An Amazing Sequence Arrangement at the 5’ Ends of Adenovirus 2 Messenger RNALouise T. Chow, Richard E. Gelinas, Thomas R. Broker and Richard J. Roberts Spliced Segments at the 5’ Terminus of Adenovirus 2 Late mRNA Susan M. Berget, Clair Moore, and Phillip A. Sharp
A Little History. . . • In 1977, research groups working on the expression of adenovirus late genes at MIT and at Cold Spring Harbor reported that the viral mRNAs were: • "mosaic molecules consisting of sequences complementary to several non-contiguous segments of the viral genome".Quote from Adenovirus amazes at Cold Spring Harbor (1977) Nature 268: 101-104. • Phil Sharp (at MIT) and Rich Roberts (at Cold Spring Harbor) led the research groups which made this discovery. They shared the Nobel Prize in Medicine (1993) for their discovery.
This “Mosaic” Property Was Soon Found to be a General Property of Euakaryotic mRNAs The notion of the cistron, the genetic unit of function that one thought corresponded to a polypeptide chain, now must be replaced by that of a transcription unit containing regions which will be lost from the mature messenger -- which I suggest we call introns (for intragenic regions) -- alternating with regions which will be expressed -- exons. The gene is a mosaic: expressed sequences held in a matrix of silent DNA, an intronic matrix.Gilbert, W. (1978) Why genes in pieces? Nature 271: 501
RNA Modification and Processing Review • The initial nuclear RNA copy of protein-coding genes transcribed in eukaryotes is known as heterogeneous nuclear RNA (but it’s too large!) • hnRNA is processed while it is being synthesized and before it leaves the nucleus en route to the ribosomes located in the cytoplasm. Three types of modification are made: • The poly(A) tail is added to the 3' end. • A cap is added to the 5' end. • Introns are spliced out.
An more extreme example is that of the dystrophin gene. The initial pre-mRNA transcript could be > 2 million nt in size but this is spliced down to an mRNA of 14,000 nt by the removal of 78 introns
The Discovery of Introns was Made Through Electron Microscopy using R-Loop Analysis
Spliced Segments at the 5’ Terminus of Adenovirus 2 Late mRNA Susan M. Berget, Clair Moore, and Phillip A. Sharp
The Researchers are Trying to Understand the Eukaryotic Transcription Process • The researchers in both studies elected to use a simple system to study this: Adenovirus 2 • Adenovirus 2 has features comparable to its eukaryotic host (human cells) namely the presence of long polyadenylated transcripts in the host nucleus, but only a small percentage of this nuclear RNA appears as polyadenylated mRNA on cytoplasmic polysomes – similar to heterogeneous nuclear RNA • Polysome =The assemblage of mRNA, ribosomes, and the growing peptide chain present during translation.
Experiments • Isolated a Late Stage Ad2 mRNA (Hexon) • Hybridized to Restriction Fragments of Ad2 DNA (Eco R1, and Hind III) • Used R-Loop technique coupled with Electron Microscopy to Identify non-hybridizing segments and map position
Observations & Conclusions • A non-hybridizing 5’ RNA tail (160 nts) was nearly always present in hexon – Hind III fragment A (which should entirely contain the hexon mRNA) R-Loops (Here hexon mRNA hybridized with ds DNA fragment) • This unexpected anomaly is still present when using ssDNA fragment, and under different conditions – strongly suggesting that the segment complementary to adjacent viral DNA sequence • The structure of hexon mRNA and EcoRI DNA hybrids suggest three short sequences are spliced into the 5’ tail of hexon mRNA during post transcription processing. • These RNA sequences originate upstream from those coding the body of hexon.
An Amazing Sequence Arrangement at the 5’ Ends of Adenovirus 2 Messenger RNALouise T. Chow, Richard E. Gelinas, Thomas R. Broker and Richard J. Roberts
Experiments • Many techniques are the same (R-loop, restriction mapping) but instead mixed late RNA is used • The hybridizations are subsequently probed with restriction fragments from the r strand and other parts of the genome to map unhybridized fragments
Observations & Conclusions • Sequences present at three separated sites on the R-strand of the Ad2 genome are complementary to a continuous sequences at the 5’ end of late Ad2 mRNAs • Additional late mRNAs show different headers from non-adjacent DNA sequence
. . . continued • These observations are inconsistent with any previous mechanism proposed for eukaryotic mRNA synthesis • Together both papers present data that helped develop understand of eukaryotic mRNA processing, the presence of introns, and splicing.