DNA, it has turned out, isn’t all it was wound up to be. In recent years we learned that the molecule of life, the discovery of the 20th century, did not-could not-by itself explain the huge differences in complexity between a human and a worm. Forced to look elsewhere, scientists turned to RNA; however, methodological problems have historically plagued the study of RNA regulation in living cells.
But now, Robert Darnell and his team at Rockefeller University have developed a genome-wide platform to study how specialized proteins regulate RNA in living cells. The platform allows researchers to identify, in a single experiment, every sequence within every strand of RNA to which proteins bind. The result is an unbiased and unprecedented look at how differences in RNA can explain how a worm and a human can each have 25,000 genes yet be so different.
In the new method, called high-throughput sequencing−cross-linking immunoprecipitation (HITS-CLIP), RNA−protein complexes are cross-linked in situ, partially digested with RNase to trim away RNA that does not directly contact the protein, and immunoprecipitated. Then, the protein-bound RNA is isolated, converted to DNA by RT-PCR, and subjected to high-throughput sequencing to produce a map of every position on every transcribed RNA where the RNA-binding protein is binding.
The investigators applied HITS-CLIP to a neuronal RNA-binding protein called Nova2. They found that depending on where Nova2 binds to RNA, they could predict and directly observe whether an exon would be included or excluded in the final transcript, and which protein version it created. In addition, the finding that Nova2 binds to sites in the 3’-UTR of RNAs led to the discovery that Nova2 regulates alternative polyadenylation in the brain. “The cell seems to be going through great trouble to regulate these RNAs in different conditions and different cell types,” says Darnell. “When RNA developed the ability to make a more stable copy of itself, DNA it didn’t write itself off as a relic for the textbooks. It stayed at the core of complex processes in the cell.” Read all about it at Nature, November 2008