RNA Processing I: Splicing notes
Eukaryotic Split Genes and Splicing
Eukaryotic genes are interrupted by noncoding DNA, unlike bacterial genes.
RNA polymerases transcribe both coding and noncoding regions.
The cell removes noncoding RNA from the primary transcript via splicing.
Eukaryotes add a 5’ cap and a 3’ poly-A tail to the transcript.
All mRNA processing events occur in the nucleus before export to the cytoplasm.
Genes in Pieces
Introns: Noncoding, intervening sequences within a gene.
Exons: Coding regions of a gene.
mRNA genes can have anywhere from 0 to 362 introns, while tRNA genes have 0 or 1.
RNA Splicing
Introns are transcribed along with exons in the primary transcript.
Splicing removes introns and joins exons to form mature RNA.
Possibility 1: Introns are never transcribed
Polymerase somehow jumps from one exon to another
Possibility 2: Introns are transcribed
Primary transcript result, an overlarge gene product is cut down by removing introns
This is correct process
Splicing Signals
Splicing must be precise.
Splicing signals in nuclear mRNA precursors are uniform.
The first two bases of introns are GU, and the last two are AG.
5’- and 3’-splice sites have consensus sequences with a branchpoint.
Yeast: 5’-AG/GUAAGU-intron-YNCURAC-YnNYAG/G-3’
Whole consensus sequences are crucial for proper splicing.
Mutations in consensus sequences can lead to abnormal splicing.
Mechanism of Splicing of Nuclear mRNA Precursors
Splicing involves a branched intermediate called a lariat.
Two-step model:
The 2’-OH group of A in the middle of the intron attacks the phosphodiester bond between the first exon and the G at the beginning of the intron, forming the lariat.
The 3’-OH left at the end of the first exon attacks the phosphodiester bond linking the intron G to the second exon, forming the exon-exon phosphodiester bond.
This releases the intron in lariat form.
Signal at the Branch
Along with consensus sequences at 5’- and 3’-ends of nuclear introns, branchpoint consensus sequences also occur.
Yeast sequence invariant: UACUAAC (e.g. yeast actin gene study).
Higher eukaryote consensus sequence is more variable (U47NC63U53R72A91C47)
Branched nucleotide is final A in the sequence.
Spliceosomes
Splicing occurs on a particle called a spliceosome.
Yeast and mammalian spliceosomes have sedimentation coefficients of 40S and 60S, respectively.
Spliceosomes contain pre-mRNA, snRNPs, and protein splicing factors.
These components recognize key splicing signals and orchestrate the splicing process
snRNPs
Small nuclear RNAs coupled to proteins are abbreviated as snRNPs (snurps), small nuclear ribonuclear proteins.
The snRNAs can be resolved on a gel:
U1, U2, U4, U5, U6
All 5 snurps join the spliceosome to play crucial roles in splicing
U1 snRNP
U1 snRNA sequence is complementary to both 5’- and 3’-splice site consensus sequences.
U1 snRNA base-pairs with these splice sites
Brings the sites together for splicing is too simple an explanation
Splicing involves a branch within the intron.
Genetic experiments have shown that base pairing between U1 snRNA and 5’-splice site of mRNA precursor is necessary but not sufficient for binding. Alternative 5’-splice sites of E1A
U6 snRNP
U6 snRNP associates with the 5’-end of the intron by base pairing through the U6 RNA.
U6 cross-links to intron at +5, suggesting that the invariant sequence ACA in U6 base-pairs with the conserved UGU (+4 to +6) of the intron.
Occurs first prior to formation of lariat intermediate but after first step in splicing.
The association between U6 and splicing substrate is essential for the splicing process.
U6 also associates with U2 during splicing (Active site).
U2 snRNP
U2 snRNA base-pairs with the conserved sequence at the splicing branchpoint.
This base pairing is essential for splicing.
U2 (nuc 23 and 26-28) also forms base pairs with U6 (nuc 56-59):
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 shows no sequence complementarity but does associate 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.
U4 snRNP
U4 base-pairs with U6 through stems I and II and does not play a role in splicing.
Its role seems to be to bind and sequester U6.
When U6 is needed in a splicing reaction, U4 is removed to allow interaction with U2.
Interaction of U6 with U2 involves some U6 bases involved in U4 stem I formation.
snRNP Involvement in mRNA Splicing
Spliceosomal complex contains (Substrate, U2, U5 & U6).
The complex ready for the 2nd step in splicing can be drawn as a group II intron (self-splicing intron) at the same stage of splicing.
Spliceosomal snRNPs substitute for elements at the center of catalytic activity of group II introns at the same stage of splicing.
Spliceosome Catalytic Activity
Catalytic center of spliceosome appears to include and a base-paired complex of 3 RNAs:
U2 snRNA
U6 snRNA (binds plays a role in catalysis)
Branchpoint region of the intron
Protein-free fragments of these RNAs can catalyze a reaction related to the first step in splicing.
Spliceosome Assembly and Function
Spliceosome is composed of many components – proteins and RNA.
These components assemble stepwise.
The spliceosome cycle includes assembly, splicing activity, and 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 (CC), a unit committed to splicing out the intron.
U2 joins, with help from ATP, to form the A complex.
Next, U4-U6 and U5 join to form the B1 complex.
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
Allows U6 to base-pair with U2
Activated spliceosome is called the B2 complex.
ATP provides the energy to allow lariat formation in the C1 complex.
Once another ATP is consumed, the second splicing step occurs resulting in the C2 complex.
Mature RNA exits the complex, leaving the intron bound to the I complex.
A Minor Spliceosome
A minor class of introns with variant but highly conserved 5’-splice sites and branchpoints can be spliced with the help of a variant class of snRNAs.
Cells can contain minor spliceosome with minor snRNAs:
U11 performs like U1
U12 acts like U2
U4atac and U6atac perform like U4 and U6 respectively
Use the same U5 as the major spliceosome
Commitment, Splice Site Selection and Alternative Splicing
snRNPs do not have enough specificity and affinity to bind exclusively and tightly at exon-intron boundaries.
Additional splicing factors are needed to help snRNPs bind.
Some splicing factors are needed to bridge across introns and exons and so define these RNA elements.
Exon and Intron Definition
The spliceosome can recognize either exons or introns in the splicing commitment process, presumably by assembling splicing factors to bridge across exons or introns.
If exons are recognized it is exon definition (higher eukaryotes).
If introns are recognized it is intron definition (yeast).
Splicing in a given organism typically uses either exon definition or intron definition.
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.
SR proteins SC35 and SF2/ASF commit splicing on human β-globin pre-mRNA and HIV tat pre-mRNA, respectively.
Part of the commitment involves attraction of U1 in some cases.
Commitment with different pre-mRNAs requires different splicing factors.
Bridging Proteins and Commitment
Because U1 is the first to bind, interacting proteins were sought out (synthetic lethality in U1 MUD2; in MUD2 MSL-5 [BBP]).
Yeast commitment complex has a branchpoint bridging protein (BBP) binds to:
U1 snRNP protein at the 5’-end of the intron
Mud2p near the 3’-end of the intron
RNA near the 3’-end of the intron
Bridges the intron and could play a role defining intron prior to splicing.
Mammalian BBP is SF1 and Mud2p is U2AF65 and may serve the same bridging function to define exons.
3’-Splice Site Selection
Splicing factor Slu7 is required for correct 3’- splicing site selection (proper AG at the 3’ of intron).
Without Slu7, splicing to correct 3’-splice site AG is suppressed and splicing to aberrant AG is activated.
U2AF is also required for 3’- splice site recognition.
65KDa U2AF subunit binds to polypyrimidine tract upstream of 3’-splice site and 35KDa subunit binds to the 3’-splice site AG.
Role of the RNA Polymerase II CTD
C-terminal domain of the Rpb1 subunit of RNA polymerase II stimulates splicing of substrates that use exon definition.
This does not apply to those that use intron definition to prepare for splicing.
CTD binds to splicing factors and could assemble the factors at the end of exons to set them off for splicing.
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 (µs and µm)
Activity and inactivity
Products of 3 genes in sex determination pathway of the fruit fly are subject to alternative splicing.
Female-specific splicing of tra transcript gives:
An active product that causes female-specific splicing of dsx pre-mRNA
This produces a female fruit fly
Male-specific splicing of tra transcript gives:
An inactive product that allows male-specific splicing of dsx pre-mRNA
This produces a male fruit fly
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
We have possible isoforms
Control of Splicing
What stimulates recognition of signals under only some circumstances?
Exons can contain different sequences
Exonic splicing enhancers (ESEs) tend to interact with SR proteins and stimulate splicing (e.g. dsx exon 4 weak 3’ SS for U2AF)
Exonic splicing silencers (ESSs) interact with hnRNP proteins and inhibit splicing (e.g. hnRNP A1)
Alternative Splicing Summary
Alternative splicing is very common in higher eukaryotes.
It represents a way to get more than one protein product out of the same gene and a way to control gene expression in cells.
Such control is exerted by splicing factors that bind to splice sites and a branchpoint, and also by proteins that interact with ESEs, ESSs and intronic splicing elements.
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
Another group, 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
Second step, first exon attacks the 3’-splice site.
Ligates 2 exons together
Releases the linear intron
Intron cyclizes twice, losing nucleotides each time (15 + 4 = 19 nt), 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.
Secondary structures of the splicing complexes involving spliceosomal systems and group II introns are very similar, suggesting a common evolutionary origin.
Nuclear pre-mRNA introns may have descended from bacterial group II introns.
Group II introns have been found in archaea, cyanobacteria and purple bacteria
Eukaryotic Split Genes and Splicing
Eukaryotic genes are interrupted by noncoding DNA, a feature absent in bacterial genes, which primarily contain continuous coding sequences.
RNA polymerases transcribe both coding and noncoding regions, necessitating post-transcriptional processing in eukaryotes.
The cell removes noncoding RNA from the primary transcript via splicing, a crucial step to produce functional mRNA.
Eukaryotes add a 5’ cap (modified guanine nucleotide) and a 3’ poly-A tail (series of adenine nucleotides) to the transcript.
All mRNA processing events occur in the nucleus before export to the cytoplasm, ensuring precise control over gene expression.
Genes in Pieces
Introns: Noncoding, intervening sequences within a gene that are removed during RNA splicing. These can regulate gene expression and play a role in evolutionary processes.
Exons: Coding regions of a gene that are retained in the mature mRNA after splicing. They contain the instructions for protein synthesis.
mRNA genes can have anywhere from 0 to 362 introns, while tRNA genes have 0 or 1, showcasing the variability in gene structure.
RNA Splicing
Introns are transcribed along with exons in the primary transcript, forming a pre-mRNA molecule.
Splicing removes introns and joins exons to form mature RNA, which is then translated into protein.
Possibility 1: Introns are never transcribed - Polymerase somehow jumps from one exon to another (Incorrect mechanism).
Possibility 2: Introns are transcribed - Primary transcript results; an overlarge gene product is cut down by removing introns.
This is the correct process involving the spliceosome.
Splicing Signals
Splicing must be precise to maintain the correct reading frame and ensure proper protein synthesis.
Splicing signals in nuclear mRNA precursors are uniform. The first two bases of introns are GU, and the last two are AG. These are highly conserved.
5’- and 3’-splice sites have consensus sequences with a branchpoint. These consensus sequences are crucial for spliceosome recognition and activity.
Yeast: 5’-AG/GUAAGU-intron-YNCURAC-YnNYAG/G-3’ (Y = pyrimidine, R = purine, N = any base). This is a typical yeast consensus sequence.
Whole consensus sequences are crucial for proper splicing. Even small mutations can disrupt splicing.
Mutations in consensus sequences can lead to abnormal splicing, resulting in non-functional or altered proteins. Examples include mutations causing genetic disorders.
Mechanism of Splicing of Nuclear mRNA Precursors
Splicing involves a branched intermediate called a lariat, which is a loop-like structure formed during the splicing process.
Two-step model:
The 2’-OH group of A in the middle of the intron attacks the phosphodiester bond between the first exon and the G at the beginning of the intron, forming the lariat. $ApGUAAGU5’-AGG-3’{47}{C63}{53}{72}{91}{47}). The variability allows for more flexibility in splicing regulation.
Branched nucleotide is final A in the sequence. This A forms the 2’-5’ phosphodiester bond in the lariat structure.
Spliceosomes
Splicing occurs on a particle called a spliceosome, a large RNA-protein complex.
Yeast and mammalian spliceosomes have sedimentation coefficients of 40S and 60S, respectively. These values indicate their size and complexity.
Spliceosomes contain pre-mRNA, snRNPs, and protein splicing factors. These components recognize key splicing signals and orchestrate the splicing process.
snRNPs
Small nuclear RNAs coupled to proteins are abbreviated as snRNPs (snurps), small nuclear ribonuclear proteins. They are fundamental components of the spliceosome.
The snRNAs can be resolved on a gel: U1, U2, U4, U5, U6. Each snRNP has a specific role in splicing.
All 5 snurps join the spliceosome to play crucial roles in splicing. They recognize splice sites, catalyze splicing, and regulate the process.
U1 snRNP
U1 snRNA sequence is complementary to both 5’- and 3’-splice site consensus sequences. This complementarity helps U1 bind to splice sites.
U1 snRNA base-pairs with these splice sites. It initiates the spliceosome assembly process.
Brings the sites together for splicing is too simple an explanation. The interaction is more complex and involves additional factors.
Splicing involves a branch within the intron.
Genetic experiments have shown that base pairing between U1 snRNA and 5’-splice site of mRNA precursor is necessary but not sufficient for binding. Alternative 5’-splice sites of E1A. Other factors are required for stable binding and commitment.
U6 snRNP
U6 snRNP associates with the 5’-end of the intron by base pairing through the U6 RNA. This interaction is crucial for catalysis.
U6 cross-links to intron at +5, suggesting that the invariant sequence ACA in U6 base-pairs with the conserved UGU (+4 to +6) of the intron. This base pairing is essential for the first step in splicing.
Occurs first prior to the formation of the lariat intermediate but after the first step in splicing. U6 plays a catalytic role in the splicing process.
The association between U6 and splicing substrate is essential for the splicing process. It helps position the splice sites for the transesterification reactions.
U6 also associates with U2 during splicing (Active site). This interaction is central to the catalytic activity of the spliceosome.
U2 snRNP
U2 snRNA base-pairs with the conserved sequence at the splicing branchpoint. This interaction is crucial for splicing. The branchpoint adenosine bulges out, facilitating the first transesterification reaction.
U2 (nuc 23 and 26-28) also forms base pairs with U6 (nuc 56-59):
This region is called helix I. It is a critical structural element in the spliceosome.
Helps orient snRNPs for splicing. Proper orientation is essential for catalysis.
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. This highlights the differences in splicing mechanisms across different organisms.
U5 snRNP
U5 snRNA shows no sequence complementarity but does associate with the last nucleotide in one exon and the first nucleotide of the next exon. It helps align the exons for splicing.
This should result in the two exons lining up for splicing. This is crucial for maintaining the correct reading frame.
U4 snRNP
U4 base-pairs with U6 through stems I and II and does not play a direct role in splicing. Its role seems to be to bind and sequester U6.
When U6 is needed in a splicing reaction, U4 is removed to allow interaction with U2. This is a key regulatory step in spliceosome activation.
Interaction of U6 with U2 involves some U6 bases involved in U4 stem I formation. This highlights the dynamic interactions within the spliceosome.
snRNP Involvement in mRNA Splicing
Spliceosomal complex contains (Substrate, U2, U5 & U6).
The complex ready for the 2nd step in splicing can be drawn as a group II intron (self-splicing intron) at the same stage of splicing. This suggests an evolutionary relationship between spliceosomes and group II introns.
Spliceosomal snRNPs substitute for elements at the center of catalytic activity of group II introns at the same stage of splicing. This further supports the evolutionary link.
Spliceosome Catalytic Activity
Catalytic center of spliceosome appears to include Mg^{2+}Mg^{2+}2^6=64