Transcript An Amazing Sequence Arrangement at the 5’ Ends of

An Amazing Sequence Arrangement at the 5’
Ends of Adenovirus 2 Messenger RNA
Louise 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
Background:
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 nonhybridizing 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 RNA
Louise 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.
Questions ?