A protocol for visual analysis of alternative splicing in RNA-Seq data using integrated genome browser.

Ultrahigh-throughput sequencing of cDNA (RNA-Seq) is an invaluable resource for investigating alternative splicing in an organism. Alternative splicing is a form of posttranscriptional regulation in which primary RNA transcripts from a single gene can be spliced in multiple ways leading to different RNA and protein products. In plants and other species, it has been shown that many genes involved in circadian regulation are alternatively spliced. As new RNA-Seq data sets become available, these data will lead to new insights into links between regulation RNA splicing and the circadian system. Analyzing RNA-Seq data sets requires software tools that can display RNA-Seq read alignments alongside gene models, enabling assessment of how treatments or developmental stages affect splicing patterns and production of novel variants. The Integrated Genome Browser (IGB) software program is a free and flexible desktop tool that enables discovery and quantification of alternative splicing. In this protocol, we use IGB and a cold-stress RNA-Seq data set to examine alternative splicing of Arabidopsis thaliana LHY, a circadian clock regulator. IGB is freely available from http://www.bioviz.org .

Mining Arabidopsis thaliana RNA-seq data with Integrated Genome Browser reveals stress-induced alternative splicing of the putative splicing regulator SR45a.

PREMISE OF THE STUDY: High-throughput sequencing of cDNA libraries prepared from diverse samples (RNA-seq) can reveal genome-wide changes in alternative splicing. Using RNA-seq data to assess splicing at the level of individual genes requires the ability to visualize read alignments alongside genomic annotations. To meet this need, we added RNA-seq visualization capability to Integrated Genome Browser (IGB), a free desktop genome visualization tool. To illustrate this capability, we present an in-depth analysis of abiotic stresses and their effects on alternative splicing of SR45a (AT1G07350), a putative splicing regulator from Arabidopsis thaliana. METHODS: cDNA libraries prepared from Arabidopsis plants that were subjected to heat and dehydration stresses were sequenced on an Illumina GAIIx sequencer, yielding more than 511 million high-quality 75-base, single-end sequence reads. Reads were aligned onto the reference genome and visualized in IGB. KEY RESULTS: Using IGB, we confirmed exon-skipping alternative splicing in SR45a. Exon-skipped variant AT1G07350.1 encodes full-length SR45a protein with intact RS and RNA recognition motifs, while nonskipped variant AT1G07350.2 lacks the C-terminal RS region due to a frameshift in the alternative exon. Heat and drought stresses increased both transcript abundance and the proportion of exon-skipped transcripts encoding the full-length protein. We identified new splice sites and observed frequent intron retention flanking the alternative exon. CONCLUSIONS: This study underlines the importance of visual inspection of RNA-seq alignments when investigating alternatively spliced genes. We showed that heat and dehydration stresses increase overall abundance of SR45a mRNA while also increasing production of transcripts encoding the full-length SR45a protein relative to other splice variants.

Daily genetic profiling indicates JAK/STAT signaling promotes early hepatic stellate cell transdifferentiation.

AIM: To identify signaling pathways and genes that initiate and commit hepatic stellate cells (HSCs) to transdifferentiation. METHODS: Primary HSCs were isolated from male Sprague-Dawley rats and cultured on plastic for 0-10 d. Gene expression was assessed daily (quiescent to day 10 culture-activation) by real time polymerase chain reaction and data clustered using AMADA software. The significance of JAK/STAT signaling to HSC transdifferentiation was determined by treating cells with a JAK2 inhibitor. RESULTS: Genetic cluster analyses, based on expression of these 21 genes, showed similar expression profiles on days 1-3, days 5 and 6, and days 7-10, while freshly isolated cells (day Q) and day 4 cells were genotypically distinct from any of the other days. Additionally, gene expression clustering revealed strong upregulation of interleukin-6, JAK2 and STAT3 mRNA in the early stages of activation. Inhibition of the JAK/STAT signaling pathway impeded the morphological transdifferentiation of HSCs which correlated with decreased mRNA expression of several profibrotic genes including collagens, α-SMA, PDGFR and TGFßR. CONCLUSION: These data demonstrate unique clustered genetic profiles during the daily progression of HSC transdifferentiation and that JAK/STAT signaling may be critical in the early stages of transdifferentiation.

Altered dopaminergic profiles: implications for the regulation of voluntary physical activity.

The biological regulating factors of physical activity in animals are not well understood. This study investigated differences in the central mRNA expression of seven dopamine genes (Drd1, Drd2, Drd3, Drd4, Drd5, TH, and DAT) between high active C57/LJ (n=17) male mice and low active C3H/HeJ (n=20) male mice, and between mice with access to a running wheel and without running wheel access within strain. Mice were housed with running wheels interfaced with a computer for 21 days with distance and duration recorded every 24 h. On day 21, the striatum and nucleus accumbens were removed during the active period (approximately 9 pm) for dopaminergic analysis. On average, the C57L/J mice with wheels ran significantly farther (10.25+/-1.37 km/day vs. 0.01+/-0.09 km/day, p<0.001), longer (329.73+/-30.52 min/day vs. 7.81+/-6.32 min/day, p<0.001), and faster (31.27+/-3.13 m/min vs. 11.81+/-1.08 m/min, p<0.001) than the C3H/HeJ mice with wheels over the 21 day period. No differences in gene expression were found between mice in either strain with wheels and those without wheels suggesting that access to running wheels did not alter dopaminergic expression. In contrast, relative expression for two dopamine genes was significantly lower in the C57L/J mice compared to the C3H/HeJ mice. These results indicate that decreased dopaminergic functioning is correlated with increased activity levels in C57L/J mice and suggests that D1-like receptors as well as tyrosine hydroxylase (an indicator of dopamine production), but not D2-like receptors may be associated with the regulation of physical activity in inbred mice.

Effects of hyperfibrinogenemia on vasculature of C57BL/6 mice with and without atherogenic diet.

OBJECTIVE: Elevated fibrinogen is correlated with severe atherosclerosis, as defined by the occurrence of ischemic events, but the mechanistic basis for this correlation remains unknown. To study this relationship, we examined spontaneous and diet-induced atherosclerosis in transgenic mice with hyperfibrinogenemia (elevated fibrinogen). METHODS AND RESULTS: Normal and transgenic mice were fed either an atherogenic diet or simple breeder chow. We measured plasma fibrinogen levels and identified an age-dependent and diet-dependent increase in fibrinogen. After 8 to 12 months, aortic sections were prepared and stained, and lipid-containing lesions were counted, measured, and assessed for maturity. Lipid-filled deposits appeared spontaneously in a small number of mice on breeder chow; typical fatty streak-type lesions appeared in almost all of the diet-fed animals. Morphometric analysis showed that neither the number nor the size of lesions was influenced by either fibrinogen level or genotype. CONCLUSIONS: Our data showed that neither fibrinogen concentration nor genotype had a statistically significant effect on the initiation, initial growth, or early progression of atherosclerotic lesions.