Photoactivation through the central dogma of molecular biology

Photoactivation is the property of a molecule of being capable of pronounced changes in its chemical properties in response to irradiation with light of a specific wavelength and intensity. This feature provides unique possibilities for the design of new strategies aimed at the spatio-temporal deciphering of molecular pathwhays occuring in living cells, organelles and intracellular molecules. Here, I spotlight two recent applications: from photoactivable nucleotides to photoactivable proteins.

RNA-binding motifs
UV-light irradiation has been used in the past to isolate RNAs bound to specific binding proteins (RBPs) but the discovery rate was inefficient because of problems in separating signal from background. Haffner and colleagues exploited photoactivable nucleosides as efficient and non-toxic crosslinkers that are well incorporated into the nascent RNA. Cells are grown in the presence of X mM of 4-thiouridine (4SU): 4SU-substituted RNA is 'activated' by UV light at 365 nm and specifically crosslinks to its RNA-binding partner proteins. Interestingly, if crosslinked and de-crosslinked (after immunoprecipitation of the RNA-bound-protein) 4SU is then mis-paired with guanine (G) instead of adenine (A) during the reverse transcription to cDNA. This allows the precise mapping of the binding sites by scoring the G/A transitions in the cDNA sequence. The researchers used this technique to define the binding motifs of 13 RBPs. (Haffner et al., Cell 2010)

Proteasome dynamics
Fluorescent reporters have already been used in the past to monitor proteosomal degradation, but effects on reporter degradation are difficult to separate from effects on reporter synthesis. Hamer and colleagues designed a reporter in which these processes can be easily distinguished. The green-to-red photoconvertible protein Dendra2 is fused to a form of ubiquitin that targets Dendra2 for proteosomal degradation. By observing only the photoconverted red form of the reporer, effects on its synthesis can be ruled out. The researchers used this reporter to monitor the cell type- and age-dependence of ubiquitin proteosome activity in living C.elegans. (Hamer et al., Nature Methods 2010)


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Hafner, M., Landthaler, M., Burger, L., Khorshid, M., Hausser, J., Berninger, P., Rothballer, A., Ascano Jr., M., Jungkamp, A., & Munschauer, M. (2010). Transcriptome-wide Identification of RNA-Binding Protein and MicroRNA Target Sites by PAR-CLIP Cell, 141 (1), 129-141 DOI: 10.1016/j.cell.2010.03.009

Hamer, G., Matilainen, O., & Holmberg, C. (2010). A photoconvertible reporter of the ubiquitin-proteasome system in vivo Nature Methods, 7 (6), 473-478 DOI: 10.1038/nmeth.1460