SPLIT GENES

mRNA Processing in Eukaryotes

Enzymes in the eukaryotic nucleus modify
pre-mRNA (RNA processing) before the
genetic messages are dispatched to the
cytoplasm
During RNA processing, both ends of the
primary transcript are usually altered
Also, usually some interior parts of the
molecule are cut out, and the other parts
spliced together
 Alteration of mRNA Ends
Each end of a pre-mRNA molecule is
modified in a particular way
The 5 end receives a modified nucleotide 5
cap
The 3 end gets a poly-A tail
These modifications share several
functions
They seem to facilitate the export of mRNA
to the cytoplasm
They protect mRNA from hydrolytic enzymes
They help ribosomes attach to the 5 end
 5 Cap of mRNA
The 5 end of mRNA is modified by addition
of 7-methylguanosine cap.
The enzymes (guanylyl transferase) for
capping is attached to the phosphorylated
CTD of RNA polymerase II.
The cap is added after addition of first 20-30
nucleotide of RNA.
Capping is initiated be addition of GTP in
reverse orientation to 5terminal of RNA.
Then methyl group is added to this guanine
and first few 5end nucleotides of RNA chain
 Polyadenylation
In eukaryotes, RNA polymerase II
transcribes the polyadenylation signal
sequence; the RNA transcript is released
1035 nucleotides past this
polyadenylation sequence.
The signal include highly conserved
hexanucleotide (AAUAAA in mammalian
cells), which is located 10-30 nucleiotide
upstream of site of polyadenylation that
end in usually CA.
 Polyadenylation
Downstream to polyadenylation site, it had
U-or UG-rich sequence element that is
recognised by endonuclease that cleaves
RNA chain.
The poly-A polymerase add about 200
nucleotides to the transcript.
The enzyme for polyadenylation are also
attached to phosphorylated CTD of RNA
polymerase II.
Polyadenylation
 Polyadenylation
The recognition of polyadenylation signal leads to
transcription termination, cleavage and
polyadenylation of transcript and degradation of
RNA downstream of site of poly A addition.
Length of poly-A tail determine translation activity
of mRNA.
Many mRNA in unfertilized egg have poly-A tail of
30-50 nucleotides and they remain stored in
inactive form
Post fertilization these tails will be lengthen and
leads to activation and translation of stored mRNA
for embryonic development.
 Split Genes and RNA Splicing
Most eukaryotic genes and their RNA
transcripts have long noncoding stretches
of nucleotides that lie between coding
regions
These noncoding regions are called
intervening sequences, or introns
The other regions are called exons
because they are eventually expressed,
usually translated into amino acid
sequences
RNA splicing removes introns and joins
exons, creating an mRNA molecule with a
continuous coding sequence
 Split Genes : Examples
Actin gene has 309-bp intron separates first
three amino acids and the other 350 or so.
Chicken pro-alpha-2 collagen gene is 40-kbp
long, with 51 exons of only 5 kbp total.
The exons range in size from 45 to 249
bases
Mechanism by which introns are excised and
exons are spliced together is complex and
must be precise
 Splicing Mechanisms
There are three critical sequence elements
of pre-mRNAs: sequence at 5splice site,
sequence at 3 splice site and sequence
within the intron at branch point, that
causes splicing reaction.
It is carried out in two steps: first cleavage
at 5 splice site (SS) and joining of 5 end
of intron to an A (adenine) within the intron
(branch point).
 Splicing Mechanisms
This reaction yield a lariat-like intermediate
in which intron will form a loop.
The second step involves cleavage at the
3splice site and simultaneous ligation o
exons, resulting in excision of intron in
lariat like structure.
The lariat is unstable; the 2'-5'
phosphodiester is quickly cleaved and
intron is degraded in the nucleus.
Splicing Mechanisms
 Splicing Mechanisms
There are two mechanism of splicing which
have been observed.
The self spicing mRNA that work as ribozyme
and doesnt need any additional RNAs or
proteins for splicing.
Splicing carried out by large complex known
as spilceosomes.
Spliceosome is composed of five small
nuclear RNAs (snRNAs) complexed with 6-10
small nuclear riboprotein particles (snRNPs).
 Self Splicing Introns
First time discovered in 28S rRNA of
Tetrahymena by Tom Cech and his
colleagues.
This RNA has a 400bp long intron which is
removed by self incubation of pre-RNA
without addition of any protein/s.
The self splicing is divided into two classes
based their reaction mechanism.
 Spliceosomes mediated Splicing
The RNA component of is made up of five
small nuclear RNAs (snRNAs) i.e. U1, U2,
U4, U5 and U6.
These snRNAs ranges in size from 50-200
nucleotides and complexed with 6-10 small
nuclear riboprotein particles (snRNPs).
The U1, U2 and U5 snRNA contain single
snRNP each, but U4 and U6 snRNA are
complexed to each other in a single snRNP.
 Spliceosomes mediated Splicing
The first step in spliceosome assembly is
binding of U1 snRNP to the 5 splice site,
consensus sequence and a complementary
sequence at the 5 end of U1snRNA.
Similarly U2snRNP bind to branch point by
complementary base pairing between U2
snRNA and branch point sequence.
A preformed complex of U4/U6 and U5
snRNP is then loaded to the complex with U5
binding to sequence upsteam of 5 splice site.
 Spliceosomes mediated Splicing
The snRNAs are rearranged, U6 replaces
U1,and U1 and U4 get dissociated from
the complex.
U6 forms complex with U2, which brings
branch point close to 5 splice site and
lariat is formed.
U5 binds to 3 splice site, maintain
alignment between 5 and 3 exon causing
their ligation and excision of intron.
Spliceosomes mediated Splicing
 Splicing Factors
There are various splicing factors such as SR
and U2AF that helps in regulation of
spliceosome assembly.
Introns contain many sequences that
resemble splice sites, these factors identify
appropriate 5/3 splice sites at intron/exon
boundary.
They direct U1 and U2 to appropriate site on
pre-mRNA by protein protein interaction.
These splicing factors are often associated
with phosphorylated CTD of RNA polymerase
II and help coupling transcription with
splicing.
Splicing Factors
 Alternative Splicing
The central role of this mechanism is
production of multiple transcripts from the
same pre-mRNA by different combination of
5 and 3 splice site.
The process of generating various mRNAs by
varying pattern of pre-mRNA splicing is called
alternative splicing.
This process is carried out with the help of
various regulators (repressor or activators)
that are produced in response to extracellular
signal or are tissue specific.
Alternative Splicing
Alternative Splicing
 Alternative Splicing: Sex
Determination in Drosophila
 Alternative Splicing : Dscam
Gene
The dscam gene is a cell surface protein in
Drosophila neuron.
It contain 24 exon out of which there are four
sets of alternative exons, with only a single
exon from each set is incorporated into
spliced mRNA.
This can be joined in any combination with
38016 potential combination can be produced
from this single gene (more than total no. of
gene in Drosophila).
Alternative Splicing : Dscam
Gene
 Alternative Splicing : Dscam
Gene
These different spliced form of Dscam
provide individual neuron with identity
code that help in establishing connection
between neurons needed at the
development of fly brain.