Since the RNA and RNA-binding protein are fused together, the researchers can really beat up the extract and rigorously purify the protein without fear of losing the RNA. At the end of the day, they are left with the RNA sequence to which the protein was bound. They can then take these sequences to Rockefeller's high throughput sequence facility, and with the help of Research Support Specialist Scott Dewell, overlay them onto the genome and see where they match. What they get is a map of every position on every transcribed RNA where the RNA binding protein is binding.
When DNA is transcribed into RNA, the primary transcript is divided into many blocks called exons, which are separated by empty spaces. In order to convert the transcript into some sort of message, all the spaces need to be removed; but if an exon is dropped, a different version of that protein, which could carry a very different message, is created. "That's RNA splicing," says first author Donny Licatalosi, a postdoctoral associate in the lab. "It is what gives rise to this massive pool of diverse and complex tissues with a relatively small number of genes."
In the past, the group used a sophisticated process of evidence and inference to make predictions of the points of regulation along the transcript. "Now, we have direct biochemical validation that these interactions occur in the brain to regulate splicing," says Licatalosi.
And as it turns out, "The observed map -- and this was amazing -- looked just like our predicted map," says Darnell.
Darnell, Licatalosi and their colleagues Aldo Mele, a research assistant, John Fak, a research assist
|Contact: Thania Benios|