Figure 3. Synergies between methodologies lead to new rules of RNA regulation.
Biochemical methods, exemplified by HITS-CLIP, can yield genome-wide footprints of direct RNA-protein interactions, but lack functional information. In contrast, microarrays or RNA-Seq are able to correlate differences in RNA profiles between tissues45 or genetic systems such as KO vs. WT animals53, but cannot distinguish direct from indirect targets. Bioinformatic analysis can also be used to identify sequence features associated with specific RNA regulatory events69, but still require biochemical validation of putative regulatory interactions. Overlaying these approaches can yield a powerful maps of functional RNA-protein interaction sites for Nova53,88,134 and Fox1/272,90,139.
Two important maps can be derived from combining these approaches, one biologic (bottom right panel), one mechanistic (bottom left panels). An assessment of the directly regulated mRNAs can address the extent to which there is a biologically coherence to the set of target RNAs—for example, the first such assessment of a genome-wide, directly regulated validated set of targets revealed that Nova regulates RNAs encoding synaptic functions53,88,95. Second, new rules of regulation can be derived from combining experiments to yield functional maps—for example it became apparent that the position of binding in a transcript determines the outcome of Nova134 or Fox1/269,72,90 to enhance or inhibit alternative exon inclusion.