Chapter 19 - RNA splicing and Processing Flashcards
There is very little mRNA processing in prokaryotes, so the primary transcript is considered a
mature mRNA
The primary transcript of a eukaryotic gene has the same organization as the gene and is called a
pre-mRNA
A eukaryotic pre-mRNA is usually … before export as a mature mRNA
capped, poly-A tailed, and spliced
Enzymes for capping, tailing, and splicing are coupled to
the transcriptional apparatus
The capping enzymes help the transcriptional apparatus
clear the promoter
TFIIH phosphorylates CTD Ser5 residues during
transition to elongation
Capping enzymes binds to
Ser5-P CTD
Elongating RNAP II associates with enzymes that phosphorylate
Ser2
Some splicing and tailing enzymes bind the
Ser2/5-P CTD
The first nucleotide in an RNA transcript is usually a
purine nucleotide triphosphate
what is the mammalian enzyme responsible for adding the cap
guanylyl-transferase
what enzyme then adds a single methyl group at the 7’ position of the terminal guanine
guanine - 7 - methyltransferase
RNAP II pauses 30 nt downstream of initiation site and
waits for capping enzymes to act
Uncapped nascent RNA is vulnerable to attack by
exonucleases
Cap is also necessary for
initiation of splicing and cytoplasmic export
Most splice sites contain
short consensus sequences
major introns are also known as
U2-type
GU-AG introns
minor introns are also known as
U12-type introns
Differences in the 5’ and 3’ splice site consensus sequences gives the splice sites
directionality
Splice sites are recognized in a
pairwise manner
splice sites are
generic
the splice site apparatus is … in every tissue
the same
splicing is temporarily coupled to
transcription
branch point
Not well conserved in multicellular eukaryotes
Preferences for purines and pyrimidines at each position
Highly conserved target adenine nucleotide
Lies 18-40 nt upstream of the 3’ splice site
Identifies the nearest 3’ splice site for interactions with a 5’ splice site
The first step in the splicing reaction is a
nucleophilic attack by the 2’-OH of the adenine in the branch point on the 5’ splice site
Free 3’-OH from end of preceding exon attacks the
phosphodiester bond at the 3’ splice site
Transesterifies to form
5’ to 3’ bond between first and second exon
small cytoplasmic RNAs
tRNA, small rRNA, miRNA
small nuclear RNAs
snRNAs
small nucleolar RNAs
snoRNAs
snRNA
important component of the spliceosome
snoRNA
involved in processing of rRNA
spliceosome
A large complex of proteins and small RNAs that comprise the splicing apparatus
splicesome contents
Contains 5 snRNAs and 41 snRNA associated proteins
Spliceosome also contains 70 proteins called splicing factors
Also contains 30 other proteins that act as an interface to other steps of gene expression
splicing factors
Associated with spliceosome assembly, RNA transcript binding, and RNA active site assembly
The spliceosome forms on the pre-RNA via several presplicing complexes of
snRNPs
small ribonucleoproteins
snRNA and proteins
Several splicing reactions require the RNAs of the snRNPs to base pair with
the RNA transcript being spliced
Spliceosome is released immediately following
final ligation of the exons
spliceosome must … at each intron
reassemble
Recognition of the splicing consensus sequences requires both
RNA and proteins
The first step in splicing is binding of
the U1 snRNP to the 5’ splice site
Binding of U1 to the RNA is stabilized by two proteins
Multimeric U2AF binds to a polypyrimidine tract located between the branch point and 3’ splice site
Branch point binding protein (BBP/SF1) interacts with the branch point
E complex
commitment complex
U1+BBP/SF1+U2AF
SR proteins
Family of splicing factors that contain RNA- and protein-binding motifs
Function in spliceosome assembly and as splicing initiators in multicellular eukaryotes
Play a key role in maintaining splicing accuracy in multicellular eukaryotes where splice site consensus is weak
intron splice site recognition
5’ and 3’ splice sites are simultaneously recognized by components of E complex
Sequential deposit of U1 and then U2AF as nascent mRNA emerges from RNAP II
Used for splicing of small, single-intron genes in unicellular eukaryotes
exon splice site recognition
Takes advantage of presence of small exons of a consistent size
Introns are long and variable in multicellular eukaryotes
Many sequences in introns resemble true splice sites
The paired recognition of splice sites flanking an intron is generally quite inefficient
U2AF binds to the 3’ splice site
U1 binds to the 5’ splice site at the beginning of the next intron
Bridges the exon
Sequential deposit of U2AF and then U1 as nascent mRNA emerges from RNAP II
Complexes are switched to link across the introns
spliceosome assembly
- U2 snRNP binds to the branch point
- A tri-snRNP complex composed of U5 and U4/U6 binds the A complex to form the B1 complex
- U1 and U4 are released resulting in formation of the B2 complex
- Several RNA rearrangements occur in the B2 complex to form the C1 complex
- The U5 snRNP positions U2 and U6 in the C2 complex for the second transesterification reaction between the flanking exons
- The snRNPs remain attached to the lariat, but are quickly released as the lariat dissociates and is degraded
U2 snRNP binds to the branch point
Facilitated by base pairing between U2 snRNA and branch point consensus
Displaces BBP/SF1 and U2AF
Requires ATP hydrolysis
Forms the A complex
B1 complex
First complex considered a true spliceosome because it contains all components needed for splicing
Upon release of U1 and U4 …
U6 pairs more extensively with U2
U4 sequesters the U6 snRNA until it is needed
U2 is already paired with the branch point
U6 now pairs with intronic sequence downstream of the 5’ splice site
U2-U6 pairing brings 5’ splice site in close contact with branch point
Assisted by interactions between U5 and upstream exon
The first transesterification reaction between the 5’ splice site and branch point to form the lariat occurs in
the C1 complex
Splicing seems very inefficient because it requires, per splicing event
100+ proteins
Five snRNA molecules
Hydrolysis of eight ATP molecules
Reassembly of the entire active site
Inefficiency may be a consequence of
overexpansion of ancient self-splicing mechanisms
Some aspects of complexity are actually needed such as
ATP hydrolysis
ATP hydrolysis reactions are used to break specific RNA-RNA base pairs
Breaking of specific base pairs is required to make others that are specifically required for the sequential assembly of the spliceosome
If the initial correct base pairs do not form, then ATP hydrolysis will not occur, and spliceosome assembly will not proceed
ATP hydrolysis is also used for kinetic proofreading
Correct base pairing is stronger than incorrect pairing
Incorrect pairing will dissociate more quickly than correct
ATP-mediated rearrangements that result in incorrect pairing will be less stable and are less likely to incorrectly proceed to the next stage of assembly than rearrangements that are correct
ATP hydrolysis is used for
kinetic proofreading and to break specific RNA-RNA base pairs
exon-junction complex
EJC
Deposited onto each exon-exon junction following splicing
Directly recruits RNA-binding proteins associated with nuclear export
nonsense-mediated decay
The EJC is also involved in proofreading of mutant mRNA transcripts in the cytoplasm
EJCs are usually displaced from the mRNA by the ribosome during the first translation event
If a nonsense mutation has occurred, the ribosome will not remove the EJCs
EJCs will recruit decapping enzymes that result in degradation of the mRNA
self spliced introns
group 1 and group 2
Require no external proteins or nucleotides to catalyze splicing in vitro
Proteins are required for folding in vivo
group I and Group II introns
Found in fungal and plant organelles, some bacteria, and the nucleus of some simple eukaryotes
Group II introns are excised via the same mechanism as
nuclear pre-mRNAs
RNA world hypothesis evidence
Existence of self-splicing RNAs and ribozymes
Ribose can be spontaneously produced from formaldehyde in abiotic conditions
Deoxyribose is not readily produced in this manner
Deoxyribose is produced from ribose in the cell using a protein enzyme, which suggests a later origin
why did we move away from RNA world
Proteins have a greater range of potential enzymatic reactions
Long double stranded DNA is a more stable hereditary material
Also more readily repaired
Over 90% of mammalian genes are
alternatively spliced
Effects of alternative splicing
Omit or include some coding sequences
Create alternative reading frames
Alternative splicing is often associated with
weak splice sites that are easily modulated
Specific exonic and intronic sequences can enhance or suppress splice site selection via interactions with
trans-acting alternative splicing regulators
The effect of splicing enhancers and silencers are mediated by
sequence-specific RNA binding proteins
The rate of transcription can also directly affect the outcome of
alternative splicing
Functions of Poly(A) tail
Protect from 3’ to 5’ exonucleases
Facilitates nuclear export
Cap stability
Tail addition
RNAP II does not terminate at specific sites
Sequences in the mRNA are recognized as targets for an endonucleolytic cut
Poly(A) tail is added at the 3’ end of the cut site
RNAP II continues after the cleavage
5’ end liberated after cleavage signals transcription termination
The cleavage/polyadenylation site is usually flanked by
conserved sequences
Cleavage stimulatory factor (CstF)
recognizes the upstream AAUAAA
Cleavage and polyadenylation specific factor (CPSF)
recognizes the downstream U/GU-rich element
CPSF and CstF
will recruit other protein factors that cleave the RNA and produce a 3’ end
Poly(A) polymerase (PAP)
binds the free 3’ end of the RNA
PAP activity
PAP has specific activity at the AAUAAA site when combined with other tailing factors
PAP first adds a short oligo(A) sequence at the 3’ end
Activity dependent on CPSF and CstF
Nuclear poly(A) binding protein (PABP)
binds the poly(A) tail
Upon binding of PABP, PAP extends the poly(A) tail to its full length of approximately 200 nt
A cytoplasmic form of PABP participate in translation
The final length of the tail is determined by
a feedback mechanism between the cooperative binding of PABPs and PAP
RNAP II continues transcription for hundreds of nucleotides after
RNA is cleaved
two factors lead to RNAP II termination
- allosteric changes
- Exonuclease torpedo
allosteric changes
Binding of cleavage factors and subsequent RNA cleavage leads to a conformational change in RNAP II
Conformational change makes the enzyme less processive and more likely to dissociate from the DNA
exonuclease torpedo
RNA cleavage produces an uncapped 5’ RNA end which is eventually bound by a 5’3’ exonuclease
Exonuclease is carried on RNAP II?
The exonuclease degrades the RNA 5’3’
When the exonuclease reaches RNAP II it destroy the RNA-DNA hybrid
RNAP II dissociates
RNAP III termination
looks for a discrete poly-T sequence in the template strand
RNAP I termination requires
Accessory terminator proteins that recognize one of two terminator sequences
Cleavage of the nascent RNA
