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Nijsje Dorman, Ph.D., Patrick C.H. Lo, Ph.D., and Kristie Nybo, Ph.D.
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Moyroud et al. 2009. The analysis of entire gene promoters by surface plasmon resonance. Plant J 59(5):851–858.

Moving into the Light

Plants respond to light for processes such as seed germination, seedling de-etiolation, and shade avoidance. They accomplish this through the action of light-absorbing proteins called phytochromes, which are sensitive to red and near-infrared light. Reporting in the journal Nature, Levskaya et al. take advantage of the light-sensing abilities of phytochromes, along with other proteins in the phytochrome signaling network, to reversibly control the translocation of proteins to the cell membrane. Their new method uses a well-characterized Arabidopsis thaliana pathway, in which phytochrome B (PhyB) binds directly to the downstream transcription factor phytochrome interaction factor 3 (PIF3) when stimulated by red light. The resulting heterodimer then moves into the nucleus to modulate transcription of light response genes. In contrast, when exposed to infrared light, PIF3 is no longer capable of binding PhyB, reversing the reaction. In order to harness this pathway for experimental use, the authors fused each of the previously reported PIF domains to yellow fluorescent protein (YFP) while attaching phytochrome domains to the m-Cherry fluorescent protein along with a membrane localization sequence. The fusion proteins were then co-expressed in NIH3T3 cells and screened for optimized light-directed spatiotemporal control in mammalian cells. The Phy-PIF pair showed translocation to the cell membrane in response to red light, while the pair moved away from the membrane when exposed to infrared light at rates an order of magnitude faster than what can be achieved by chemically induced translocation systems. In a proof-of-concept experiment, controlled actin remodeling was demonstrated by fusing the Phy-PIF constructs to catalytic modules of guanine nucleotide exchange factors (GEF) for Rho-family GTPases. In response to light, the GEF catalytic domains were recruited to the membrane to regulate the actin cytoskelelon at the polarized edges of motile cells. These interactions were stable, showing no decrease in membrane recruitment capacity even following more than 100 cycles of light exposure. This level of light control allows for unprecedented precision in the spatial and temporal manipulation of proteins when studying cell signaling events.

Levskaya et al. Spatiotemporal control of cell signaling using a light-switchable protein interaction. Nature. 2009 Sep 13. [Epub ahead of print; doi 10.1038/nature08446]

Taking the Direct Route

Transcriptomics typically depends on the analysis of cDNA using microarrays or next-generation sequencing. These methods, however, suffer from artifacts and biases caused by the error-prone and inefficient reverse transcriptases needed to convert RNA into cDNA. To circumvent these problems, Ozsolak and colleagues describe in Nature a new method for large-scale, massively parallel direct sequencing of RNA molecules that eliminates the need for cDNA synthesis and any ligation/amplification steps. Direct RNA sequencing (DRS) is an extension of the single-molecule DNA sequencing technology developed by Helicos Biosciences. Instead of DNA, individual RNA molecules serve as the templates for step-wise synthesis of a complementary DNA strand that is attached to a planar surface and imaged. The authors identified the optimal polymerase with reverse transcriptase activity that could efficiently incorporate their proprietary Virtual Terminator (VT) nucleotides which contain a fluorescent dye and chemically cleavable blocking group. The first step in DRS is the addition of a short poly(A) tail to RNA molecules lacking one. The 3′ end of the tail is then blocked by incorporation of 3′-deoxyATP to prevent further nucleotide addition, and the treated RNAs are hybridized to a poly(dT)-coated planar surface. Sequencing is primed by a fill-in reaction with unlabeled dTTP and polymerase, and then “locked in” with the incorporation of either a VT-A, VT-C, or VT-G nucleotide, ensuring that sequencing starts in the unique RNA sequence next to the poly(A) tail. The surface is washed to remove unincorporated VT nucleotides, imaged to locate the hybridized templates, and then the fluorescent dye and blocking group are cleaved off, which allow the next round of VT nucleotide incorporation. Successive alternating rounds of extension, imaging, and cleavage allows the determination of the RNA sequence. DRS of 2 femtomoles of Saccharomyces cerevisiae poly(A)+ RNA yielded around 40,000 reads, of which 48% aligned to the yeast genome with an average read length of 28 nucleotides. Comparison with a yeast expressed sequence tag (EST) database demonstrated the majority of reads were near the 3′ ends of ESTs. Intriguingly, they identified reads corresponding to the 3′ ends of mature rRNAs and snoRNAs, suggesting that a fraction of these RNAs are polyadenylated. The simplicity, sensitivity, and accuracy of DRS should prove extremely useful in transcriptomic studies, particularly with low-quantity samples or samples with very short RNAs.

Ozsolak et al. Direct RNA sequencing. Nature. 2009 Sep 23. [Epub ahead of print; doi:10.1038/nature08390]

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