Discovery of RNA splicing While working in Phillip Sharp's lab in 1976, Berget started investigating RNA in the cellular
cytoplasm and how they were connected to the structure of the genome of
adenovirus. She used
electron microscopy to visually inspect the structural differences. The lab's
technician, Claire Moore, began using
R-loop analysis to be able to map a string of RNA on a DNA template and hybridize them, allowing for the isolation of what genes the RNA was sequenced from. Using adenovirus to infect human cells, Berget then purified the
messenger RNA (mRNA) from the virus replicating in the cells and hybridized them with the cellular human DNA with Moore and the R-loop analysis process. The R-loop
micrographs had an unexpected discrepancy however, with the normal R-loops featuring pieces of RNA extending out from them. Other scientists had found that adenovirus RNA in the
cell nucleus is generally longer than the RNA produced in the cytoplasm, so Berget, Moore, and Sharp decided they must just be artifacts that were added on during the hybridization process. To fix this, they removed the opposite side of the DNA strand so the RNA sequence would have no competition in binding to its DNA strand counterpart, but the extended tails persisted. Multiple months and experiments to try and remove the tails by perfecting other parts of the hybridization process failed. But Berget's compilation of the data they had collected suggested that perhaps the tail sequences were from a different part of the adenovirus sequence, prompting them to use a longer DNA sequence from the human cells. This was successful, causing the tails to bind to a further part of the DNA and forming the R-loops, proving the discovery of
RNA splicing and
split genes. Berget, Moore, and Sharp had found out that the reason why nuclear mRNA is longer is because the cytoplasmic mRNA has
introns spliced out to allow for
protein synthesis. They published this finding in
PNAS in August 1977.
Exons and splice sites After establishing her own laboratory, Berget began work investigating the deeper features of RNA splicing and how introns and exons are processed and what biochemical mechanisms are involved. Using
uridine triphosphate marked with a
radioisotope, her lab was able to produce multiple radioactive RNA
substrates for study each week, along with using
HeLa cells to obtain nuclear DNA extracts. In the late 1980s, she found that by destroying specific
snurps involved in splicing, she could prevent the process from happening at all. Berget's lab proposed in a 1990 paper that for organisms with longer stretches of introns between each exon, such as
vertebrates, that there must be some other system capable of identifying the exon sequences themselves. The paper noted that the existence of a
downstream splicing location was necessary for an upstream intron to be spliced, giving credence to some sort of recognition complex of proteins. They expanded, in 1998, on the mechanisms of differential splicing choices, such as between the pre-mRNA for
calcitonin versus
CGRP, by showing that there is some sort of "splicing factor" that binds to the splicing site in order to cause
polyadenylation upstream of that binding location. ==Organizations==