A survey of fly and nematode small RNAs by deep sequencing

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2008."Suppl. materials for chapters 2 + 4"--handwritten on CDROM. Vita.Includes bibliographical references.Small RNAs of -22 nt length play a variety of roles in the biology of animals by repressing the translation or stimulating the degradation of complementary messenger RNAs. Depending on the structure of their precursors, they can be categorized as either microRNAs (miRNAs) or small interfering RNAs (siRNAs). In animals, miRNAs derive from characteristic hairpins in primary transcripts through two sequential RNase III-mediated cleavages; Drosha cleaves near the base of the stem to liberate a pre-miRNA hairpin, then Dicer cleaves near the loop to generate a miRNA:miRNA* duplex. Large-scale sequencing of cDNAs derived from endogenously expressed small RNAs is used here to examine the small RNAs of the nematode Caenorhabditis elegans and the fly Drosophila melanogaster, revealing a number of previously unidentified miRNA genes from each organism. These data also revealed a novel miRNA biogenesis pathway, the mirtron pathway, in which debranched introns mimic the structural features of pre-miRNAs to enter the miRNA-processing pathway without Drosha-mediated cleavage. Mirtrons were identified in both D. melanogaster and C. elegans, some of which exhibit patterns of sequence conservation suggesting important regulatory functions. Sequencing was performed across a time-course of D. melanogaster development, permitting refinement of preexisting miRNA annotations and providing insights into miRNA biogenesis and expression. Conserved miRNAs were typically expressed more broadly and robustly than non conserved miRNAs, and miRNAs with more restricted expression tended to have fewer predicted targets. Insights were also provided into miRNA gene evolution.(cont.) Finally, two possible sources of endogenous siRNAs were revealed: antisense transcription and endogenous hpRNAs. Besides miRNAs, sequencing from C. elegans revealed thousands of endogenous siRNAs generated by RNA-directed RNA polymerases acting preferentially on spermatogenesis- and transposon-associated transcripts. A third class of nematode small RNAs, called 21U-RNAs, was also discovered. 21U-RNAs are precisely 21 nucleotides long and begin with a uridine but are diverse in their remaining 20 nucleotides. 21U-RNAs originate from >5700 genomic loci dispersed in two broad regions of chromosome IV. These loci share an upstream motif that enables accurate prediction of additional 21U-RNAs. The motif is conserved in other nematodes, presumably because of its importance for producing these diverse, autonomously expressed, small RNAs.by J. Graham Ruby.Ph.D

Formation, regulation and evolution of Caenorhabditis elegans 3'UTRs

Post-transcriptional gene regulation frequently occurs through elements in mRNA 3′ untranslated regions (UTRs)1, 2. Although crucial roles for 3′UTR-mediated gene regulation have been found in Caenorhabditis elegans3, 4, 5, most C. elegans genes have lacked annotated 3′UTRs6, 7. Here we describe a high-throughput method for reliable identification of polyadenylated RNA termini, and we apply this method, called poly(A)-position profiling by sequencing (3P-Seq), to determine C. elegans 3′UTRs. Compared to standard methods also recently applied to C. elegans UTRs8, 3P-Seq identified 8,580 additional UTRs while excluding thousands of shorter UTR isoforms that do not seem to be authentic. Analysis of this expanded and corrected data set suggested that the high A/U content of C. elegans 3′UTRs facilitated genome compaction, because the elements specifying cleavage and polyadenylation, which are A/U rich, can more readily emerge in A/U-rich regions. Indeed, 30% of the protein-coding genes have mRNAs with alternative, partially overlapping end regions that generate another 10,480 cleavage and polyadenylation sites that had gone largely unnoticed and represent potential evolutionary intermediates of progressive UTR shortening. Moreover, a third of the convergently transcribed genes use palindromic arrangements of bidirectional elements to specify UTRs with convergent overlap, which also contributes to genome compaction by eliminating regions between genes. Although nematode 3′UTRs have median length only one-sixth that of mammalian 3′UTRs, they have twice the density of conserved microRNA sites, in part because additional types of seed-complementary sites are preferentially conserved. These findings reveal the influence of cleavage and polyadenylation on the evolution of genome architecture and provide resources for studying post-transcriptional gene regulation.National Institutes of Health (U.S.) (Grant number GM067031)National Science Foundation (U.S.). Predoctural FellowshipUnited States. Dept. of Energy. Computational Science Graduate Fellowship (Krell Institute

Transcriptome Sequencing Demonstrates that Human Papillomavirus Is Not Active in Cutaneous Squamous Cell Carcinoma

β-Human papillomavirus (β-HPV) DNA is present in some cutaneous squamous cell carcinomas (cuSCCs), but no mechanism of carcinogenesis has been determined. We used ultra-high-throughput sequencing of the cancer transcriptome to assess whether papillomavirus transcripts are present in these cancers. In all, 67 cuSCC samples were assayed for β-HPV DNA by PCR, and viral loads were measured with type-specific quantitative PCR. A total of 31 SCCs were selected for whole transcriptome sequencing. Transcriptome libraries were prepared in parallel from the HPV18-positive HeLa cervical cancer cell line and HPV16-positive primary cervical and periungual SCCs. Of the tumors, 30% (20/67) were positive for β-HPV DNA, but there was no difference in β-HPV viral load between tumor and normal tissue (P=0.310). Immunosuppression and age were significantly associated with higher viral load (P=0.016 for immunosuppression; P=0.0004 for age). Transcriptome sequencing failed to identify papillomavirus expression in any of the skin tumors. In contrast, HPV16 and HPV18 mRNA transcripts were readily identified in primary cervical and periungual cancers and HeLa cells. These data demonstrate that papillomavirus mRNA expression is not a factor in the maintenance of cuSCCs

Comparative Analysis of Tandem Repeats from Hundreds of Species Reveals Unique Insights into Centromere Evolution

Centromeres are essential for chromosome segregation, yet their DNA sequences evolve rapidly. In most animals and plants that have been studied, centromeres contain megabase-scale arrays of tandem repeats. Despite their importance, very little is known about the degree to which centromere tandem repeats share common properties between different species across different phyla. We used bioinformatic methods to identify high-copy tandem repeats from 282 species using publicly available genomic sequence and our own data. The assumption that the most abundant tandem repeat is the centromere DNA was true for most species whose centromeres have been previously characterized, suggesting this is a general property of genomes. Our methods are compatible with all current sequencing technologies. Long Pacific Biosciences sequence reads allowed us to find tandem repeat monomers up to 1,419 bp. High-copy centromere tandem repeats were found in almost all animal and plant genomes, but repeat monomers were highly variable in sequence composition and in length. Furthermore, phylogenetic analysis of sequence homology showed little evidence of sequence conservation beyond ~50 million years of divergence. We find that despite an overall lack of sequence conservation, centromere tandem repeats from diverse species showed similar modes of evolution, including the appearance of higher order repeat structures in which several polymorphic monomers make up a larger repeating unit. While centromere position in most eukaryotes is epigenetically determined, our results indicate that tandem repeats are highly prevalent at centromeres of both animals and plants. This suggests a functional role for such repeats, perhaps in promoting concerted evolution of centromere DNA across chromosomes

The Macronuclear Genome of \u3cem\u3eStentor coeruleus\u3c/em\u3e Reveals Tiny Introns in a Giant Cell

The giant, single-celled organism Stentor coeruleus has a long history as a model system for studying pattern formation and regeneration in single cells. Stentor [1, 2] is a heterotrichous ciliate distantly related to familiar ciliate models, such as Tetrahymena or Paramecium. The primary distinguishing feature of Stentor is its incredible size: a single cell is 1 mm long. Early developmental biologists, including T.H. Morgan [3], were attracted to the system because of its regenerative abilities—if large portions of a cell are surgically removed, the remnant reorganizes into a normal-looking but smaller cell with correct proportionality [2, 3]. These biologists were also drawn to Stentor because it exhibits a rich repertoire of behaviors, including light avoidance, mechanosensitive contraction, food selection, and even the ability to habituate to touch, a simple form of learning usually seen in higher organisms [4]. While early microsurgical approaches demonstrated a startling array of regenerative and morphogenetic processes in this single-celled organism, Stentor was never developed as a molecular model system. We report the sequencing of the Stentor coeruleus macronuclear genome and reveal key features of the genome. First, we find that Stentor uses the standard genetic code, suggesting that ciliate-specific genetic codes arose after Stentor branched from other ciliates. We also discover that ploidy correlates with Stentor’s cell size. Finally, in the Stentor genome, we discover the smallest spliceosomal introns reported for any species. The sequenced genome opens the door to molecular analysis of single-cell regeneration in Stentor

Correction: Correction: A Novel Rhabdovirus Associated with Acute Hemorrhagic Fever in Central Africa.

[This corrects the article DOI: 10.1371/journal.ppat.1002924.]

Attitudes to in vitro meat:A survey of potential consumers in the United States

Positivity towards meat consumption remains strong, despite evidence of negative environmental and ethical outcomes. Although awareness of these repercussions is rising, there is still public resistance to removing meat from our diets. One potential method to alleviate these effects is to produce in vitro meat: meat grown in a laboratory that does not carry the same environmental or ethical concerns. However, there is limited research examining public attitudes towards in vitro meat, thus we know little about the capacity for it be accepted by consumers. This study aimed to examine perceptions of in vitro meat and identify potential barriers that might prevent engagement. Through conducting an online survey with US participants, we identified that although most respondents were willing to try in vitro meat, only one third were definitely or probably willing to eat in vitro meat regularly or as a replacement for farmed meat. Men were more receptive to it than women, as were politically liberal respondents compared with conservative ones. Vegetarians and vegans were more likely to perceive benefits compared to farmed meat, but they were less likely to want to try it than meat eaters. The main concerns were an anticipated high price, limited taste and appeal and a concern that the product was unnatural. It is concluded that people in the USA are likely to try in vitro meat, but few believed that it would replace farmed meat in their diet

A randomized controlled trial of nonoperative treatment versus open reduction and internal fixation for stable, displaced, partial articular fractures of the radial head: The RAMBO trial

Background: The choice between operative or nonoperative treatment is questioned for partial articular fractures of the radial head that have at least 2 millimeters of articular step-off on at least one radiograph (defined as displaced), but less than 2 millimeter of gap between the fragments (defined as stable) and that are not associated with an elbow dislocation, interosseous ligament injury, or other fractures. These kinds of fractures are often classified as Mason type-2 fractures. Retrospective comparative studies suggest that operative treatment might be better than nonoperative treatment, but the long-term results of nonoperative treatment are very good. Most experts agree that problems like reduced range of motion, painful crepitation, nonunion or bony ankylosis are infrequent with both nonoperative and operative treatment of an isolated displaced partial articular fracture of the radial head, but determining which patients will have problems is difficult. A prospective, randomized comparison would help minimize bias and determine the balance between operative and nonoperative risks and benefits. Methods/Design. The RAMBO trial (Radial Head - Amsterdam - Amphia - Boston - Others) is an international prospective, randomized, multicenter trial. The primary objective of this study is to compare patient related outcome defined by the \u27Disabilities of Arm, Shoulder and Hand (DASH) score\u27 twelve months after injury between operative and nonoperative treated patients. Adult patients with partial articular fractures of the radial head that comprise at least 1/3rd of the articular surface, have ≥ 2 millimeters of articular step-off but less than 2 millimeter of gap between the fragments will be enrolled. Secondary outcome measures will be the Mayo Elbow Performance Index (MEPI), the Oxford Elbow Score (OES), pain intensity through the \u27Numeric Rating Scale\u27, range of motion (flexion arc and rotational arc), radiographic appearance of the fracture (heterotopic ossification, radiocapitellar and ulnohumeral arthrosis, fracture healing, and signs of implant loosening or breakage) and adverse events (infection, nerve injury, secondary interventions) after one year. Discussion. The successful completion of this trial will provide evidence on the best treatment for stable, displaced, partial articular fractures of the radial head. Trial registration. The trial is registered at the Dutch Trial Register: NTR3413. © 2014Bruinsma et al.; licensee BioMed Central Ltd

The Long March: A Sample Preparation Technique that Enhances Contig Length and Coverage by High-Throughput Short-Read Sequencing

High-throughput short-read technologies have revolutionized DNA sequencing by drastically reducing the cost per base of sequencing information. Despite producing gigabases of sequence per run, these technologies still present obstacles in resequencing and de novo assembly applications due to biased or insufficient target sequence coverage. We present here a simple sample preparation method termed the “long march” that increases both contig lengths and target sequence coverage using high-throughput short-read technologies. By incorporating a Type IIS restriction enzyme recognition motif into the sequencing primer adapter, successive rounds of restriction enzyme cleavage and adapter ligation produce a set of nested sub-libraries from the initial amplicon library. Sequence reads from these sub-libraries are offset from each other with enough overlap to aid assembly and contig extension. We demonstrate the utility of the long march in resequencing of the Plasmodium falciparum transcriptome, where the number of genomic bases covered was increased by 39%, as well as in metagenomic analysis of a serum sample from a patient with hepatitis B virus (HBV)-related acute liver failure, where the number of HBV bases covered was increased by 42%. We also offer a theoretical optimization of the long march for de novo sequence assembly