RNA-Seq analysis of splicing in Plasmodium falciparum uncovers new splice junctions, alternative splicing and splicing of antisense transcripts.

Over 50% of genes in Plasmodium falciparum, the deadliest human malaria parasite, contain predicted introns, yet experimental characterization of splicing in this organism remains incomplete. We present here a transcriptome-wide characterization of intraerythrocytic splicing events, as captured by RNA-Seq data from four timepoints of a single highly synchronous culture. Gene model-independent analysis of these data in conjunction with publically available RNA-Seq data with HMMSplicer, an in-house developed splice site detection algorithm, revealed a total of 977 new 5' GU-AG 3' and 5 new 5' GC-AG 3' junctions absent from gene models and ESTs (11% increase to the current annotation). In addition, 310 alternative splicing events were detected in 254 (4.5%) genes, most of which truncate open reading frames. Splicing events antisense to gene models were also detected, revealing complex transcriptional arrangements within the parasite's transcriptome. Interestingly, antisense introns overlap sense introns more than would be expected by chance, perhaps indicating a functional relationship between overlapping transcripts or an inherent organizational property of the transcriptome. Independent experimental validation confirmed over 30 new antisense and alternative junctions. Thus, this largest assemblage of new and alternative splicing events to date in Plasmodium falciparum provides a more precise, dynamic view of the parasite's transcriptome.

Recovery of divergent avian bornaviruses from cases of proventricular dilatation disease: identification of a candidate etiologic agent.

BACKGROUND: Proventricular dilatation disease (PDD) is a fatal disorder threatening domesticated and wild psittacine birds worldwide. It is characterized by lymphoplasmacytic infiltration of the ganglia of the central and peripheral nervous system, leading to central nervous system disorders as well as disordered enteric motility and associated wasting. For almost 40 years, a viral etiology for PDD has been suspected, but to date no candidate etiologic agent has been reproducibly linked to the disease. RESULTS: Analysis of 2 PDD case-control series collected independently on different continents using a pan-viral microarray revealed a bornavirus hybridization signature in 62.5% of the PDD cases (5/8) and none of the controls (0/8). Ultra high throughput sequencing was utilized to recover the complete viral genome sequence from one of the virus-positive PDD cases. This revealed a bornavirus-like genome organization for this agent with a high degree of sequence divergence from all prior bornavirus isolates. We propose the name avian bornavirus (ABV) for this agent. Further specific ABV PCR analysis of an additional set of independently collected PDD cases and controls yielded a significant difference in ABV detection rate among PDD cases (71%, n = 7) compared to controls (0%, n = 14) (P = 0.01; Fisher's Exact Test). Partial sequence analysis of a total of 16 ABV isolates we have now recovered from these and an additional set of cases reveals at least 5 distinct ABV genetic subgroups. CONCLUSION: These studies clearly demonstrate the existence of an avian reservoir of remarkably diverse bornaviruses and provide a compelling candidate in the search for an etiologic agent of PDD.

HMMSplicer: a tool for efficient and sensitive discovery of known and novel splice junctions in RNA-Seq data.

BACKGROUND: High-throughput sequencing of an organism's transcriptome, or RNA-Seq, is a valuable and versatile new strategy for capturing snapshots of gene expression. However, transcriptome sequencing creates a new class of alignment problem: mapping short reads that span exon-exon junctions back to the reference genome, especially in the case where a splice junction is previously unknown. METHODOLOGY/PRINCIPAL FINDINGS: Here we introduce HMMSplicer, an accurate and efficient algorithm for discovering canonical and non-canonical splice junctions in short read datasets. HMMSplicer identifies more splice junctions than currently available algorithms when tested on publicly available A. thaliana, P. falciparum, and H. sapiens datasets without a reduction in specificity. CONCLUSIONS/SIGNIFICANCE: HMMSplicer was found to perform especially well in compact genomes and on genes with low expression levels, alternative splice isoforms, or non-canonical splice junctions. Because HHMSplicer does not rely on pre-built gene models, the products of inexact splicing are also detected. For H. sapiens, we find 3.6% of 3' splice sites and 1.4% of 5' splice sites are inexact, typically differing by 3 bases in either direction. In addition, HMMSplicer provides a score for every predicted junction allowing the user to set a threshold to tune false positive rates depending on the needs of the experiment. HMMSplicer is implemented in Python. Code and documentation are freely available at http://derisilab.ucsf.edu/software/hmmsplicer.

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.

Unbiased selection of localization elements reveals cis-acting determinants of mRNA bud localization in Saccharomyces cerevisiae.

Cytoplasmic mRNA localization is a mechanism used by many organisms to generate asymmetry and sequester protein activity. In the yeast Saccharomyces cerevisiae, mRNA transport to bud tips of dividing cells is mediated by the binding of She2p, She3p, and Myo4p to coding regions of the RNA. To date, 24 bud-localized mRNAs have been identified, yet the RNA determinants that mediate localization remain poorly understood. Here, we used nonhomologous random recombination to generate libraries of sequences that could be selected for their ability to bind She-complex proteins, thereby providing an unbiased approach for minimizing and mapping localization elements in several transported RNAs. Analysis of the derived sequences and predicted secondary structures revealed short sequence motifs that mediate binding to the She complex and RNA localization to the bud tip in vivo. A predicted single-stranded core CG dinucleotide appears to be an important component of the RNA-protein interface, although other nucleotides contribute in a context-dependent manner. Our findings further our understanding of RNA recognition by the She complex, and the methods used here should be applicable for elucidating minimal RNA motifs involved in many other types of interactions.

Characterization of the transmembrane channel-like (TMC) gene family: functional clues from hearing loss and epidermodysplasia verruciformis.

Mutations of TMC1 cause deafness in humans and mice. TMC1 and a related gene, TMC2, are the founding members of a novel gene family. Here we describe six additional TMC paralogs (TMC3 to TMC8) in humans and mice, as well as homologs in other species. cDNAs spanning the full length of the predicted open reading frames of the mammalian genes were cloned and sequenced. All are strongly predicted to encode proteins with 6 to 10 transmembrane domains and a novel conserved 120-amino-acid sequence that we termed the TMC domain. TMC1, TMC2, and TMC3 comprise a distinct subfamily expressed at low levels, whereas TMC4 to TMC8 are expressed at higher levels in multiple tissues. TMC6 and TMC8 are identical to the EVER1 and EVER2 genes implicated in epidermodysplasia verruciformis, a recessive disorder comprising susceptibility to cutaneous human papilloma virus infections and associated nonmelanoma skin cancers, providing additional genetic and tissue systems in which to study the TMC gene family.