ABSTRACT Representatives of the freshwater red algal family Thoreaceae were studied to resolve th... more ABSTRACT Representatives of the freshwater red algal family Thoreaceae were studied to resolve their taxonomic and phylogenetic status. Three specimens of Nemalionopsis and five collections of Thorea were examined for pit plug ultrastructure and analyzed for the sequences of the genes coding for the large subunit of RUBISCO (rbcL) and the small subunit of rRNA (18S rRNA). The phylogenetic trees generated from the two genes, and a combined tree all showed the Thoreaceae to be contained in a well-supported monophyletic clade that is separate from the other two families currently classified in the Batrachospermales, the Batrachospermaceae and the Lemaneaceae. In addition, secondary structure elements of the 18S rRNA gene were observed at positions 650 and 1145 (Escherichia coli numbering system) that are not present in other members of the Rhodophyta. The pit plugs of the gametophytic and chantransia stages of the Thoreaceae contain two cap layers, the outer one of which is typically plate-like, though occasionally inflated ones have been seen. No pit plug cap membrane has been observed. These findings indicate the Thoreaceae has been misclassified in the Batrachospermales and should be placed in its own order, the Thoreales. This order is characterized by having freshwater representatives with multiaxial gametophytes, a uniaxial chantransia stage, and pit plugs with two cap layers, the outer one of which is usually plate-like.
The elucidation of 16S and 23S rRNA Higher-Order Structure has been addressed by Comparative Sequ... more The elucidation of 16S and 23S rRNA Higher-Order Structure has been addressed by Comparative Sequence Methods for more than a decade. During these years our comparative methods have evolved as the number of complete 16S and 23S rRNA sequences have increased significantly, resulting in the maturation of the higher-order structure models for 16S and 23S rRNA.
The ribosomal RNAs, along with their substrates the transfer RNAs, contain the most highly conser... more The ribosomal RNAs, along with their substrates the transfer RNAs, contain the most highly conserved nucleotides in all of biology. We have assembled a database containing structure-based alignments of sequences of the small-subunit rRNAs from organisms that span the entire phylogenetic spectrum, to identify the nucleotides that are universally conserved. In its simplest (bacterial and archaeal) forms, the small-subunit rRNA has ∼1500 nt, of which we identify 140 that are absolutely invariant among the 1961 species in our alignment. We examine the positions and detailed structural and functional interactions of these universal nucleotides in the context of a half century of biochemical and genetic studies and high-resolution structures of ribosome functional complexes. The vast majority of these nucleotides are exposed on the subunit interface surface of the small subunit, where the functional processes of the ribosome take place. However, only 40 of them have been directly implicated in specific ribosomal functions, such as contacting the tRNAs, mRNA, or translation factors. The roles of many other invariant nucleotides may serve to constrain the positions and orientations of those nucleotides that are directly involved in function. Yet others can be rationalized by participation in unusual noncanonical tertiary structures that may uniquely allow correct folding of the rRNA to form a functional ribosome. However, there remain at least 50 nt whose universal conservation is not obvious, serving as a metric for the incompleteness of our understanding of ribosome structure and function.
An increasing number of recognition mechanisms in RNA are found to involve G.U base pairs. In ord... more An increasing number of recognition mechanisms in RNA are found to involve G.U base pairs. In order to detect new functional sites of this type, we exhaustively analyzed the sequence alignments and secondary structures of eubacterial and chloroplast 16S and 23S rRNA, seeking positions with high levels of G.U pairs. Approximately 120 such sites were identified and classified according to their secondary structure and sequence environment. Overall biases in the distribution of G.U pairs are consistent with previously proposed structural rules: the side of the wobble pair that is subject to a loss of stacking is preferentially exposed to a secondary structure loop, where stacking is not as essential as in helical regions. However, multiple sites violate these rules and display highly conserved G.U pairs in orientations that could cause severe stacking problems. In addition, three motifs displaying a conserved G.U pair in a specific sequence/structure environment occur at an unusually high frequency. These motifs, of which two had not been reported before, involve sequences 5'UG3' 3'GA5' and 5'UG3' 3'GU5', as well as G.U pairs flanked by a bulge loop 3' of U. The possible structures and functions of these recurrent motifs are discussed.
Structure probing combined with next-generation sequencing (NGS) has provided novel insights into... more Structure probing combined with next-generation sequencing (NGS) has provided novel insights into RNA structure–function relationships. To date, such studies have focused largely on bacteria and eukaryotes, with little attention given to the third domain of life, archaea. Furthermore, functional RNAs have not been extensively studied in archaea, leaving open questions about RNA structure and function within this domain of life. With archaeal species being diverse and having many similarities to both bacteria and eukaryotes, the archaea domain has the potential to be an evolutionary bridge. In this study, we introduce a method for probing RNA structure in vivo in the archaea domain of life. We investigated the structure of ribosomal RNA (rRNA) fromMethanosarcina acetivorans, a well-studied anaerobic archaeal species, grown with either methanol or acetate. After probing the RNA in vivo with dimethyl sulfate (DMS), Structure-seq2 libraries were generated, sequenced, and analyzed. We mapped the reactivity of DMS onto the secondary structure of the ribosome, which we determined independently with comparative analysis, and confirmed the accuracy of DMS probing inM. acetivorans. Accessibility of the rRNA to DMS in the two carbon sources was found to be quite similar, although some differences were found. Overall, this study establishes the Structure-seq2 pipeline in the archaea domain of life and informs about ribosomal structure withinM. acetivorans.
RNAcentral is a comprehensive database of non-coding RNA (ncRNA) sequences that provides a single... more RNAcentral is a comprehensive database of non-coding RNA (ncRNA) sequences that provides a single access point to 44 RNA resources and >18 million ncRNA sequences from a wide range of organisms and RNA types. RNAcentral now also includes secondary (2D) structure information for >13 million sequences, making RNAcentral the world’s largest RNA 2D structure database. The 2D diagrams are displayed using R2DT, a new 2D structure visualization method that uses consistent, reproducible and recognizable layouts for related RNAs. The sequence similarity search has been updated with a faster interface featuring facets for filtering search results by RNA type, organism, source database or any keyword. This sequence search tool is available as a reusable web component, and has been integrated into several RNAcentral member databases, including Rfam, miRBase and snoDB. To allow for a more fine-grained assignment of RNA types and subtypes, all RNAcentral sequences have been annotated with S...
Non-coding RNAs (ncRNA) are essential for all life, and the functions of many ncRNAs depend on th... more Non-coding RNAs (ncRNA) are essential for all life, and the functions of many ncRNAs depend on their secondary (2D) and tertiary (3D) structure. Despite proliferation of 2D visualisation software, there is a lack of methods for automatically generating 2D representations in consistent, reproducible, and recognisable layouts, making them difficult to construct, compare and analyse. Here we present R2DT, a comprehensive method for visualising a wide range of RNA structures in standardised layouts. R2DT is based on a library of 3,632 templates representing the majority of known structured RNAs, from small RNAs to the large subunit ribosomal RNA. R2DT has been applied to ncRNA sequences from the RNAcentral database and produced >13 million diagrams, creating the world’s largest RNA 2D structure dataset. The software is freely available at https://github.com/rnacentral/R2DT and a web server is found at https://rnacentral.org/r2dt.
<p>The coloring scheme is the same as in <a href="http://www.plosone.org/article/in... more <p>The coloring scheme is the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038203#pone-0038203-g001" target="_blank">Fig. 1</a>. The helices shared by the bacterial 16S and eukaryotic 18S rRNAs are shown in red, while the eukaryotic-specific and bacterial-specific helices are shown in green and in blue, respectively.</p
<p>The base pairings, helices other RNA structural elements, and relevant ribosomal protein... more <p>The base pairings, helices other RNA structural elements, and relevant ribosomal proteins in the <i>T. thermophila</i> 40S crystal structure are mapped onto the <i>T. thermophila</i> SSU rRNA comparative secondary structure diagram <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038203#pone.0038203-Cannone1" target="_blank">[7]</a>. The nucleotides in the eukaryotic-specific structural elements are colored green, while the nucleotides colored red are structural elements in the variable regions that are analogous to helices in the bacterial structure. The V2, V4, and V6 variable regions are colored red while the bacterial-specific helix in V6 is shown in blue on the bacterial <i>Thermus thermophilus</i> SSU rRNA (inset). The long-range tertiary contacts maintained in both the eukaryotic and the bacterial SSU rRNA are shown with red lines, while those specific for the eukaryotic 18S rRNA in green lines; the tertiary contacts specifically associated with V2, V4, and V6 are shown with thicker lines. The ribosomal proteins common between eukaryotes and bacteria are shown in red, with their bacterial equivalents in parentheses, while those present only in eukaryotes in green and those present in archaea and eukaryotes in purple. The sequence insertions in the eukaryotic SSU rRNAs are highlighted in green with green arrows and numbers indicating the number of inserted nucleotides.</p
Helices are an essential element in defining the three-dimensional architecture of structured RNA... more Helices are an essential element in defining the three-dimensional architecture of structured RNAs. While internal basepairs in a canonical helix stack on both sides, the ends of the helix stack on only one side and are exposed to the loop side, thus susceptible to fraying unless they are protected. While coaxial stacking has long been known to stabilize helix ends by directly stacking two canonical helices coaxially, based on analysis of helix-loop junctions in RNA crystal structures, herein we describe helix capping, topological stacking of a helix end with a basepair or an unpaired nucleotide from the loop side, which in turn protects helix ends. Beyond the topological protection of helix ends against fraying, helix capping should confer greater stability onto the resulting composite helices. Our analysis also reveals that this general motif is associated with the formation of tertiary structure interactions. Greater knowledge about the dynamics at the helix-junctions in the secondary structure should enhance the prediction of RNA secondary structure with a richer set of energetic rules and help better understand the folding of a secondary structure into its three-dimensional structure. These together suggest that helix capping likely play a fundamental role in driving RNA folding.
ABSTRACT Representatives of the freshwater red algal family Thoreaceae were studied to resolve th... more ABSTRACT Representatives of the freshwater red algal family Thoreaceae were studied to resolve their taxonomic and phylogenetic status. Three specimens of Nemalionopsis and five collections of Thorea were examined for pit plug ultrastructure and analyzed for the sequences of the genes coding for the large subunit of RUBISCO (rbcL) and the small subunit of rRNA (18S rRNA). The phylogenetic trees generated from the two genes, and a combined tree all showed the Thoreaceae to be contained in a well-supported monophyletic clade that is separate from the other two families currently classified in the Batrachospermales, the Batrachospermaceae and the Lemaneaceae. In addition, secondary structure elements of the 18S rRNA gene were observed at positions 650 and 1145 (Escherichia coli numbering system) that are not present in other members of the Rhodophyta. The pit plugs of the gametophytic and chantransia stages of the Thoreaceae contain two cap layers, the outer one of which is typically plate-like, though occasionally inflated ones have been seen. No pit plug cap membrane has been observed. These findings indicate the Thoreaceae has been misclassified in the Batrachospermales and should be placed in its own order, the Thoreales. This order is characterized by having freshwater representatives with multiaxial gametophytes, a uniaxial chantransia stage, and pit plugs with two cap layers, the outer one of which is usually plate-like.
The elucidation of 16S and 23S rRNA Higher-Order Structure has been addressed by Comparative Sequ... more The elucidation of 16S and 23S rRNA Higher-Order Structure has been addressed by Comparative Sequence Methods for more than a decade. During these years our comparative methods have evolved as the number of complete 16S and 23S rRNA sequences have increased significantly, resulting in the maturation of the higher-order structure models for 16S and 23S rRNA.
The ribosomal RNAs, along with their substrates the transfer RNAs, contain the most highly conser... more The ribosomal RNAs, along with their substrates the transfer RNAs, contain the most highly conserved nucleotides in all of biology. We have assembled a database containing structure-based alignments of sequences of the small-subunit rRNAs from organisms that span the entire phylogenetic spectrum, to identify the nucleotides that are universally conserved. In its simplest (bacterial and archaeal) forms, the small-subunit rRNA has ∼1500 nt, of which we identify 140 that are absolutely invariant among the 1961 species in our alignment. We examine the positions and detailed structural and functional interactions of these universal nucleotides in the context of a half century of biochemical and genetic studies and high-resolution structures of ribosome functional complexes. The vast majority of these nucleotides are exposed on the subunit interface surface of the small subunit, where the functional processes of the ribosome take place. However, only 40 of them have been directly implicated in specific ribosomal functions, such as contacting the tRNAs, mRNA, or translation factors. The roles of many other invariant nucleotides may serve to constrain the positions and orientations of those nucleotides that are directly involved in function. Yet others can be rationalized by participation in unusual noncanonical tertiary structures that may uniquely allow correct folding of the rRNA to form a functional ribosome. However, there remain at least 50 nt whose universal conservation is not obvious, serving as a metric for the incompleteness of our understanding of ribosome structure and function.
An increasing number of recognition mechanisms in RNA are found to involve G.U base pairs. In ord... more An increasing number of recognition mechanisms in RNA are found to involve G.U base pairs. In order to detect new functional sites of this type, we exhaustively analyzed the sequence alignments and secondary structures of eubacterial and chloroplast 16S and 23S rRNA, seeking positions with high levels of G.U pairs. Approximately 120 such sites were identified and classified according to their secondary structure and sequence environment. Overall biases in the distribution of G.U pairs are consistent with previously proposed structural rules: the side of the wobble pair that is subject to a loss of stacking is preferentially exposed to a secondary structure loop, where stacking is not as essential as in helical regions. However, multiple sites violate these rules and display highly conserved G.U pairs in orientations that could cause severe stacking problems. In addition, three motifs displaying a conserved G.U pair in a specific sequence/structure environment occur at an unusually high frequency. These motifs, of which two had not been reported before, involve sequences 5'UG3' 3'GA5' and 5'UG3' 3'GU5', as well as G.U pairs flanked by a bulge loop 3' of U. The possible structures and functions of these recurrent motifs are discussed.
Structure probing combined with next-generation sequencing (NGS) has provided novel insights into... more Structure probing combined with next-generation sequencing (NGS) has provided novel insights into RNA structure–function relationships. To date, such studies have focused largely on bacteria and eukaryotes, with little attention given to the third domain of life, archaea. Furthermore, functional RNAs have not been extensively studied in archaea, leaving open questions about RNA structure and function within this domain of life. With archaeal species being diverse and having many similarities to both bacteria and eukaryotes, the archaea domain has the potential to be an evolutionary bridge. In this study, we introduce a method for probing RNA structure in vivo in the archaea domain of life. We investigated the structure of ribosomal RNA (rRNA) fromMethanosarcina acetivorans, a well-studied anaerobic archaeal species, grown with either methanol or acetate. After probing the RNA in vivo with dimethyl sulfate (DMS), Structure-seq2 libraries were generated, sequenced, and analyzed. We mapped the reactivity of DMS onto the secondary structure of the ribosome, which we determined independently with comparative analysis, and confirmed the accuracy of DMS probing inM. acetivorans. Accessibility of the rRNA to DMS in the two carbon sources was found to be quite similar, although some differences were found. Overall, this study establishes the Structure-seq2 pipeline in the archaea domain of life and informs about ribosomal structure withinM. acetivorans.
RNAcentral is a comprehensive database of non-coding RNA (ncRNA) sequences that provides a single... more RNAcentral is a comprehensive database of non-coding RNA (ncRNA) sequences that provides a single access point to 44 RNA resources and >18 million ncRNA sequences from a wide range of organisms and RNA types. RNAcentral now also includes secondary (2D) structure information for >13 million sequences, making RNAcentral the world’s largest RNA 2D structure database. The 2D diagrams are displayed using R2DT, a new 2D structure visualization method that uses consistent, reproducible and recognizable layouts for related RNAs. The sequence similarity search has been updated with a faster interface featuring facets for filtering search results by RNA type, organism, source database or any keyword. This sequence search tool is available as a reusable web component, and has been integrated into several RNAcentral member databases, including Rfam, miRBase and snoDB. To allow for a more fine-grained assignment of RNA types and subtypes, all RNAcentral sequences have been annotated with S...
Non-coding RNAs (ncRNA) are essential for all life, and the functions of many ncRNAs depend on th... more Non-coding RNAs (ncRNA) are essential for all life, and the functions of many ncRNAs depend on their secondary (2D) and tertiary (3D) structure. Despite proliferation of 2D visualisation software, there is a lack of methods for automatically generating 2D representations in consistent, reproducible, and recognisable layouts, making them difficult to construct, compare and analyse. Here we present R2DT, a comprehensive method for visualising a wide range of RNA structures in standardised layouts. R2DT is based on a library of 3,632 templates representing the majority of known structured RNAs, from small RNAs to the large subunit ribosomal RNA. R2DT has been applied to ncRNA sequences from the RNAcentral database and produced >13 million diagrams, creating the world’s largest RNA 2D structure dataset. The software is freely available at https://github.com/rnacentral/R2DT and a web server is found at https://rnacentral.org/r2dt.
<p>The coloring scheme is the same as in <a href="http://www.plosone.org/article/in... more <p>The coloring scheme is the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038203#pone-0038203-g001" target="_blank">Fig. 1</a>. The helices shared by the bacterial 16S and eukaryotic 18S rRNAs are shown in red, while the eukaryotic-specific and bacterial-specific helices are shown in green and in blue, respectively.</p
<p>The base pairings, helices other RNA structural elements, and relevant ribosomal protein... more <p>The base pairings, helices other RNA structural elements, and relevant ribosomal proteins in the <i>T. thermophila</i> 40S crystal structure are mapped onto the <i>T. thermophila</i> SSU rRNA comparative secondary structure diagram <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038203#pone.0038203-Cannone1" target="_blank">[7]</a>. The nucleotides in the eukaryotic-specific structural elements are colored green, while the nucleotides colored red are structural elements in the variable regions that are analogous to helices in the bacterial structure. The V2, V4, and V6 variable regions are colored red while the bacterial-specific helix in V6 is shown in blue on the bacterial <i>Thermus thermophilus</i> SSU rRNA (inset). The long-range tertiary contacts maintained in both the eukaryotic and the bacterial SSU rRNA are shown with red lines, while those specific for the eukaryotic 18S rRNA in green lines; the tertiary contacts specifically associated with V2, V4, and V6 are shown with thicker lines. The ribosomal proteins common between eukaryotes and bacteria are shown in red, with their bacterial equivalents in parentheses, while those present only in eukaryotes in green and those present in archaea and eukaryotes in purple. The sequence insertions in the eukaryotic SSU rRNAs are highlighted in green with green arrows and numbers indicating the number of inserted nucleotides.</p
Helices are an essential element in defining the three-dimensional architecture of structured RNA... more Helices are an essential element in defining the three-dimensional architecture of structured RNAs. While internal basepairs in a canonical helix stack on both sides, the ends of the helix stack on only one side and are exposed to the loop side, thus susceptible to fraying unless they are protected. While coaxial stacking has long been known to stabilize helix ends by directly stacking two canonical helices coaxially, based on analysis of helix-loop junctions in RNA crystal structures, herein we describe helix capping, topological stacking of a helix end with a basepair or an unpaired nucleotide from the loop side, which in turn protects helix ends. Beyond the topological protection of helix ends against fraying, helix capping should confer greater stability onto the resulting composite helices. Our analysis also reveals that this general motif is associated with the formation of tertiary structure interactions. Greater knowledge about the dynamics at the helix-junctions in the secondary structure should enhance the prediction of RNA secondary structure with a richer set of energetic rules and help better understand the folding of a secondary structure into its three-dimensional structure. These together suggest that helix capping likely play a fundamental role in driving RNA folding.
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