Upload
ferdinand-golden
View
217
Download
0
Embed Size (px)
Citation preview
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 result-
an 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 twice- losing 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.
Disclaimer• 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.