8
"Chapter Presentation 21 !
RNA Splicing and Processing!
Topics!
! Alternative splicing, Drosophila Sex determination pathway!
! Mis-splicing and cancer!
! Trans-Splicing reactions use small RNAs!
! 3 end formation !
! 3 mRNA end processing is critical for transcriptional termination!
! T-RNA splicing!
! Introns early Introns late models!
Optional reading!
Pg 47!
1!
Fas receptor
(also known as tumor necrosis factor receptor [TNFR])!
Multiple isoforms of the Fas receptor protein are produced by alternative splicing. !
Two normally occurring isoforms in humans are produced by an exon-skipping mechanism. !
An mRNA including exon 6 encodes the membrane-bound form of the Fas receptor, which
promotes apoptosis (programmed cell death). [Increased expression of Fas receptor in skin cells chronically exposed
to the sun, and absence of expression in skin cancer cells, suggests that this mechanism may be important in elimination of precancerous cells in humans.] !
If exon 6 is skipped, the resulting mRNA encodes a soluble Fas protein that does not promote
apoptosis.!
The inclusion or skipping of the exon depends on two antagonistic proteins, TIA-1 and
polypyrimidine tract-binding protein (PTB).
!
The 5' donor site in the intron
downstream from exon 6 in the premRNA has a weak agreement with
the consensus sequence, and is not
bound usually by the U1 snRNP. !
If U1 does not bind, the exon is
skipped.!
2
Fas receptor!
Binding of TIA-1 protein to an
intronic splicing enhancer site
stabilizes binding of the U1 snRNP.!
Exon 6 contains a pyrimidine-rich
exonic splicing silencer, ure6, where
PTB can bind. !
The resulting 5' donor site complex
assists in binding of the splicing
factor U2AF to the 3' splice site
upstream of the exon.!
If PTB binds, it inhibits the effect
of the 5' donor complex on the
binding of U2AF to the acceptor
site, resulting in exon skipping.!
3
In addition!
To!
GUAG!
Splicing!
Depends on: !
ESE !
Exonic!
Splicing!
Enhancers!
SR !
Splicing!
Regulatory!
proteins!
4
!
DNA sequence motif consisting of 6 bases within an exon that directs, or enhances, accurate splicing of pre-mRNA into messenger RNA (mRNA).!
Pathway of Sex Determination in D. melanogaster!
Uses alternative splicing patterns to specify male and female sexes.!
ratio of X chromosomes to autosomes determines the expression of the sex-lethal gene (sxl)!
female:
male:
"2 X chromosomes : 2 of each autosome
"1 X chromosome : 2 of each autosome
EXON 3 of sxl gene "! contains a STOP codon
"! ratio = 1!
"! ratio = 0.5!
"! no functional protein made.!
Exon 3 is included in male sxl mRNA, but not female sxl mRNA !
Thus only females make the sex-lethal protein!
5!
Sxl protein ! RNA binding protein!
! Is involved in splicing of the Transformer Gene mRNA (Tra)!
! To produce a female specic transcript!
! Makes a protein that is also a splicing regulator!
! Acts to produce Tra 2 protein!
"Tra + Tra 2 ! turn ON the double sex gene
! Results in female specic protein pattern!
Males: default pathway, !
no doublesex protein!
therefore "!
! male specic expression!
6!
Repress yolk protein
gene expression "
"
"
"
"
"Activates transcription !
"of yolk protein genes!
7!
FIGURE 21.23 Sex determination in D. melanogaster!
Mis-splicing and Cancer!
Mis-splicing due to Mutations in:!
Splice sites!
Exonic Enhancer Sequences!
Splicing Regulatory Factors !
pre-mRNA!
{!
**!
v1 - v10!
CD44 = transmembrane receptor important for cell-cell interactions, cell adhesion and !
migration, it binds hyaluronic acid collagens and others.!
Many variant isoforms found in normal cells due to alternative splicing.!
9!
<
<!!
<!
<!
<!
<!
<!
<!
<!
**!
mis-spliced!
CD44!
mRNA!
with!
v4 + v5!
Inclusion of exons v4, and v5 correlate with tumorigenesis and metastatis of breast cancer. !
Overexpression of these abnormal variants have been associated with breast cancer and !
poor prognosis.!
Cause of CD44 mis-splicing in breast cancer?!
!High level of the Tra2-! mRNA and Tra2-! protein levels
(human homologous of Drosophila Tra2)!
!Tra2-! protein was bound to the exonic enhancers found on the CD44 exon v4 and v5 !
sequences, " inducing mis-splicing and inclusion of exons v4 and v5!
Thus, high levels of the Tra2-! splicing factor may explain the observed alternative splicing!
of C D44 isoforms associated with tumor progression and metastasis during breast cancer!
development.!
10!
The human slo gene codes for a membrane protein that regulates the entry an exit of potassium ions!
into and out of cells.!
The gene has 35 exons!
Eight of which are involved in alternative splicing !
500 different proteins are produced by this gene!
(with 8! = 40,320 possible mRNAs)!
The slo gene is expressed in hair cells of the inner ear, !
These cells respond to sound frequencies between 20 Hz and 20,000 Hz!
And their capabilities in are part due to the properties!
Of the slo proteins.!
Alternative Splicing As a Strategy to Create Protein Variability!
11!
!
!
!
Figure 21.26
Splicing reactions usually
occur only in cis between
splice junctions on the same
molecule of RNA.!
Trans splicing can occur when
special constructs are made
that support base pairing
between introns.!
12!
trans-splicing Reactions Use
Small RNAs!
SL RNA is trans-spliced!
*!
Figure 21.27
13!
RNA polymerase II!
The 3! Ends of mRNAs Are Generated
by Cleavage and Polyadenylation!
!
The sequence AAUAAA is a signal
for cleavage to generate a 3! end
of mRNA that is polyadenylated.!
"[deletion of this sequence prevents generation
of the polyA 3 end]!
!
Additionally, a downstream GUrich sequence is involved.!
!
The poly(A) tail controls mRNA
stability and inuences translation.!
!
Uncapped remaining mRNA signals
transcriptional termination.!
3 5 !
Figure 21.29!
RNA polymerase II!
*
Figure 21.29 !
RNA polymerase II!
3 end formation of Pol II transcripts!
*
Figure 21.32 !
*!
*!
0.1%, use U12 spliceosome; similar to GU-AG introns!
tRNA splicing occurs by successive cleavage and ligation reactions!
S. Cerevisiae!
59 of the 272 nuclear tRNA genes are interrupted!
! a single intron,!
! located just one nucleotide beyond the 3 side of the anticodon. !
! vary in length from 14-60 bp!
! no consensus sequence !
! also true of interrupted nuclear tRNA genes of plants, amphibians, and mammals.!
17!
Yeast tRNA Splicing Involves Cutting and
Rejoining!
! tRNA splicing occurs by successive cleavage and ligation reactions.!
*!
Figures 21.34 and 21.35
18!
The Splicing Endonuclease Recognizes
tRNA!
*!
Figure 21.36
19!
tRNA splicing occurs by successive cleavage and ligation reactions!
S. Cerevisiae!
!a temperature-sensitive mutant of yeast!
! fails to remove the introns, !
!interrupted precursors accumulate in the nucleus.!
Protein encoded enzymatic activities:!
function in tRNA splicing!
Ribonuclease!
Phosphodiesterase!
Kinase!
RNA ligase !
Generate a phosphodiester bond !
between the two exon sequences !
generated by cleavage of the intron.!
20!
How Did Interrupted Genes Evolve?!
!
Observations: Prokaryotes have few introns!
!
!
! Eukaryotes have many!
!
The major evolutionary question is: !
! Did genes originate as sequences interrupted by introns?!
! introns early!
! Or were they originally uninterrupted?!
! introns late!
!
Most protein-coding genes probably originated in an interrupted form!
!
Interrupted genes that code for RNA may have originally been uninterrupted.!
21
!
! A special class of introns is mobile and can insert
itself into genes.!
Chromosomal DNA!
splicing!
Processed mRNA!
with 2 Exons!
Mobile element DNA!
Insertion into gene!
Chromosomal DNA!
New pattern of mRNA!
with 3 Exons!
Figure 8.12
Some Exons Can Be Equated with Protein
Functions!
! Facts suggesting that exons were the
building blocks of evolution and the rst
genes were interrupted are:!
1.! Gene structure is conserved between genes in
very distant species.!
23
!
2. !Many exons can be equated with coding for protein
sequences that have particular functions.!
Spliced !
mRNAs!
Figure 4.16
24
!
3. !Related exons are found in different genes.!
introns!
25
!
Figure 4.17
The Members of a Gene Family Have a
Common Organization!
!
A common feature in a set of genes is assumed to identify a property that preceded their
separation in evolution.!
!
All globin genes have a common form of organization with three exons and two introns;!
! This suggests that they are descended from a single ancestral gene.!
Leghemoglobin- binds oxygen,
similar to myoglobin (an ancient
hemoglobin).!
Not clear if the central Exon of
myoglobin derived by fusion of two
exons of the anscestral gene, !
or if the plant gene [leghemoglobin]
suffered an insertion to split that
exon at the start of plant evolution.!
26
!
Figure 4.18
Summary:!
Introns Early!
Introns Late debate:!
Eukaryotes: introns!
Prokaryotes: very few introns!
Were introns added to eukaryotic genomes, or were introns lost from prokaryotes?!
introns early model: mosaic structure of genes is a remnant of an ancient approach to the construction of
genes to make novel proteins. Separate coding sequences, reorganized and juxtaposed different polypeptides to
build up new proteins. Splicing utilized to combine the polypeptides into a single polypeptide.!
Same for organelle genomes, some have introns, (choroplast genes with homology to E. coli), but the E.coli
genomes have lost its introns.!
Introns late model: (most likely scenario for structural RNAs) !
Alternative forms of rRNA and tRNAs are sometimes found, with and without introns. The idea here is that for
tRNAs for which all molecules adhere to a similar form, it is unlikely that nature brought together the two
regions of the gene. The different regions are involved in base pairing that gives signicance to the structure.!