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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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Secondary structure of entire SRA lncRNA, based on DMS and RNase V1 experiments. DMS probing detects single-stranded regions; RNase V1 digestion detects double stranded helical regions. Uncircled nucleotides, normalized DMS reactivity < 0.3; grey circled nucleotides, 0.3 < normalized DMS reactivity < 0.5; yellow circled nucleotides, 0.5 < normalized DMS reactivity < 0.7; orange circled nucleotides, normalized DMS reactivity > 0.7; blue arrows, normalized RNase V1 cleavage > 0.5; green nucleotides, no probing data. Helices are indicated by H1, … , H25. INSET 1: Raw capillary electropherograms of RNA region containing helices H12–H13 of domain II. Brown, DMS reactivity; blue, RNase V1 cleavage; green, raw blank trace. INSET 2: Raw capillary electropherograms of RNA region containing helices H19-H21 of domain III. Brown, DMS reactivity; blue, RNase V1 cleavage; green, raw blank trace.
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gks071-F3: Secondary structure of entire SRA lncRNA, based on DMS and RNase V1 experiments. DMS probing detects single-stranded regions; RNase V1 digestion detects double stranded helical regions. Uncircled nucleotides, normalized DMS reactivity < 0.3; grey circled nucleotides, 0.3 < normalized DMS reactivity < 0.5; yellow circled nucleotides, 0.5 < normalized DMS reactivity < 0.7; orange circled nucleotides, normalized DMS reactivity > 0.7; blue arrows, normalized RNase V1 cleavage > 0.5; green nucleotides, no probing data. Helices are indicated by H1, … , H25. INSET 1: Raw capillary electropherograms of RNA region containing helices H12–H13 of domain II. Brown, DMS reactivity; blue, RNase V1 cleavage; green, raw blank trace. INSET 2: Raw capillary electropherograms of RNA region containing helices H19-H21 of domain III. Brown, DMS reactivity; blue, RNase V1 cleavage; green, raw blank trace.

Mentions: Figures 2 and 3 summarize our experimental findings and display our experimentally derived secondary structure, annotated with either SHAPE and in-line reactivities or DMS and RNase V1 reactivities, respectively. The lncRNA appears to possess a complex secondary structure organization consisting of four major domains, comprising 25 helices in total. To simplify the following discussion, we also outline the helix nomenclature of the lncRNA based on several conventions previously applied to the ribosomal RNA secondary structure. First, the numbering of the helices was sequential, starting from the 5′-end to the 3′-end. Second, helices were differentiated when they were separated by either (i) a junction, (ii) a large internal loop (>12 nt in total), or (iii) a highly asymmetric internal loop with zero residues present on one side and a large number of single-stranded nucleotides (>6 nt) on the other side.Figure 2.


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

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

Secondary structure of entire SRA lncRNA, based on DMS and RNase V1 experiments. DMS probing detects single-stranded regions; RNase V1 digestion detects double stranded helical regions. Uncircled nucleotides, normalized DMS reactivity < 0.3; grey circled nucleotides, 0.3 < normalized DMS reactivity < 0.5; yellow circled nucleotides, 0.5 < normalized DMS reactivity < 0.7; orange circled nucleotides, normalized DMS reactivity > 0.7; blue arrows, normalized RNase V1 cleavage > 0.5; green nucleotides, no probing data. Helices are indicated by H1, … , H25. INSET 1: Raw capillary electropherograms of RNA region containing helices H12–H13 of domain II. Brown, DMS reactivity; blue, RNase V1 cleavage; green, raw blank trace. INSET 2: Raw capillary electropherograms of RNA region containing helices H19-H21 of domain III. Brown, DMS reactivity; blue, RNase V1 cleavage; green, raw blank trace.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3367176&req=5

gks071-F3: Secondary structure of entire SRA lncRNA, based on DMS and RNase V1 experiments. DMS probing detects single-stranded regions; RNase V1 digestion detects double stranded helical regions. Uncircled nucleotides, normalized DMS reactivity < 0.3; grey circled nucleotides, 0.3 < normalized DMS reactivity < 0.5; yellow circled nucleotides, 0.5 < normalized DMS reactivity < 0.7; orange circled nucleotides, normalized DMS reactivity > 0.7; blue arrows, normalized RNase V1 cleavage > 0.5; green nucleotides, no probing data. Helices are indicated by H1, … , H25. INSET 1: Raw capillary electropherograms of RNA region containing helices H12–H13 of domain II. Brown, DMS reactivity; blue, RNase V1 cleavage; green, raw blank trace. INSET 2: Raw capillary electropherograms of RNA region containing helices H19-H21 of domain III. Brown, DMS reactivity; blue, RNase V1 cleavage; green, raw blank trace.
Mentions: Figures 2 and 3 summarize our experimental findings and display our experimentally derived secondary structure, annotated with either SHAPE and in-line reactivities or DMS and RNase V1 reactivities, respectively. The lncRNA appears to possess a complex secondary structure organization consisting of four major domains, comprising 25 helices in total. To simplify the following discussion, we also outline the helix nomenclature of the lncRNA based on several conventions previously applied to the ribosomal RNA secondary structure. First, the numbering of the helices was sequential, starting from the 5′-end to the 3′-end. Second, helices were differentiated when they were separated by either (i) a junction, (ii) a large internal loop (>12 nt in total), or (iii) a highly asymmetric internal loop with zero residues present on one side and a large number of single-stranded nucleotides (>6 nt) on the other side.Figure 2.

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