mRNA Deep Sequencing Research Articles

Disruption of signaling in a fungal-grass symbiosis leads to pathogenesis.

Symbiotic associations between plants and fungi are a dominant feature of many terrestrial ecosystems, yet relatively little is known about the signaling, and associated transcriptome profiles, that define the symbiotic metabolic state. Using the Epichloë festucae-perennial ryegrass (Lolium perenne) association as a model symbiotic experimental system, we show an essential role for the fungal stress-activated mitogen-activated protein kinase (sakA) in the establishment and maintenance of this mutualistic interaction. Deletion of sakA switches the fungal interaction with the host from mutualistic to pathogenic. Infected plants exhibit loss of apical dominance, premature senescence, and dramatic changes in development, including the formation of bulb-like structures at the base of tillers that lack anthocyanin pigmentation. A comparison of the transcriptome of wild-type and sakA associations using high-throughput mRNA sequencing reveals dramatic changes in fungal gene expression consistent with the transition from restricted to proliferative growth, including a down-regulation of several clusters of secondary metabolite genes and up-regulation of a large set of genes that encode hydrolytic enzymes and transporters. Analysis of the plant transcriptome reveals up-regulation of host genes involved in pathogen defense and transposon activation as well as dramatic changes in anthocyanin and hormone biosynthetic/responsive gene expression. These results highlight the fine balance between mutualism and antagonism in a plant-fungal interaction and the power of deep mRNA sequencing to identify candidate sets of genes underlying the symbiosis.

MRNA deep sequencing reveals 75 new genes and a complex transcriptional landscape in Mimivirus

Mimivirus, a virus infecting Acanthamoeba, is the prototype of the Mimiviridae, the latest addition to the nucleocytoplasmic large DNA viruses. The Mimivirus genome encodes close to 1000 proteins, many of them never before encountered in a virus, such as four amino-acyl tRNA synthetases. To explore the physiology of this exceptional virus and identify the genes involved in the building of its characteristic intracytoplasmic "virion factory," we coupled electron microscopy observations with the massively parallel pyrosequencing of the polyadenylated RNA fractions of Acanthamoeba castellanii cells at various time post-infection. We generated 633,346 reads, of which 322,904 correspond to Mimivirus transcripts. This first application of deep mRNA sequencing (454 Life Sciences [Roche] FLX) to a large DNA virus allowed the precise delineation of the 5' and 3' extremities of Mimivirus mRNAs and revealed 75 new transcripts including several noncoding RNAs. Mimivirus genes are expressed across a wide dynamic range, in a finely regulated manner broadly described by three main temporal classes: early, intermediate, and late. This RNA-seq study confirmed the AAAATTGA sequence as an early promoter element, as well as the presence of palindromes at most of the polyadenylation sites. It also revealed a new promoter element correlating with late gene expression, which is also prominent in Sputnik, the recently described Mimivirus "virophage." These results-validated genome-wide by the hybridization of total RNA extracted from infected Acanthamoeba cells on a tiling array (Agilent)--will constitute the foundation on which to build subsequent functional studies of the Mimivirus/Acanthamoeba system.

Deep mRNA sequencing for in vivo functional analysis of cardiac transcriptional regulators: application to Galphaq.

Transcriptional profiling can detect subclinical heart disease and provide insight into disease etiology and functional status. Current microarray-based methods are expensive and subject to artifact. To develop RNA sequencing methodologies using next generation massively parallel platforms for high throughput comprehensive analysis of individual mouse cardiac transcriptomes. To compare the results of sequencing- and array-based transcriptional profiling in the well-characterized Galphaq transgenic mouse hypertrophy/cardiomyopathy model. The techniques for preparation of individually bar-coded mouse heart RNA libraries for Illumina Genome Analyzer II resequencing are described. RNA sequencing showed that 234 high-abundance transcripts (>60 copies/cell) comprised 55% of total cardiac mRNA. Parallel transcriptional profiling of Galphaq transgenic and nontransgenic hearts by Illumina RNA sequencing and Affymetrix Mouse Gene 1.0 ST arrays revealed superior dynamic range for mRNA expression and enhanced specificity for reporting low-abundance transcripts by RNA sequencing. Differential mRNA expression in Galphaq and nontransgenic hearts correlated well between microarrays and RNA sequencing for highly abundant transcripts. RNA sequencing was superior to arrays for accurately quantifying lower-abundance genes, which represented the majority of the regulated genes in the Galphaq transgenic model. RNA sequencing is rapid, accurate, and sensitive for identifying both abundant and rare cardiac transcripts, and has significant advantages in time- and cost-efficiencies over microarray analysis.

Deep Sequencing Raises Known Mimivirus Gene Count Close to 1,000

Polyadenylated RNA pyrosequencing has helped reveal 75 additional genes in the genetically well-endowed Acanthamoeba–infecting mimivirus, according to Jean-Michel Claverie and Chantal Abergel at the Mediterranean Institute of Microbiology in Marseille and the Institut de Genomique in Evry, France, and their collaborators. In addition to raising the mimiviral gene count to almost 1,000, this use of deep mRNA sequencing in a large DNA virus also confirms the presence of 900 predicted protein-coding genes and maps their location with a 1-nucleotide resolution.

Deep sequencing of mRNA to understand function of splicing regulatory factors

Splicing regulatory elements (SREs) and the associated splicing factors play central roles in the control of pre‐mRNA splicing and in exon evolution. We are studying in depth the regulatory properties of representative splicing factors, including the widely expressed factor hnRNP H, and the muscle‐ and brain‐specific factors of the CELF and Muscleblind families, using knockout or knockdown followed by deep sequencing of mRNAs (KO‐Seq or KD‐Seq), cross‐linking/immunoprecipitation/sequencing (CLIP‐Seq), and other approaches. In this talk I will discuss our progress towards understanding the functions of these factors, including the finding that the activity of G‐run intronic splicing enhancers (ISEs) and hnRNP H is highly dependent on the strength of the adjacent 5' splice site sequence, and the functional and evolutionary implications of this finding.Our work is supported by the NHGRI and the NIGMS.