The structures of RNA molecules are often important for their function and regulation1-6, yet there are no experimental techniques for genome-scale measurement of RNA structure. Here, we describe a novel strategy termed Parallel Analysis of RNA Structure (PARS), which is based on deep sequencing fragments of RNAs that were treated with structure-specific enzymes, thus providing simultaneous in-vitro profiling of the secondary structure of thousands of RNA species at single nucleotide resolution. We apply PARS to profile the secondary structure of the mRNAs of the budding yeast S. cerevisiae and obtain structural profiles for over 3000 distinct transcripts. Analysis of these profiles reveals several RNA structural properties of yeast transcripts, including the existence of more secondary structure over coding regions compared to untranslated regions, a three-nucleotide periodicity of secondary structure across coding regions, and a relationship between the efficiency with which an mRNA is translated and the lack of structure over its translation start site. PARS is readily applicable to other organisms and to profiling RNA structure in diverse conditions, thus enabling studies of the dynamics of secondary structure at a genomic scale.

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RNA structural transitions are important in the function and regulation of RNAs. Here, we reveal a layer of transcriptome organization in the form of RNA folding energies. By probing yeast RNA structures at different temperatures, we obtained relative melting temperatures (Tm) for RNA structures in over 4000 transcripts. Specific signatures of RNA Tm demarcated the polarity of mRNA open reading frames, and highlighted numerous candidate regulatory RNA motifs in 3′ untranslated regions. RNA Tm distinguished non-coding versus coding RNAs, identified mRNAs with distinct cellular functions. We identified thousands of putative RNA thermometers, and their presence is predictive of the pattern of RNA decay in vivo during heat shock. The exosome complex recognizes unpaired bases during heat shock to degrade these RNAs, coupling intrinsic structural stabilities to gene regulation. Thus, genome-wide structural dynamics of RNA can parse functional elements of the transcriptome and reveal diverse biological insights.

Abnormalities of genomic methylation patterns are lethal or cause disease, but the cues that normally designate CpG dinucleotides for methylation are poorly understood. We have developed a new method of methylation profiling that has single-CpG resolution and can address the methylation status of repeated sequences. We have used this method to determine the methylation status of >275 million CpG sites in human and mouse DNA from breast and brain tissues. Methylation density at most sequences was found to increase linearly with CpG density and to fall sharply at very high CpG densities, but transposons remained densely methylated even at higher CpG densities. The presence of histone H2A.Z and histone H3 di-or trimethylated at lysine 4 correlated strongly with unmethylated DNA and occurred primarily at promoter regions. We conclude that methylation is the default state of most CpG dinucleotides in the mammalian genome and that a combination of local dinucleotide frequencies, the interaction of repeated sequences, and the presence or absence of histone variants or modifications shields a population of CpG sites (most of which are in and around promoters) from DNA methyltransferases that lack intrinsic sequence specificity.

The complete nucleotide sequence of the maize chlorotic mottle virus (MCMV) genome has been determined to be 4437 nucleotides. The viral genome has four long open reading frames (ORFs) which could encode polypeptides of 31.6, 50, 8.9 and 25.1 kd. If the termination codons, for the polypeptides encoded by the 50 and 8.9 kd ORFs are suppressed, readthrough products of 111 and 32.7 kd result. The 31.6 and 50 kd ORFs overlap for nearly the entire length of the 31.6 kd ORF. Striking amino acid homology has been observed between two potential polypeptides encoded by MCMV and polypeptides encoded by carnation mottle virus (CarMV) and turnip crinkle virus (TCV). The 25.1 kd ORF most likely encodes the capsid protein. The similar genome organization and amino acid sequence homology of MCMV with CarMV and TCV suggest an evolutionary relationship with these members of the carmovirus group.

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The Ti plasmid sequences (T-DNA) from the octopine-producing crown gall tumor A6S/2 were isolated by molecular cloning, using the bacteriophage A vector Charon 4A. Analysis of the clone DNA segments indicates that the Ti plasmid sequences are covalently joined to plant nuclear DNA. These data demonstrate that genetic recombination between a eukaryote and a prokaryote can occur as a natural phenomenon.Crown gall is a disease of dicotyledonous plants caused by Agrobacterium tumefaciens. The virulence trait of A. tumefaciens is carried on diverse tumor-inducing (Ti) plasmids, which range in size between about 90 and 150 X 106 daltons (1-3). In the course of infection a portion of the Ti plasmid, the T-DNA, is stably transferred to the plant (4) and causes two fundamental changes in the physiology of the plant cells. First, the cells become transformed: whereas normal plant tissue grows in callus culture only when auxin and cytokinin are added to the medium, the growth of crown gall tissue is phytohormone independent (5). Second, crown gall tissues characteristically synthesize opines, primarily octopine or nopaline, which are not synthesized by normal plant tissues (6, 7). The particular opine produced is coded by the Ti plasmid (8, 9). The Ti plasmid also codes for the catabolism of the corresponding opine (8, 9). Thus the A. tumefaciens-plant interaction is one in which a prokaryote "genetically engineers" a eukaryote to synthesize a compound the bacterium can use as a carbon, nitrogen, and energy source.We have recently described the organization of T-DNA in four independent crown gall tumor lines incited by three closely related octopine-type Ti plasmids (10). It was shown that each tumor line contains a "core" T-DNA segment that: (i) is apparently responsible for maintaining the transformed state; (ii) is colinear with the Ti plasmid; and (iii) contains the Ti plasmid sequences termed "common DNA"-sequences found in most Ti plasmids and thought to have a central role in crown gall tumorigenesis (11,12). We also presented data suggesting that the T-DNA is integrated into plant DNA, that preferred regions of the Ti plasmid serve as the points of attachment to plant DNA, and that T-DNA can be linked to more than one site in the plant genome (10). Here we report the molecular cloning of the T-DNA and adjacent plant sequences from the crown gall tumor A6S/2. MATERIALS AND METHODSTobacco Cell Lines. The derivation of the cloned octopine-producing tumor line A6S/2 has been described (13). Nicotiana tabacum strain WBSR was derived from a single root A. tumefaciens (19) and E. coli (10, 20), and chloroplast DNA from tobacco leaves (21) were isolated as previously described. Phage DNA was isolated from CsCl-banded phage (15).In Vitro Labeling of DNA and Hybridization Conditions. DNA samples were labeled with 32P by a slight modification (10) of the nick-translation procedure (22). Hybridization of nitrocellulose filters containing bound DNA with the 32P-labeled probes and autoradiography were as described (10)....

Massively parallel, tag-based sequencing systems, such as the SOLiD system, hold the promise of revolutionizing the study of whole genome gene expression due to the number of data points that can be generated in a simple and cost-effective manner. We describe the development of a 5′–end transcriptome workflow for the SOLiD system and demonstrate the advantages in sensitivity and dynamic range offered by this tag-based application over traditional approaches for the study of whole genome gene expression. 5′-end transcriptome analysis was used to study whole genome gene expression within a colon cancer cell line, HT-29, treated with the DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine (5Aza). More than 20 million 25-base 5′-end tags were obtained from untreated and 5Aza-treated cells and matched to sequences within the human genome. Seventy three percent of the mapped unique tags were associated with RefSeq cDNA sequences, corresponding to approximately 14,000 different protein-coding genes in this single cell type. The level of expression of these genes ranged from 0.02 to 4,704 transcripts per cell. The sensitivity of a single sequence run of the SOLiD platform was 100–1,000 fold greater than that observed from 5′end SAGE data generated from the analysis of 70,000 tags obtained by Sanger sequencing. The high-resolution 5′end gene expression profiling presented in this study will not only provide novel insight into the transcriptional machinery but should also serve as a basis for a better understanding of cell biology.