Genes in Pieces

Download Report

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
– Polymerase somehow
jumps from one exon to
• 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
• 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 –
• 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
• Splicing takes place on a particle called a
• 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
• 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
– 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
• Occurs first prior to
formation of lariat
• 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
• 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
• This should result in the
two exons lining up for
snRNP Involvement in mRNA
• Spliceosomal complex
All snRNP are made up of
same seven set of proteins
called Sm proteins
Spliceosome Assembly and
• 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 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
• 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 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
– 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
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
– Group II introns also have some self-splicing
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
– Releases the first exon
Linear Introns
• Second step- first exon
attacks the 3’-splice
– Ligates 2 exons together
– Releases the linear
• 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
This project is funded by a grant awarded under the President’s Community Based Job Training Grant as
implemented by the U.S. Department of Labor’s Employment and Training Administration (CB-15-162-06-60).
NCC is an equal opportunity employer and does not discriminate on the following basis:
against any individual in the United States, on the basis of race, color, religion, sex, national origin, age disability,
political affiliation or belief; and
against any beneficiary of programs financially assisted under Title I of the Workforce Investment Act of 1998
(WIA), on the basis of the beneficiary’s citizenship/status as a lawfully admitted immigrant authorized to work in
the United States, or his or her participation in any WIA Title I-financially assisted program or activity.
• This workforce solution was funded by a grant awarded under the
President’s Community-Based Job Training Grants as implemented
by the U.S. Department of Labor’s Employment and Training
Administration. The solution was created by the grantee and does
not necessarily reflect the official position of the U.S. Department of
Labor. The Department of Labor makes no guarantees, warranties,
or assurances of any kind, express or implied, with respect to such
information, including any information on linked sites and including,
but not limited to, accuracy of the information or its completeness,
timeliness, usefulness, adequacy, continued availability, or
ownership. This solution is copyrighted by the institution that
created it. Internal use by an organization and/or personal use by
an individual for non-commercial purposes is permissible. All other
uses require the prior authorization of the copyright owner.