit A) binds the tRNA’s acceptor stem and catalyzes aminoacylation, while the intermediate α-helical and C-terminal domains of the other subunit (subunit B) bind the tRNA’s anticodon and variable arms. Although TyrRS homodimers contain two active sites, only a single tRNATyr is bound and charged, a phenomenon known as “half-sites reactivity” (12). Mt TyrRSs are structurally homologous to the bacterial enzymes, and CYT-18 uses both its N-terminal catalytic and C-terminal tRNA-binding domains to bind group I intron RNAs (13–15). However, group I intron splicing activity has been found only for the mt TyrRSs of Pezizomycotina, filamentous fungi that includes the model organisms Neurospora crassa, Aspergillus nidulans, and Podospora anserina, as well as important human pathogens, such as Histoplasma capsulatum, Coccidioides posadasii, and Aspergillus fumigatus (2). The acquisition of group I intron splicing activity by the mt TyrRSs of these fungi can be traced to a series of structural adaptations in different regions of the protein, including a number of small “insertions”, which occurred during or after the divergence of Pezizomycotina from yeast (2, 16). As illustrated in Figure 1 for several representative examples, these Pezizomycotina-specific insertions include an α-helical N-terminal extension (H0), two small insertions in the catalytic domain (Ins1 and Ins2), three additional insertions in the C-terminal domain (Ins3, 4, and 5), and a variable length C-terminal extension (CTE). Studies with CYT-18 showed that the N-terminal domain insertions H0, Ins1 and Ins2 are required for group I intron splicing but not TyrRS activity and form part of a new group I intron-binding site distinct from that which binds tRNATyr (14, 16, 17). A co-crystal structure of a splicing active C-terminally truncated CYT-18 protein (CYT-18/Δ424–669) with a group I intron RNA (the bacteriophage Twort orf142-I2 ribozyme) revealed key features of the RNA-protein interface and showed that H0, Ins1, and Ins2 bind directly to the group I intron catalytic core in position to stabilize key tertiary interactions (18). Figure 1 Comparison of splicing-active Pezizomycotina mt TyrRSs with non-splicing bacterial and mt TyrRSs. The Pezizomycotina mt TyrRSs are distinguished by a series of insertions, including an α-helical N-terminal extension H0, Ins1 and Ins2 in the catalytic ... Thus far, there has been relatively little information about how CYT-18’s C-terminal domain contributes to group I intron splicing or about the structure and function of the C-terminal domain insertions. Although a C-terminally truncated CYT-18 protein consisting of the N-terminal catalytic and intermediate α-helical domains can splice many group I introns, the C-terminal domain contributes to group I intron binding and is essential for splicing some introns, e.g., the N. crassa mt large ribosomal subunit (Nc mt LSU) intron (14). Genetic studies showed that CYT-18’s C-terminal domain is needed to compensate for certain structural mutations that impair self-splicing of group I intron RNAs, including mutations that weaken two key long-range tertiary interactions: L9-P5, which helps establish the correct relative orientation of the two catalytic core domains, and L2-P8, which helps position the P1 helix containing the 5’-splice site at the RNA’s active site (15). Additionally, small deletions within the catalytic domain’s Ins2, which binds near the L9-P5 tetraloop-tetraloop receptor interaction, have more severe effects in the C-terminally truncated CYT-18/Δ424–669 protein than in the full-length protein, suggesting that C-terminal domain binding can compensate for loss of some N-terminal domain interactions (17). Together, these findings suggest that CYT-18’s N- and C-terminal domains both contribute to group I intron binding, with greater or lesser dependence on the C-terminal domain reflecting different structural defects that must be compensated for in each intron. Site-directed hydroxyl-radical cleavage experiments showed that CYT-18’s C-terminal domain binds near P6–P6a, P3–P8 and P5 in the Nc ND1 intron and near P6–P6a, P2, P4 and P5 in the Nc mt LSU intron (19). While CYT-18’s C-terminal domain contributes to the splicing of a number of group I introns, it inhibits the second step of splicing in some heterologous group I introns, suggesting that it co-evolved to function optimally with N. crassa mt group I introns (20). The C-terminal domain of CYT-18, like those of most bacterial TyrRSs, has been intractable to X-ray crystallography in the full-length protein, presumably due to its attachment via a flexible linker that impedes crystallization in a specific orientation (8). Here, we used heteronuclear multidimensional NMR to determine the solution structure of the closely related but smaller C-terminal domain of the splicing-active Aspergillus nidulans (An) mt TyrRS. The structure confirmed the S4-like fold, but with Pezizomycotina-specific features, including the C-terminal domain insertions, which potentially contribute to novel functions. Modeling indicated that flexible attachment of the C-terminal domain is critical for its ability to interact with mt tRNATyr and group I intron RNAs on opposite sides of the catalytic domain. Surprisingly, however, NMR experiments showed that the C-terminal domains of the full-length An mt TyrRS and GeoBacillus stearothermophilus (Gs) TyrRSs do not tumble independently, implying that their attachment to the remainder of the protein is less flexible than was believed previously.

  • Identification and evolution of fungal mitochondrial tyrosyl-tRNA synthetases with group I intron splicing activity
    Proceedings of the National Academy of Sciences of the United States of America, 2008
    Co-Authors: Paul J. Paukstelis, Alan M. Lambowitz
    Abstract:

    The bifunctional Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) both aminoacylates mitochondrial tRNATyr and acts as a structure-stabilizing splicing cofactor for group I introns. Previous studies showed that CYT-18 has distinct tRNATyr and group I intron-binding sites, with the latter formed by three small “insertions” in the nucleotide-binding fold and other structural adaptations compared with nonsplicing bacterial tyrosyl-tRNA synthetases. Here, analysis of genomic sequences shows that mitochondrial tyrosyl-tRNA synthetases with structural adaptations similar to CYT-18's are uniquely characteristic of fungi belonging to the subphylum Pezizomycotina, and biochemical assays confirm group I intron splicing activity for the enzymes from several of these organisms, including Aspergillus nidulans and the human pathogens Coccidioides posadasii and Histoplasma capsulatum. By combining multiple sequence alignments with a previously determined cocrystal structure of a CYT-18/group I intron RNA complex, we identify conserved features of the Pezizomycotina enzymes related to group I intron and tRNA interactions. Our results suggest that mitochondrial tyrosyl-tRNA synthetases with group I intron splicing activity evolved during or after the divergence of the fungal subphyla Pezizomycotina and Saccharomycotina by a mechanism involving the concerted differentiation of preexisting protein loop regions. The unique group I intron splicing activity of these fungal enzymes may provide a new target for antifungal drugs.

Paul J. Paukstelis - One of the best experts on this subject based on the ideXlab platform.

  • Structural divergence of the group I intron binding surface in fungal mitochondrial tyrosyl-tRNA synthetases that function in RNA splicing
    The Journal of biological chemistry, 2016
    Co-Authors: Lilian T. Lamech, M.m. Saoji, Paul J. Paukstelis, Alan M. Lambowitz
    Abstract:

    The mitochondrial tyrosyl-tRNA synthetases (mtTyrRSs) of Pezizomycotina fungi, a subphylum that includes many pathogenic species, are bifunctional proteins that both charge mitochondrial tRNA(Tyr) and act as splicing cofactors for autocatalytic group I introns. Previous studies showed that one of these proteins, Neurospora crassa CYT-18, binds group I introns by using both its N-terminal catalytic and C-terminal anticodon binding domains and that the catalytic domain uses a newly evolved group I intron binding surface that includes an N-terminal extension and two small insertions (insertions 1 and 2) with distinctive features not found in non-splicing mtTyrRSs. To explore how this RNA binding surface diverged to accommodate different group I introns in other Pezizomycotina fungi, we determined x-ray crystal structures of C-terminally truncated Aspergillus nidulans and Coccidioides posadasii mtTyrRSs. Comparisons with previous N. crassa CYT-18 structures and a structural model of the Aspergillus fumigatus mtTyrRS showed that the overall topology of the group I intron binding surface is conserved but with variations in key intron binding regions, particularly the Pezizomycotina-specific insertions. These insertions, which arose by expansion of flexible termini or internal loops, show greater variation in structure and amino acids potentially involved in group I intron binding than do neighboring protein core regions, which also function in intron binding but may be more constrained to preserve mtTyrRS activity. Our results suggest a structural basis for the intron specificity of different Pezizomycotina mtTyrRSs, highlight flexible terminal and loop regions as major sites for enzyme diversification, and identify targets for therapeutic intervention by disrupting an essential RNA-protein interaction in pathogenic fungi.

  • nmr structure of the c terminal domain of a tyrosyl trna synthetase that functions in group i intron splicing
    Biochemistry, 2011
    Co-Authors: Paul J. Paukstelis, Alan M. Lambowitz, Nandini S Chari, David W Hoffman
    Abstract:

    The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (mt TyrRS; CYT-18 protein) and those of other fungi of the subphylum Pezizomycotina are bifunctional proteins that both aminoacylate mt tRNATyr and promote the splicing of mt group I introns (1, 2). Previous studies showed that CYT-18 recognizes conserved structural features of the group I intron catalytic core and promotes splicing by stabilizing the catalytically active RNA structure (3–5). The group I intron catalytic core consists of two extended helical domains: P4–P6 consisting of stacked helices P5, P4, P6, and P6a/b, and P3–P9 consisting of helices P9, P7, P3, and P8 (6). The two domains interact via a series of tertiary contacts, with the P3–P9 domain wrapping around the P4–P6 domain forming a cleft that contains the intron RNA’s active site. This active site binds the splice sites and guanosine cofactor and uses specifically bound Mg2+ ions to catalyze splicing via guanosine-initiated transesterification reactions. Biochemical and genetic experiments suggested that CYT-18 binds first to the P4–P6 domain to promote its assembly and then makes additional contacts with the P3–P9 domain that stabilize the active RNA structure relative to alternative non-native structures (3–5, 7). The structural stabilization of the group I intron core afforded by CYT-18 compensates for RNA structural defects that impair self-splicing (3). Bacterial TyrRSs are comprised of an N-terminal nucleotide-binding fold or catalytic domain, an intermediate α-helical domain, and a C-terminal tRNA-binding domain. The latter has a fold similar to that of ribosomal protein S4 and is attached to the remainder of the protein via a flexible linker (8, 9). The functional TyrRS is a homodimer, which binds tRNATyr asymmetrically across the two subunits (10, 11). The catalytic domain of one subunit (subunit A) binds the tRNA’s acceptor stem and catalyzes aminoacylation, while the intermediate α-helical and C-terminal domains of the other subunit (subunit B) bind the tRNA’s anticodon and variable arms. Although TyrRS homodimers contain two active sites, only a single tRNATyr is bound and charged, a phenomenon known as “half-sites reactivity” (12). Mt TyrRSs are structurally homologous to the bacterial enzymes, and CYT-18 uses both its N-terminal catalytic and C-terminal tRNA-binding domains to bind group I intron RNAs (13–15). However, group I intron splicing activity has been found only for the mt TyrRSs of Pezizomycotina, filamentous fungi that includes the model organisms Neurospora crassa, Aspergillus nidulans, and Podospora anserina, as well as important human pathogens, such as Histoplasma capsulatum, Coccidioides posadasii, and Aspergillus fumigatus (2). The acquisition of group I intron splicing activity by the mt TyrRSs of these fungi can be traced to a series of structural adaptations in different regions of the protein, including a number of small “insertions”, which occurred during or after the divergence of Pezizomycotina from yeast (2, 16). As illustrated in Figure 1 for several representative examples, these Pezizomycotina-specific insertions include an α-helical N-terminal extension (H0), two small insertions in the catalytic domain (Ins1 and Ins2), three additional insertions in the C-terminal domain (Ins3, 4, and 5), and a variable length C-terminal extension (CTE). Studies with CYT-18 showed that the N-terminal domain insertions H0, Ins1 and Ins2 are required for group I intron splicing but not TyrRS activity and form part of a new group I intron-binding site distinct from that which binds tRNATyr (14, 16, 17). A co-crystal structure of a splicing active C-terminally truncated CYT-18 protein (CYT-18/Δ424–669) with a group I intron RNA (the bacteriophage Twort orf142-I2 ribozyme) revealed key features of the RNA-protein interface and showed that H0, Ins1, and Ins2 bind directly to the group I intron catalytic core in position to stabilize key tertiary interactions (18). Figure 1 Comparison of splicing-active Pezizomycotina mt TyrRSs with non-splicing bacterial and mt TyrRSs. The Pezizomycotina mt TyrRSs are distinguished by a series of insertions, including an α-helical N-terminal extension H0, Ins1 and Ins2 in the catalytic ... Thus far, there has been relatively little information about how CYT-18’s C-terminal domain contributes to group I intron splicing or about the structure and function of the C-terminal domain insertions. Although a C-terminally truncated CYT-18 protein consisting of the N-terminal catalytic and intermediate α-helical domains can splice many group I introns, the C-terminal domain contributes to group I intron binding and is essential for splicing some introns, e.g., the N. crassa mt large ribosomal subunit (Nc mt LSU) intron (14). Genetic studies showed that CYT-18’s C-terminal domain is needed to compensate for certain structural mutations that impair self-splicing of group I intron RNAs, including mutations that weaken two key long-range tertiary interactions: L9-P5, which helps establish the correct relative orientation of the two catalytic core domains, and L2-P8, which helps position the P1 helix containing the 5’-splice site at the RNA’s active site (15). Additionally, small deletions within the catalytic domain’s Ins2, which binds near the L9-P5 tetraloop-tetraloop receptor interaction, have more severe effects in the C-terminally truncated CYT-18/Δ424–669 protein than in the full-length protein, suggesting that C-terminal domain binding can compensate for loss of some N-terminal domain interactions (17). Together, these findings suggest that CYT-18’s N- and C-terminal domains both contribute to group I intron binding, with greater or lesser dependence on the C-terminal domain reflecting different structural defects that must be compensated for in each intron. Site-directed hydroxyl-radical cleavage experiments showed that CYT-18’s C-terminal domain binds near P6–P6a, P3–P8 and P5 in the Nc ND1 intron and near P6–P6a, P2, P4 and P5 in the Nc mt LSU intron (19). While CYT-18’s C-terminal domain contributes to the splicing of a number of group I introns, it inhibits the second step of splicing in some heterologous group I introns, suggesting that it co-evolved to function optimally with N. crassa mt group I introns (20). The C-terminal domain of CYT-18, like those of most bacterial TyrRSs, has been intractable to X-ray crystallography in the full-length protein, presumably due to its attachment via a flexible linker that impedes crystallization in a specific orientation (8). Here, we used heteronuclear multidimensional NMR to determine the solution structure of the closely related but smaller C-terminal domain of the splicing-active Aspergillus nidulans (An) mt TyrRS. The structure confirmed the S4-like fold, but with Pezizomycotina-specific features, including the C-terminal domain insertions, which potentially contribute to novel functions. Modeling indicated that flexible attachment of the C-terminal domain is critical for its ability to interact with mt tRNATyr and group I intron RNAs on opposite sides of the catalytic domain. Surprisingly, however, NMR experiments showed that the C-terminal domains of the full-length An mt TyrRS and GeoBacillus stearothermophilus (Gs) TyrRSs do not tumble independently, implying that their attachment to the remainder of the protein is less flexible than was believed previously.

  • NMR structure of the C-terminal domain of a tyrosyl-tRNA synthetase that functions in group I intron splicing †
    Biochemistry, 2011
    Co-Authors: Paul J. Paukstelis, Alan M. Lambowitz, Nandini S Chari, David W Hoffman
    Abstract:

    The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (mt TyrRS; CYT-18 protein) and those of other fungi of the subphylum Pezizomycotina are bifunctional proteins that both aminoacylate mt tRNATyr and promote the splicing of mt group I introns (1, 2). Previous studies showed that CYT-18 recognizes conserved structural features of the group I intron catalytic core and promotes splicing by stabilizing the catalytically active RNA structure (3–5). The group I intron catalytic core consists of two extended helical domains: P4–P6 consisting of stacked helices P5, P4, P6, and P6a/b, and P3–P9 consisting of helices P9, P7, P3, and P8 (6). The two domains interact via a series of tertiary contacts, with the P3–P9 domain wrapping around the P4–P6 domain forming a cleft that contains the intron RNA’s active site. This active site binds the splice sites and guanosine cofactor and uses specifically bound Mg2+ ions to catalyze splicing via guanosine-initiated transesterification reactions. Biochemical and genetic experiments suggested that CYT-18 binds first to the P4–P6 domain to promote its assembly and then makes additional contacts with the P3–P9 domain that stabilize the active RNA structure relative to alternative non-native structures (3–5, 7). The structural stabilization of the group I intron core afforded by CYT-18 compensates for RNA structural defects that impair self-splicing (3). Bacterial TyrRSs are comprised of an N-terminal nucleotide-binding fold or catalytic domain, an intermediate α-helical domain, and a C-terminal tRNA-binding domain. The latter has a fold similar to that of ribosomal protein S4 and is attached to the remainder of the protein via a flexible linker (8, 9). The functional TyrRS is a homodimer, which binds tRNATyr asymmetrically across the two subunits (10, 11). The catalytic domain of one subunit (subunit A) binds the tRNA’s acceptor stem and catalyzes aminoacylation, while the intermediate α-helical and C-terminal domains of the other subunit (subunit B) bind the tRNA’s anticodon and variable arms. Although TyrRS homodimers contain two active sites, only a single tRNATyr is bound and charged, a phenomenon known as “half-sites reactivity” (12). Mt TyrRSs are structurally homologous to the bacterial enzymes, and CYT-18 uses both its N-terminal catalytic and C-terminal tRNA-binding domains to bind group I intron RNAs (13–15). However, group I intron splicing activity has been found only for the mt TyrRSs of Pezizomycotina, filamentous fungi that includes the model organisms Neurospora crassa, Aspergillus nidulans, and Podospora anserina, as well as important human pathogens, such as Histoplasma capsulatum, Coccidioides posadasii, and Aspergillus fumigatus (2). The acquisition of group I intron splicing activity by the mt TyrRSs of these fungi can be traced to a series of structural adaptations in different regions of the protein, including a number of small “insertions”, which occurred during or after the divergence of Pezizomycotina from yeast (2, 16). As illustrated in Figure 1 for several representative examples, these Pezizomycotina-specific insertions include an α-helical N-terminal extension (H0), two small insertions in the catalytic domain (Ins1 and Ins2), three additional insertions in the C-terminal domain (Ins3, 4, and 5), and a variable length C-terminal extension (CTE). Studies with CYT-18 showed that the N-terminal domain insertions H0, Ins1 and Ins2 are required for group I intron splicing but not TyrRS activity and form part of a new group I intron-binding site distinct from that which binds tRNATyr (14, 16, 17). A co-crystal structure of a splicing active C-terminally truncated CYT-18 protein (CYT-18/Δ424–669) with a group I intron RNA (the bacteriophage Twort orf142-I2 ribozyme) revealed key features of the RNA-protein interface and showed that H0, Ins1, and Ins2 bind directly to the group I intron catalytic core in position to stabilize key tertiary interactions (18). Figure 1 Comparison of splicing-active Pezizomycotina mt TyrRSs with non-splicing bacterial and mt TyrRSs. The Pezizomycotina mt TyrRSs are distinguished by a series of insertions, including an α-helical N-terminal extension H0, Ins1 and Ins2 in the catalytic ... Thus far, there has been relatively little information about how CYT-18’s C-terminal domain contributes to group I intron splicing or about the structure and function of the C-terminal domain insertions. Although a C-terminally truncated CYT-18 protein consisting of the N-terminal catalytic and intermediate α-helical domains can splice many group I introns, the C-terminal domain contributes to group I intron binding and is essential for splicing some introns, e.g., the N. crassa mt large ribosomal subunit (Nc mt LSU) intron (14). Genetic studies showed that CYT-18’s C-terminal domain is needed to compensate for certain structural mutations that impair self-splicing of group I intron RNAs, including mutations that weaken two key long-range tertiary interactions: L9-P5, which helps establish the correct relative orientation of the two catalytic core domains, and L2-P8, which helps position the P1 helix containing the 5’-splice site at the RNA’s active site (15). Additionally, small deletions within the catalytic domain’s Ins2, which binds near the L9-P5 tetraloop-tetraloop receptor interaction, have more severe effects in the C-terminally truncated CYT-18/Δ424–669 protein than in the full-length protein, suggesting that C-terminal domain binding can compensate for loss of some N-terminal domain interactions (17). Together, these findings suggest that CYT-18’s N- and C-terminal domains both contribute to group I intron binding, with greater or lesser dependence on the C-terminal domain reflecting different structural defects that must be compensated for in each intron. Site-directed hydroxyl-radical cleavage experiments showed that CYT-18’s C-terminal domain binds near P6–P6a, P3–P8 and P5 in the Nc ND1 intron and near P6–P6a, P2, P4 and P5 in the Nc mt LSU intron (19). While CYT-18’s C-terminal domain contributes to the splicing of a number of group I introns, it inhibits the second step of splicing in some heterologous group I introns, suggesting that it co-evolved to function optimally with N. crassa mt group I introns (20). The C-terminal domain of CYT-18, like those of most bacterial TyrRSs, has been intractable to X-ray crystallography in the full-length protein, presumably due to its attachment via a flexible linker that impedes crystallization in a specific orientation (8). Here, we used heteronuclear multidimensional NMR to determine the solution structure of the closely related but smaller C-terminal domain of the splicing-active Aspergillus nidulans (An) mt TyrRS. The structure confirmed the S4-like fold, but with Pezizomycotina-specific features, including the C-terminal domain insertions, which potentially contribute to novel functions. Modeling indicated that flexible attachment of the C-terminal domain is critical for its ability to interact with mt tRNATyr and group I intron RNAs on opposite sides of the catalytic domain. Surprisingly, however, NMR experiments showed that the C-terminal domains of the full-length An mt TyrRS and GeoBacillus stearothermophilus (Gs) TyrRSs do not tumble independently, implying that their attachment to the remainder of the protein is less flexible than was believed previously.

  • Identification and evolution of fungal mitochondrial tyrosyl-tRNA synthetases with group I intron splicing activity
    Proceedings of the National Academy of Sciences of the United States of America, 2008
    Co-Authors: Paul J. Paukstelis, Alan M. Lambowitz
    Abstract:

    The bifunctional Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (CYT-18 protein) both aminoacylates mitochondrial tRNATyr and acts as a structure-stabilizing splicing cofactor for group I introns. Previous studies showed that CYT-18 has distinct tRNATyr and group I intron-binding sites, with the latter formed by three small “insertions” in the nucleotide-binding fold and other structural adaptations compared with nonsplicing bacterial tyrosyl-tRNA synthetases. Here, analysis of genomic sequences shows that mitochondrial tyrosyl-tRNA synthetases with structural adaptations similar to CYT-18's are uniquely characteristic of fungi belonging to the subphylum Pezizomycotina, and biochemical assays confirm group I intron splicing activity for the enzymes from several of these organisms, including Aspergillus nidulans and the human pathogens Coccidioides posadasii and Histoplasma capsulatum. By combining multiple sequence alignments with a previously determined cocrystal structure of a CYT-18/group I intron RNA complex, we identify conserved features of the Pezizomycotina enzymes related to group I intron and tRNA interactions. Our results suggest that mitochondrial tyrosyl-tRNA synthetases with group I intron splicing activity evolved during or after the divergence of the fungal subphyla Pezizomycotina and Saccharomycotina by a mechanism involving the concerted differentiation of preexisting protein loop regions. The unique group I intron splicing activity of these fungal enzymes may provide a new target for antifungal drugs.

David W Hoffman - One of the best experts on this subject based on the ideXlab platform.

  • nmr structure of the c terminal domain of a tyrosyl trna synthetase that functions in group i intron splicing
    Biochemistry, 2011
    Co-Authors: Paul J. Paukstelis, Alan M. Lambowitz, Nandini S Chari, David W Hoffman
    Abstract:

    The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (mt TyrRS; CYT-18 protein) and those of other fungi of the subphylum Pezizomycotina are bifunctional proteins that both aminoacylate mt tRNATyr and promote the splicing of mt group I introns (1, 2). Previous studies showed that CYT-18 recognizes conserved structural features of the group I intron catalytic core and promotes splicing by stabilizing the catalytically active RNA structure (3–5). The group I intron catalytic core consists of two extended helical domains: P4–P6 consisting of stacked helices P5, P4, P6, and P6a/b, and P3–P9 consisting of helices P9, P7, P3, and P8 (6). The two domains interact via a series of tertiary contacts, with the P3–P9 domain wrapping around the P4–P6 domain forming a cleft that contains the intron RNA’s active site. This active site binds the splice sites and guanosine cofactor and uses specifically bound Mg2+ ions to catalyze splicing via guanosine-initiated transesterification reactions. Biochemical and genetic experiments suggested that CYT-18 binds first to the P4–P6 domain to promote its assembly and then makes additional contacts with the P3–P9 domain that stabilize the active RNA structure relative to alternative non-native structures (3–5, 7). The structural stabilization of the group I intron core afforded by CYT-18 compensates for RNA structural defects that impair self-splicing (3). Bacterial TyrRSs are comprised of an N-terminal nucleotide-binding fold or catalytic domain, an intermediate α-helical domain, and a C-terminal tRNA-binding domain. The latter has a fold similar to that of ribosomal protein S4 and is attached to the remainder of the protein via a flexible linker (8, 9). The functional TyrRS is a homodimer, which binds tRNATyr asymmetrically across the two subunits (10, 11). The catalytic domain of one subunit (subunit A) binds the tRNA’s acceptor stem and catalyzes aminoacylation, while the intermediate α-helical and C-terminal domains of the other subunit (subunit B) bind the tRNA’s anticodon and variable arms. Although TyrRS homodimers contain two active sites, only a single tRNATyr is bound and charged, a phenomenon known as “half-sites reactivity” (12). Mt TyrRSs are structurally homologous to the bacterial enzymes, and CYT-18 uses both its N-terminal catalytic and C-terminal tRNA-binding domains to bind group I intron RNAs (13–15). However, group I intron splicing activity has been found only for the mt TyrRSs of Pezizomycotina, filamentous fungi that includes the model organisms Neurospora crassa, Aspergillus nidulans, and Podospora anserina, as well as important human pathogens, such as Histoplasma capsulatum, Coccidioides posadasii, and Aspergillus fumigatus (2). The acquisition of group I intron splicing activity by the mt TyrRSs of these fungi can be traced to a series of structural adaptations in different regions of the protein, including a number of small “insertions”, which occurred during or after the divergence of Pezizomycotina from yeast (2, 16). As illustrated in Figure 1 for several representative examples, these Pezizomycotina-specific insertions include an α-helical N-terminal extension (H0), two small insertions in the catalytic domain (Ins1 and Ins2), three additional insertions in the C-terminal domain (Ins3, 4, and 5), and a variable length C-terminal extension (CTE). Studies with CYT-18 showed that the N-terminal domain insertions H0, Ins1 and Ins2 are required for group I intron splicing but not TyrRS activity and form part of a new group I intron-binding site distinct from that which binds tRNATyr (14, 16, 17). A co-crystal structure of a splicing active C-terminally truncated CYT-18 protein (CYT-18/Δ424–669) with a group I intron RNA (the bacteriophage Twort orf142-I2 ribozyme) revealed key features of the RNA-protein interface and showed that H0, Ins1, and Ins2 bind directly to the group I intron catalytic core in position to stabilize key tertiary interactions (18). Figure 1 Comparison of splicing-active Pezizomycotina mt TyrRSs with non-splicing bacterial and mt TyrRSs. The Pezizomycotina mt TyrRSs are distinguished by a series of insertions, including an α-helical N-terminal extension H0, Ins1 and Ins2 in the catalytic ... Thus far, there has been relatively little information about how CYT-18’s C-terminal domain contributes to group I intron splicing or about the structure and function of the C-terminal domain insertions. Although a C-terminally truncated CYT-18 protein consisting of the N-terminal catalytic and intermediate α-helical domains can splice many group I introns, the C-terminal domain contributes to group I intron binding and is essential for splicing some introns, e.g., the N. crassa mt large ribosomal subunit (Nc mt LSU) intron (14). Genetic studies showed that CYT-18’s C-terminal domain is needed to compensate for certain structural mutations that impair self-splicing of group I intron RNAs, including mutations that weaken two key long-range tertiary interactions: L9-P5, which helps establish the correct relative orientation of the two catalytic core domains, and L2-P8, which helps position the P1 helix containing the 5’-splice site at the RNA’s active site (15). Additionally, small deletions within the catalytic domain’s Ins2, which binds near the L9-P5 tetraloop-tetraloop receptor interaction, have more severe effects in the C-terminally truncated CYT-18/Δ424–669 protein than in the full-length protein, suggesting that C-terminal domain binding can compensate for loss of some N-terminal domain interactions (17). Together, these findings suggest that CYT-18’s N- and C-terminal domains both contribute to group I intron binding, with greater or lesser dependence on the C-terminal domain reflecting different structural defects that must be compensated for in each intron. Site-directed hydroxyl-radical cleavage experiments showed that CYT-18’s C-terminal domain binds near P6–P6a, P3–P8 and P5 in the Nc ND1 intron and near P6–P6a, P2, P4 and P5 in the Nc mt LSU intron (19). While CYT-18’s C-terminal domain contributes to the splicing of a number of group I introns, it inhibits the second step of splicing in some heterologous group I introns, suggesting that it co-evolved to function optimally with N. crassa mt group I introns (20). The C-terminal domain of CYT-18, like those of most bacterial TyrRSs, has been intractable to X-ray crystallography in the full-length protein, presumably due to its attachment via a flexible linker that impedes crystallization in a specific orientation (8). Here, we used heteronuclear multidimensional NMR to determine the solution structure of the closely related but smaller C-terminal domain of the splicing-active Aspergillus nidulans (An) mt TyrRS. The structure confirmed the S4-like fold, but with Pezizomycotina-specific features, including the C-terminal domain insertions, which potentially contribute to novel functions. Modeling indicated that flexible attachment of the C-terminal domain is critical for its ability to interact with mt tRNATyr and group I intron RNAs on opposite sides of the catalytic domain. Surprisingly, however, NMR experiments showed that the C-terminal domains of the full-length An mt TyrRS and GeoBacillus stearothermophilus (Gs) TyrRSs do not tumble independently, implying that their attachment to the remainder of the protein is less flexible than was believed previously.

  • NMR structure of the C-terminal domain of a tyrosyl-tRNA synthetase that functions in group I intron splicing †
    Biochemistry, 2011
    Co-Authors: Paul J. Paukstelis, Alan M. Lambowitz, Nandini S Chari, David W Hoffman
    Abstract:

    The Neurospora crassa mitochondrial tyrosyl-tRNA synthetase (mt TyrRS; CYT-18 protein) and those of other fungi of the subphylum Pezizomycotina are bifunctional proteins that both aminoacylate mt tRNATyr and promote the splicing of mt group I introns (1, 2). Previous studies showed that CYT-18 recognizes conserved structural features of the group I intron catalytic core and promotes splicing by stabilizing the catalytically active RNA structure (3–5). The group I intron catalytic core consists of two extended helical domains: P4–P6 consisting of stacked helices P5, P4, P6, and P6a/b, and P3–P9 consisting of helices P9, P7, P3, and P8 (6). The two domains interact via a series of tertiary contacts, with the P3–P9 domain wrapping around the P4–P6 domain forming a cleft that contains the intron RNA’s active site. This active site binds the splice sites and guanosine cofactor and uses specifically bound Mg2+ ions to catalyze splicing via guanosine-initiated transesterification reactions. Biochemical and genetic experiments suggested that CYT-18 binds first to the P4–P6 domain to promote its assembly and then makes additional contacts with the P3–P9 domain that stabilize the active RNA structure relative to alternative non-native structures (3–5, 7). The structural stabilization of the group I intron core afforded by CYT-18 compensates for RNA structural defects that impair self-splicing (3). Bacterial TyrRSs are comprised of an N-terminal nucleotide-binding fold or catalytic domain, an intermediate α-helical domain, and a C-terminal tRNA-binding domain. The latter has a fold similar to that of ribosomal protein S4 and is attached to the remainder of the protein via a flexible linker (8, 9). The functional TyrRS is a homodimer, which binds tRNATyr asymmetrically across the two subunits (10, 11). The catalytic domain of one subunit (subunit A) binds the tRNA’s acceptor stem and catalyzes aminoacylation, while the intermediate α-helical and C-terminal domains of the other subunit (subunit B) bind the tRNA’s anticodon and variable arms. Although TyrRS homodimers contain two active sites, only a single tRNATyr is bound and charged, a phenomenon known as “half-sites reactivity” (12). Mt TyrRSs are structurally homologous to the bacterial enzymes, and CYT-18 uses both its N-terminal catalytic and C-terminal tRNA-binding domains to bind group I intron RNAs (13–15). However, group I intron splicing activity has been found only for the mt TyrRSs of Pezizomycotina, filamentous fungi that includes the model organisms Neurospora crassa, Aspergillus nidulans, and Podospora anserina, as well as important human pathogens, such as Histoplasma capsulatum, Coccidioides posadasii, and Aspergillus fumigatus (2). The acquisition of group I intron splicing activity by the mt TyrRSs of these fungi can be traced to a series of structural adaptations in different regions of the protein, including a number of small “insertions”, which occurred during or after the divergence of Pezizomycotina from yeast (2, 16). As illustrated in Figure 1 for several representative examples, these Pezizomycotina-specific insertions include an α-helical N-terminal extension (H0), two small insertions in the catalytic domain (Ins1 and Ins2), three additional insertions in the C-terminal domain (Ins3, 4, and 5), and a variable length C-terminal extension (CTE). Studies with CYT-18 showed that the N-terminal domain insertions H0, Ins1 and Ins2 are required for group I intron splicing but not TyrRS activity and form part of a new group I intron-binding site distinct from that which binds tRNATyr (14, 16, 17). A co-crystal structure of a splicing active C-terminally truncated CYT-18 protein (CYT-18/Δ424–669) with a group I intron RNA (the bacteriophage Twort orf142-I2 ribozyme) revealed key features of the RNA-protein interface and showed that H0, Ins1, and Ins2 bind directly to the group I intron catalytic core in position to stabilize key tertiary interactions (18). Figure 1 Comparison of splicing-active Pezizomycotina mt TyrRSs with non-splicing bacterial and mt TyrRSs. The Pezizomycotina mt TyrRSs are distinguished by a series of insertions, including an α-helical N-terminal extension H0, Ins1 and Ins2 in the catalytic ... Thus far, there has been relatively little information about how CYT-18’s C-terminal domain contributes to group I intron splicing or about the structure and function of the C-terminal domain insertions. Although a C-terminally truncated CYT-18 protein consisting of the N-terminal catalytic and intermediate α-helical domains can splice many group I introns, the C-terminal domain contributes to group I intron binding and is essential for splicing some introns, e.g., the N. crassa mt large ribosomal subunit (Nc mt LSU) intron (14). Genetic studies showed that CYT-18’s C-terminal domain is needed to compensate for certain structural mutations that impair self-splicing of group I intron RNAs, including mutations that weaken two key long-range tertiary interactions: L9-P5, which helps establish the correct relative orientation of the two catalytic core domains, and L2-P8, which helps position the P1 helix containing the 5’-splice site at the RNA’s active site (15). Additionally, small deletions within the catalytic domain’s Ins2, which binds near the L9-P5 tetraloop-tetraloop receptor interaction, have more severe effects in the C-terminally truncated CYT-18/Δ424–669 protein than in the full-length protein, suggesting that C-terminal domain binding can compensate for loss of some N-terminal domain interactions (17). Together, these findings suggest that CYT-18’s N- and C-terminal domains both contribute to group I intron binding, with greater or lesser dependence on the C-terminal domain reflecting different structural defects that must be compensated for in each intron. Site-directed hydroxyl-radical cleavage experiments showed that CYT-18’s C-terminal domain binds near P6–P6a, P3–P8 and P5 in the Nc ND1 intron and near P6–P6a, P2, P4 and P5 in the Nc mt LSU intron (19). While CYT-18’s C-terminal domain contributes to the splicing of a number of group I introns, it inhibits the second step of splicing in some heterologous group I introns, suggesting that it co-evolved to function optimally with N. crassa mt group I introns (20). The C-terminal domain of CYT-18, like those of most bacterial TyrRSs, has been intractable to X-ray crystallography in the full-length protein, presumably due to its attachment via a flexible linker that impedes crystallization in a specific orientation (8). Here, we used heteronuclear multidimensional NMR to determine the solution structure of the closely related but smaller C-terminal domain of the splicing-active Aspergillus nidulans (An) mt TyrRS. The structure confirmed the S4-like fold, but with Pezizomycotina-specific features, including the C-terminal domain insertions, which potentially contribute to novel functions. Modeling indicated that flexible attachment of the C-terminal domain is critical for its ability to interact with mt tRNATyr and group I intron RNAs on opposite sides of the catalytic domain. Surprisingly, however, NMR experiments showed that the C-terminal domains of the full-length An mt TyrRS and GeoBacillus stearothermophilus (Gs) TyrRSs do not tumble independently, implying that their attachment to the remainder of the protein is less flexible than was believed previously.

Jun-ichi Maruyama - One of the best experts on this subject based on the ideXlab platform.

  • A novel Pezizomycotina-specific protein with gelsolin domains regulates contractile actin ring assembly and constriction in perforated septum formation.
    Molecular microbiology, 2020
    Co-Authors: Abdulla Al Mamun, Takuya Katayama, Wei Cao, Shugo Nakamura, Jun-ichi Maruyama
    Abstract:

    Septum formation in fungi is equivalent to cytokinesis. It differs mechanistically in filamentous ascomycetes (Pezizomycotina) from that of ascomycete yeasts by the retention of a central septal pore in the former group. However, septum formation in both groups is accomplished by contractile actin ring (CAR) assembly and constriction. The specific components regulating septal pore organization during septum formation are poorly understood. In this study, a novel Pezizomycotina-specific actin regulatory protein GlpA containing gelsolin domains was identified using bioinformatics. A glpA deletion mutant exhibited increased distances between septa, abnormal septum morphology and defective regulation of septal pore closure. In glpA deletion mutant hyphae, overaccumulation of actin filament (F-actin) was observed, and the CAR was abnormal with improper assembly and failure in constriction. In wild-type cells, GlpA was found at the septum formation site similarly to the CAR. The N-terminal 329 residues of GlpA are required for its localization to the septum formation site and essential for proper septum formation, while its C-terminal gelsolin domains are required for the regular CAR dynamics during septum formation. Finally, in this study we elucidated a novel Pezizomycotina-specific actin modulating component, which participates in septum formation by regulating the CAR dynamics.

  • Inter-strain expression of sequence-diverse HET domain genes severely inhibits growth of Aspergillus oryzae
    Bioscience biotechnology and biochemistry, 2019
    Co-Authors: Noriko Mori, Takuya Katayama, Ryota Saito, Kazuhiro Iwashita, Jun-ichi Maruyama
    Abstract:

    In the Pezizomycotina (filamentous ascomycete) species, genes that encode proteins with an HET domain (Pfam: PF06985) are reportedly involved in heterokaryon incompatibility (HI) in which cell death or growth defects are induced after fusion of cells that are genetically incompatible owing to diversities in their nucleotide sequence. HET domain genes are commonly found in Pezizomycotina genomes and are functionally characterized in only a few species. Here, we compared 44 HET domain genes between an incompatible strain pair of Aspergillus oryzae RIB40 and RIB128 and performed inter-strain expression of 37 sequence-diverse genes for mimicking HI. Four HET domain genes were identified to cause severe growth inhibition in a strain- or sequence-specific manner. Furthermore, SNPs responsible for the inhibition of cell growth were identified. This study provides an important insight into the physiological significance of sequence diversity of HET domain genes and their potential functions in HI of A. oryzae.

  • The Woronin Body: A Fungal Organelle Regulating Multicellularity
    Biology of the Fungal Cell, 2019
    Co-Authors: Jun-ichi Maruyama, Katsuhiko Kitamoto
    Abstract:

    In filamentous fungal species belonging to the Phyla Ascomycota and Basidiomycota, hyphae are compartmentalized into distinct cells by the formation of a septum. However, the septum does not completely separate hyphae due to the presence of a septal pore, which is a perforated structure that allows the exchange of the cytoplasmic constituents between adjacent hyphal cells. Cell-to-cell connectivity through the septal pore is associated with the catastrophic risk of cytoplasmic loss by cells adjacent to individually damaged hyphae. Pezizomycotina (filamentous Ascomycota) species have evolved to possess a specialized organelle called the Woronin body around the septum. The primary function of Woronin bodies is the prevention of excessive cytoplasmic loss from cells adjacent to damaged or lysed cells. Hex1, a major structural protein of Woronin bodies, is conserved in Pezizomycotina species. Woronin bodies differentiate from the peroxisomes and are typically tethered to the septum. Recent studies have identified additional septum-related components such as proteins containing intrinsically disordered regions and those involved in hyphal fusion/sexual reproduction and mitosis, which further elucidates molecular machineries governing the septal pore closure besides Woronin bodies.

  • Inter-strain expression of sequence-diverse HET domain genes severely inhibits growth of Aspergillus oryzae
    2019
    Co-Authors: Noriko Mori, Takuya Katayama, Ryota Saito, Kazuhiro Iwashita, Jun-ichi Maruyama
    Abstract:

    In the Pezizomycotina (filamentous ascomycete) species, genes that encode proteins with an HET domain (Pfam: PF06985) are reportedly involved in heterokaryon incompatibility (HI) in which cell death or growth defects are induced after fusion of cells that are genetically incompatible owing to diversities in their nucleotide sequence. HET domain genes are commonly found in Pezizomycotina genomes and are functionally characterized in only a few species. Here, we compared 44 HET domain genes between an incompatible strain pair of Aspergillus oryzae RIB40 and RIB128 and performed inter-strain expression of 37 sequence-diverse genes for mimicking HI. Four HET domain genes were identified to cause severe growth inhibition in a strain- or sequence-specific manner. Furthermore, SNPs responsible for the inhibition of cell growth were identified. This study provides an important insight into the physiological significance of sequence diversity of HET domain genes and their potential functions in HI of A. oryzae. Inter-strain expression of sequence-diverse HET domain genes severely inhibited growth in an incompatible strain pair of Aspergillus oryzae, and SNPs responsible for the growth inhibition were identified.

David J. Mclaughlin - One of the best experts on this subject based on the ideXlab platform.

  • Short title: Neolecta septal pore ultrastructure Functional and phylogenetic implications of septal pore ultrastructure in the ascoma of Neolecta vitellina
    2013
    Co-Authors: Rosanne A. Healy, David Hewitt, Arun Kumar, David J. Mclaughlin
    Abstract:

    Functional and phylogenetic implications of septal pore ultrastructure in the ascoma of Neolecta vitellina Rosanne A. Healy Department of Plant Biology, University of Minnesota, St Paul, Minnesota 55108 T.K. Arun Kumar The Zamorin's Guruvayurappan College, Calicut, Kerala 673014, India David A. Hewitt Department of Botany, Academy of Natural Sciences, Philadelphia, Pennsylvania 19103 David J. McLaughlin Department of Plant Biology, University of Minnesota, St Paul, Minnesota 55108 Abstract: Neolecta represents the earliest derived extant ascomycete lineage (Taphrinomycotina) to produce ascomata. For this reason the genus has been of interest with regard to ascoma evolution in ascomycetes. However, the evidence is equivocal regarding whether the Neolecta ascoma is homologous or analogous to ascomata produced in the later derived ascomycete lineages (Pezizomycotina). We investigated phylogenetically informative septal pore ultrastructure of Neolecta vitellina to compare with Pezizomycotina. We found that crystalline bodies that block nonascogenous septal pores in Neolecta differ from Woronin bodies, a synapomorphy for the Pezizomycotina, in three ways: (i) vacuolar origin, (ii) associated material and ( iii) being loosely membrane bound. We also observed a unique type of membranous material within the septal pore, as well as distant from the septal pore, that appears to be associated with the endoplasmic reticulum. The vacuolar crystals and membranous material might have a function analagous to septal pore structures (e.g. In Press at Mycologia, preliminary version published on May 24, 2013 as doi:10.3852/12-347

  • Orbilia ultrastructure, character evolution and phylogeny of Pezizomycotina
    Mycologia, 2011
    Co-Authors: T. K. Arun Kumar, Joseph W. Spatafora, Rosanne A. Healy, Meredith Blackwell, David J. Mclaughlin
    Abstract:

    Molecular phylogenetic analyses indicate that the monophyletic classes Orbiliomycetes and Pezizomycetes are among the earliest diverging branches of Pezizomycotina, the largest subphylum of the Ascomycota. Although Orbiliomycetes is re- solved as the most basal lineage in some analyses, molecular support for the node resolving the relation- ships between the two classes is low and topologies are unstable. We provide ultrastructural evidence to inform the placement of Orbiliomycetes by studying an Orbilia, a member of the only order (Orbiliales) of the class. The truncate ascus apex in the Orbilia is thin- walled except at the margin, and an irregular wall rupture of the apex permits ascospore discharge. Ascus, ascogenous and non-ascogenous hyphae were simple septate, with septal pores plugged by unelabo- rated electron-dense, non-membranous occlusions. Globose Woronin bodies were located on both sides of the septum. Nuclear division was characterized by the retention of an intact nuclear envelope, and a two- layered disk-shaped spindle pole body. The less differentiated nature of the spore discharge apparatus and septal pore organization supports an earliest diverging position of Orbiliomycetes within the sub- phylum, while the closed nuclear division and disk- shaped spindle pole body are interpreted as ancestral state characters for Ascomycota.