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Structural architecture of the human long non-coding RNA, steroid receptor RNA activator.

Novikova IV, Hennelly SP, Sanbonmatsu KY - Nucleic Acids Res. (2012)

Bottom Line: Our experimental findings (SHAPE, in-line, DMS and RNase V1 probing) reveal that this lncRNA has a complex structural organization, consisting of four domains, with a variety of secondary structure elements.Rapid evolutionary stabilization of RNA structure, combined with frame-disrupting mutations in conserved regions, suggests that evolutionary pressure preserves the RNA structural core rather than its translational product.We perform similar experiments on alternatively spliced SRA isoforms to assess their structural features.

View Article: PubMed Central - PubMed

Affiliation: Theoretical Biology and Biophysics, Theoretical Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA.

ABSTRACT
While functional roles of several long non-coding RNAs (lncRNAs) have been determined, the molecular mechanisms are not well understood. Here, we report the first experimentally derived secondary structure of a human lncRNA, the steroid receptor RNA activator (SRA), 0.87 kB in size. The SRA RNA is a non-coding RNA that coactivates several human sex hormone receptors and is strongly associated with breast cancer. Coding isoforms of SRA are also expressed to produce proteins, making the SRA gene a unique bifunctional system. Our experimental findings (SHAPE, in-line, DMS and RNase V1 probing) reveal that this lncRNA has a complex structural organization, consisting of four domains, with a variety of secondary structure elements. We examine the coevolution of the SRA gene at the RNA structure and protein structure levels using comparative sequence analysis across vertebrates. Rapid evolutionary stabilization of RNA structure, combined with frame-disrupting mutations in conserved regions, suggests that evolutionary pressure preserves the RNA structural core rather than its translational product. We perform similar experiments on alternatively spliced SRA isoforms to assess their structural features.

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Alternatively spliced isoforms of SRA. ncSRA, non-coding isoform of SRA; cSRA, coding isoform of SRA; intSRA, isoform of SRA that contains an intron not present in ncSRA and cSRA. (A) Experimentally determined secondary structure of the region of the coding isoform of SRA (cSRA) that differs from the non-coding isoform (ncSRA). This region is called the 5′-extension. Annotation is same as Figure 2. The cSRA and intSRA have identical secondary structures in this region. Upper right, schematic of alternatively spliced isoforms of SRA. The ncSRA comprises fully spliced core of SRA gene (black line). The cSRA possesses an extended exon-1 (blue line) with two initiation codons for SRAP synthesis (green bars). The intSRA retains a portion of an intron that is spliced out of ncSRA, located between exon-1 and exon-2 of SRA gene (red). (B) Processed SHAPE and DMS reactivities for cSRA and intSRA 5′-end extensions.
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gks071-F7: Alternatively spliced isoforms of SRA. ncSRA, non-coding isoform of SRA; cSRA, coding isoform of SRA; intSRA, isoform of SRA that contains an intron not present in ncSRA and cSRA. (A) Experimentally determined secondary structure of the region of the coding isoform of SRA (cSRA) that differs from the non-coding isoform (ncSRA). This region is called the 5′-extension. Annotation is same as Figure 2. The cSRA and intSRA have identical secondary structures in this region. Upper right, schematic of alternatively spliced isoforms of SRA. The ncSRA comprises fully spliced core of SRA gene (black line). The cSRA possesses an extended exon-1 (blue line) with two initiation codons for SRAP synthesis (green bars). The intSRA retains a portion of an intron that is spliced out of ncSRA, located between exon-1 and exon-2 of SRA gene (red). (B) Processed SHAPE and DMS reactivities for cSRA and intSRA 5′-end extensions.

Mentions: We chose three key SRA systems, which represent the majority of transcripts determined to date in humans: the original non-coding form, the coding form, and a second, longer non-coding form (Figure 7). System 1 (Figures 1–4) is the non-coding form (ncSRA, NCBI ID: AF092038), which is lacking initiation codons for the translational machinery and is 222 nt shorter than the coding form. ncSRA is the first SRA transcript determined and proven to act as a non-coding RNA (17). This transcript possesses an interesting 4-nt mutation relative to the coding sequence: positions 1–4 of ncSRA have the sequence CGCU rather than the GAGA sequence present in the coding form cSRA (Figures 7–8). This nucleotide substitution silences the initiation codon AUG located between positions 4–6 by modifying its sequence to UUG. In addition, the substituted nucleotides are able to participate in the formation of helix H1. System 2 is the coding isoform of SRA (cSRA, NCBI id: AF293024). In this isoform, exon 1 is extended by 222 nt on the 5′-side relative to the ncSRA isoform. This 5′-extension contains two initiation AUG codons, both utilized in the production of 224 and 236 amino acid SRAP proteins (29,70). System 3 is an alternatively spliced non-coding transcript of SRA. This isoform (intSRA, NCBI id: DQ286291) contains a portion of an intron that is normally spliced out of ncSRA. This intron portion in intSRA exists between positions 27 and 28 in ncSRA numbering (Figure 7, upper right). This SRA variant has the same 5′-extension as coding isoform (cSRA); however, a pre-mature stop codon in the intron sequence aborts translation (43).Figure 7.


Structural architecture of the human long non-coding RNA, steroid receptor RNA activator.

Novikova IV, Hennelly SP, Sanbonmatsu KY - Nucleic Acids Res. (2012)

Alternatively spliced isoforms of SRA. ncSRA, non-coding isoform of SRA; cSRA, coding isoform of SRA; intSRA, isoform of SRA that contains an intron not present in ncSRA and cSRA. (A) Experimentally determined secondary structure of the region of the coding isoform of SRA (cSRA) that differs from the non-coding isoform (ncSRA). This region is called the 5′-extension. Annotation is same as Figure 2. The cSRA and intSRA have identical secondary structures in this region. Upper right, schematic of alternatively spliced isoforms of SRA. The ncSRA comprises fully spliced core of SRA gene (black line). The cSRA possesses an extended exon-1 (blue line) with two initiation codons for SRAP synthesis (green bars). The intSRA retains a portion of an intron that is spliced out of ncSRA, located between exon-1 and exon-2 of SRA gene (red). (B) Processed SHAPE and DMS reactivities for cSRA and intSRA 5′-end extensions.
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gks071-F7: Alternatively spliced isoforms of SRA. ncSRA, non-coding isoform of SRA; cSRA, coding isoform of SRA; intSRA, isoform of SRA that contains an intron not present in ncSRA and cSRA. (A) Experimentally determined secondary structure of the region of the coding isoform of SRA (cSRA) that differs from the non-coding isoform (ncSRA). This region is called the 5′-extension. Annotation is same as Figure 2. The cSRA and intSRA have identical secondary structures in this region. Upper right, schematic of alternatively spliced isoforms of SRA. The ncSRA comprises fully spliced core of SRA gene (black line). The cSRA possesses an extended exon-1 (blue line) with two initiation codons for SRAP synthesis (green bars). The intSRA retains a portion of an intron that is spliced out of ncSRA, located between exon-1 and exon-2 of SRA gene (red). (B) Processed SHAPE and DMS reactivities for cSRA and intSRA 5′-end extensions.
Mentions: We chose three key SRA systems, which represent the majority of transcripts determined to date in humans: the original non-coding form, the coding form, and a second, longer non-coding form (Figure 7). System 1 (Figures 1–4) is the non-coding form (ncSRA, NCBI ID: AF092038), which is lacking initiation codons for the translational machinery and is 222 nt shorter than the coding form. ncSRA is the first SRA transcript determined and proven to act as a non-coding RNA (17). This transcript possesses an interesting 4-nt mutation relative to the coding sequence: positions 1–4 of ncSRA have the sequence CGCU rather than the GAGA sequence present in the coding form cSRA (Figures 7–8). This nucleotide substitution silences the initiation codon AUG located between positions 4–6 by modifying its sequence to UUG. In addition, the substituted nucleotides are able to participate in the formation of helix H1. System 2 is the coding isoform of SRA (cSRA, NCBI id: AF293024). In this isoform, exon 1 is extended by 222 nt on the 5′-side relative to the ncSRA isoform. This 5′-extension contains two initiation AUG codons, both utilized in the production of 224 and 236 amino acid SRAP proteins (29,70). System 3 is an alternatively spliced non-coding transcript of SRA. This isoform (intSRA, NCBI id: DQ286291) contains a portion of an intron that is normally spliced out of ncSRA. This intron portion in intSRA exists between positions 27 and 28 in ncSRA numbering (Figure 7, upper right). This SRA variant has the same 5′-extension as coding isoform (cSRA); however, a pre-mature stop codon in the intron sequence aborts translation (43).Figure 7.

Bottom Line: Our experimental findings (SHAPE, in-line, DMS and RNase V1 probing) reveal that this lncRNA has a complex structural organization, consisting of four domains, with a variety of secondary structure elements.Rapid evolutionary stabilization of RNA structure, combined with frame-disrupting mutations in conserved regions, suggests that evolutionary pressure preserves the RNA structural core rather than its translational product.We perform similar experiments on alternatively spliced SRA isoforms to assess their structural features.

View Article: PubMed Central - PubMed

Affiliation: Theoretical Biology and Biophysics, Theoretical Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA.

ABSTRACT
While functional roles of several long non-coding RNAs (lncRNAs) have been determined, the molecular mechanisms are not well understood. Here, we report the first experimentally derived secondary structure of a human lncRNA, the steroid receptor RNA activator (SRA), 0.87 kB in size. The SRA RNA is a non-coding RNA that coactivates several human sex hormone receptors and is strongly associated with breast cancer. Coding isoforms of SRA are also expressed to produce proteins, making the SRA gene a unique bifunctional system. Our experimental findings (SHAPE, in-line, DMS and RNase V1 probing) reveal that this lncRNA has a complex structural organization, consisting of four domains, with a variety of secondary structure elements. We examine the coevolution of the SRA gene at the RNA structure and protein structure levels using comparative sequence analysis across vertebrates. Rapid evolutionary stabilization of RNA structure, combined with frame-disrupting mutations in conserved regions, suggests that evolutionary pressure preserves the RNA structural core rather than its translational product. We perform similar experiments on alternatively spliced SRA isoforms to assess their structural features.

Show MeSH
Related in: MedlinePlus