Transcript Genes in Pieces

The mechanism of splicing of
nuclear mRNA precursors
Chapter 14
Evidence for Split Genes
• Most higher eukaryotic genes coding for
mRNA and tRNA are interrupted by unrelated
regions called introns
• Exons are present surrounding the introns
• Exons contain the sequences that finally
appear in the mature RNA product
– Genes for mRNAs have been found with anywhere
from 0 to 362 exons
– tRNA genes have either 0 or 1 exon
How do introns not find its way into mature
RNA products of the genes? - RNA Splicing
• Introns are never
transcribed
– Polymerase somehow
jumps from one exon to
another
• Introns are transcribed
– Primary transcript resultan overlarge gene
product is cut down by
removing introns
– This is correct process
RNA splicing
• Process of cutting introns
out of immature RNAs and
stitching together the exons
to form final product is
RNA splicing
• Introns are transcribed
along with exons in the
primary transcript
• Introns are removed as the
exons are spliced together
Stages of RNA Splicing
• Messenger RNA synthesis in eukaryotes occurs in
stages
• First stage:
– Synthesis of primary transcript product
– This is an mRNA precursor containing introns copied from
the gene if present
– Precursor is part of a pool of large nuclear RNAs –
hnRNAs
• Second stage:
– mRNA maturation
– Removal of introns in a process called splicing
Splicing Signals
• Splicing signals in nuclear mRNA precursors are
remarkably uniform (exon/GU-intron-AG/exon)
– First 2 bases of introns are GU
– Last 2 are AG
• 5’- and 3’-splice sites have consensus sequences
extending beyond GU and AG motifs
• Whole consensus sequences are important to proper
splicing (Look at mammalian and yeast consensus
sequences on page 403)
• Abnormal splicing can occur when the consensus
sequences are mutated
Mechanism of Splicing of Nuclear
mRNA Precursors
• Intermediate in nuclear mRNA precursor splicing is
branched – looks like a lariat
• 2-step model
– 2’-OH group of adenosine nucleotide in middle of
intron attacks phosphodiester bond between 1st exon
and G beginning of intron
• Forms loop of the lariat
• Separates first exon from intron
– 3’-OH left at end of 1st exon attacks phosphodiester
bond linking intron to 2nd exon
• Forms the exon-exon phosphodiester bond
• Releases intron in lariat form at same time
Simplified Mechanism of Splicing
Spliceosomes
• Splicing takes place on a particle called a
spliceosome
• Yeast and mammalian spliceosomes have
sedimentation coefficients of 40S and 60S
• Spliceosomes contain the pre-mRNA
– Along with snRNPs and protein splicing factors
– These recognize key splicing signals and
orchestrate the splicing process
snRNPs
• Small nuclear RNAs coupled to proteins are
abbreviated as snRNPs - small nuclear
ribonuclear proteins
• The snRNAs (small nuclear RNAs) can be
resolved on a gel:
– U1, U2, U4, U5, U6
– All 5 snRNAs join the spliceosome to play crucial
roles in splicing
U1 snRNP
• U1 snRNA sequence is
complementary to 5’splice site consensus
sequences
– U1 snRNA base-pairs with
these splice sites
• Splicing involves a branch
within the intron
U6 snRNP
• U6 snRNP associates with
the 5’-end of the intron by
base pairing through the U6
RNA
• Occurs first prior to
formation of lariat
intermediate
• U6 also associates with U2
during splicing
U2 snRNP
• U2 snRNA base-pairs with
the conserved sequence at
the splicing branchpoint
• U2 also forms base pairs
with U6
– This region is called helix I
– Helps orient snRNPs for
splicing
• 5’-end of U2 interacts with
3’-end of U6
– This interaction forms a
region called helix II
– This region is important in
splicing in mammalian cells,
not in yeast cells
U5 snRNP
• U5 snRNA associates
with the last nucleotide
in one exon and the first
nucleotide of the next
exon
• This should result in the
two exons lining up for
splicing
snRNP Involvement in mRNA
Splicing
• Spliceosomal complex
contains:
–
–
–
–
–
Substrate
U2
U5
U6
All snRNP are made up of
same seven set of proteins
called Sm proteins
Spliceosome Assembly and
Function
• Spliceosome is composed of many components
– proteins and RNA
• These components assemble stepwise
• The spliceosome cycle:
– Assembly
– Function
– Disassembly
• By controlling assembly of the spliceosome - a
cell can regulate quality and quantity of
splicing and so regulate gene expression
Spliceosome Cycle
• Assembly begins with binding of U1 to splicing
substrate forming a commitment complex - a unit
committed to splicing out the intron
• U2 joins the complex next - followed by the others
• U2 binding requires ATP
• U6 dissociates from U4 and displaces U1 at the 5’splice site
– This step is ATP-dependent
– Activates the spliceosome
– Allows U1 and U4 to be released
Commitment
• Commitment to splice at a given site is
determined by an RNA-binding protein
• This protein binds to splicing substrate and
recruits other spliceosomal components
• The first component to follow is U1
Yeast Two-Hybrid Assay
Intron-Bridging Protein-Protein
Interactions
• Branchpoint bridging
protein binds to U1
snRNP protein
• Comparison of yeast to
mammalian complexes
is seen at right
Role of the RNA Polymerase II
CTD
• CTD binds to splicing factors and could
assemble the factors at the end of exons to
set them off for splicing (figure 14.37)
• Questions 27, 28 and 31 - Homework
Alternative Splicing
• Transcripts of many eukaryotic genes are
subject to alternative splicing
– This splicing can have profound effects on the
protein products of a gene
– Can make a difference between:
• Secreted or membrane-bound protein
• Activity and inactivity
Alternative Splicing Patterns-Pg 432
• Alternative splicing of the same pre-mRNA gives rise
to very different products
– Alternative splicing patterns occur in over half of human
genes
– Many genes have more than 2 splicing patterns - some
have thousands
What stimulates recognition of signals under only some
circumstances? - Silencing of Splicing
• Exons can contain
sequences –
– Exonic splicing
enhancers (ESEs)
stimulate splicing
– Exonic splicing
silencers (ESSs) inhibit
splicing
Self-Splicing RNAs
• Some RNAs could splice themselves without
aid from a spliceosome or any other protein
• Tetrahymena 26S rRNA gene has an intron,
splices itself in vitro
– Group I introns are a group of self-splicing
RNAs
– Group II introns also have some self-splicing
members
Group I Introns
• Group I introns can be
removed in vitro with no
help from protein
• Reaction begins with attack
by a guanine nucleotide on
the 5’-splice site
– Adds G to the 5’-end of the
intron
– Releases the first exon
Linear Introns
• Second step- first exon
attacks the 3’-splice
site
– Ligates 2 exons together
– Releases the linear
intron
• Intron cyclizes twicelosing nucleotides each
time - then linearizes a
last time
Group II Introns
• RNAs containing group II introns self-splice
by a pathway using an A-branched lariat
intermediate - like spliceosome lariats
Types of Alternative Splicing
• Begin transcripts at alternative promoters
• Some exons can simply be ignored resulting in
deletion of the exon
• Alternative 5’-splice sites can lead to inclusion or
deletion of part of an exon
• Alternative 3’-splice sites can lead to inclusion or
deletion of part of an exon
• A retained intron can be retained in the mRNA if it is
not recognized as an intron
• Polyadenylation causes cleavage of pre-mRNA and
loss of downstream exons
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