博士学位論文内容の要旨及び審査の結果の要旨 : 平成23年9月28日授与分

Full details on spike-in control oligos. (PDF 226 kb

Additional file 1: of Long genes and genes with multiple splice variants are enriched in pathways linked to cancer and other multigenic diseases

Supplementary Notes S1 and S2, Tables S1-S4 and Figures S1-S7. The file is in the PDF format. The content is individually referred in the main text. (PDF 1637 kb

Left: frequency distributions of loops of lengths 1–7 bases for the entire human genome

<p><b>Copyright information:</b></p><p>Taken from "Prevalence of quadruplexes in the human genome"</p><p>Nucleic Acids Research 2005;33(9):2908-2916.</p><p>Published online 24 May 2005</p><p>PMCID:PMC1140081.</p><p>© The Author 2005. Published by Oxford University Press. All rights reserved</p> Right: percentage excesses of loop 2 counts over the averages of loops 1 and 3 for the entire human genome

Additional file 2 of Single genome retrieval of context-dependent variability in mutation rates for human germline

Supplementary numerical data. The raw data on the substitution rate constants in the reference L1 sequence (data 1), the resulting Trek database processed with (data 2) and without (data 3) the strand-symmetry considerations, the k-mer content for the masked and unmasked human genomes (data 4), the full set of 7-mer sequences with the respective cancer enrichment scores and basal substitution propensity values (data 5), and the GBM parameters that were minimising the error of the tree-based test models (data 6). The file is in the plain TXT format. (TXT 3266 kb

The Kinetics and Folding Pathways of Intramolecular G‑Quadruplex Nucleic Acids

The folding kinetics of G-quadruplex forming sequences is critical to their capacity to influence biological function. While G-quadruplex structure and stability have been relatively well studied, little is known about the kinetics of their folding. We employed a stopped-flow mixing technique to systematically investigate the potassium-dependent folding kinetics of telomeric RNA and DNA G-quadruplexes and RNA G-quadruplexes containing only two G-quartets formed from sequences r­[(GGA)₃GG] and r­[(GGUUA)₃GG]. Our findings suggest a folding mechanism that involves two kinetic steps with initial binding of a single K⁺, irrespective of the number of G-quartets involved or whether the G-quadruplex is formed from RNA or DNA. The folding rates for telomeric RNA and DNA G-quadruplexes are comparable at near physiological [K⁺] (90 mM) (τ = ∼60 ms). The folding of a 2-quartet RNA G-quadruplex with single nucleotide A loops is considerably slower (τ = ∼700 ms), and we found that the time required to fold a UUA looped variant (τ > 100 s, 500 mM K⁺) exceeds the lifetimes of some regulatory RNAs. We discuss the implications of these findings with respect to the fundamental properties of G-quadruplexes and their potential functions in biology

An RNA Hairpin to G‑Quadruplex Conformational Transition

RNA molecules can fold into noncanonical structures such as the four-stranded structures known as G-quadruplexes. G-quadruplexes in the transcriptome have recently emerged as relevant regulatory elements of gene expression. Conformational transitions in RNA molecules offer an important way to regulate their biological functions. Here we report on the competition between a canonical hairpin structure and a G-quadruplex structure within an RNA molecule. We show that the conformational preference strongly depends on the relative amounts of mono- and divalent metal ions present in solution. In our system, the G-quadruplex, whose formation is not predicted by available predictive RNA folding programs, is the major conformer at physiologically relevant K⁺ and Mg²⁺ concentrations. Furthermore, we show that a synthetic small molecule can displace the structural dynamic equilibrium in favor of the hairpin conformer. This work highlights a new and important level of complexity in RNA folding that could be relevant to the biological functions and targeting of RNAs comprising G-quadruplex motifs

An Intramolecular G-Quadruplex Structure Is Required for Binding of Telomeric Repeat-Containing RNA to the Telomeric Protein TRF2

Telomeric repeat-containing RNA (TERRA) is important for telomere regulation, but the structural basis for how TERRA localizes to chromosome ends is unknown. Here we report on studies exploring whether the TERRA G-quadruplex structure is critical for binding to telomeres. We demonstrate that the telomeric protein TRF2 binds TERRA via interactions that necessitate the formation of a G-quadruplex structure rather than the TERRA sequence per se. We also show that TRF2 simultaneously binds TERRA and telomeric duplex or G-quadruplex DNA. These observations suggest that the TERRA G-quadruplex is a key feature of telomere organization

Elevated Levels of G-Quadruplex Formation in Human Stomach and Liver Cancer Tissues

<div><p>Four-stranded G-quadruplex DNA secondary structures have recently been visualized in the nuclei of human cultured cells. Here, we show that BG4, a G-quadruplex-specific antibody, can be used to stain DNA G-quadruplex structures in patient-derived tissues using immunohistochemistry. We observe a significantly elevated number of G-quadruplex-positive nuclei in human cancers of the liver and stomach as compared to background non-neoplastic tissue. Our results suggest that G-quadruplex formation can be detected and measured in patient-derived material and that elevated G-quadruplex formation may be a characteristic of some cancers.</p></div

Increased incidence of G-quadruplex-positive cell nuclei across human stomach cancers.

<p>Confirmation of the increased presence of BG4-positive nuclei in a range of stomach cancer tissues compared to background non-neoplastic tissues including unmatched cases. <b>A</b>. The nuclei of non-neoplastic stomach tissues are largely BG4-negative. Cell nuclei were counterstained with haematoxylin (blue). Scale bar corresponds to 50 µm. <b>B</b>. Gastric adenocarcinoma tissue shows the extensive presence of BG4-positive nuclei (brown). <b>C</b>. Signet ring cell carcinoma shows many BG4-positive nuclei. <b>D</b>. Gastrointestinal stromal tumors (GIST) are largely BG4-positive (for the survey of stomach cancer only unmatched GIST tissues were available). <b>E</b>. Quantification of the number of BG4-positive nuclei in individual stomach tissues including unmatched TMA cores. Non-neoplastic stomach tissue shows less BG4-positive nuclei compared to adenocarcinoma, signet ring carcinoma or GIST. Each column corresponds to a single tissue sample and error bars represent the s.e.m. calculated when duplicate tissue samples were available. <b>F</b>. Overall quantification of the number of BG4-positive nuclei in all non-neoplastic and stomach liver tissues. Error bars represent the s.e.m. **P<0.001, n = 10 and 34 for non-neoplastic and cancer cores, respectively. These results confirm the generality of more extensive G-quadruplex formation in the stomach cancers compared t­o non-neoplastic tissues.</p

Binding Interactions between Long Noncoding RNA HOTAIR and PRC2 Proteins

Long noncoding RNAs (lncRNAs) play a key role in the epigenetic regulation of cells. Many of these lncRNAs function by interacting with histone repressive proteins of the Polycomb group (PcG) family, recruiting them to gene loci to facilitate silencing. Although there are now many RNAs known to interact with the PRC2 complex, little is known about the details of the molecular interactions. Here, we show that the PcG protein heterodimer EZH2-EED is necessary and sufficient for binding to the lncRNA HOTAIR. We also show that protein recognition occurs within a folded 89-mer domain of HOTAIR. This 89-mer represents a minimal binding motif, as further deletion of nucleotides results in substantial loss of affinity for PRC2. These findings provide molecular insights into an important system involved in epigenetic regulation