Pre-mRNA (precursor mRNA):
Mature mRNA:
Pre-mRNA splicing:
mRNA isoforms:
Spliceosome:
Splice sites:
Pre-mRNA (precursor mRNA): full length unprocessed RNA transcript
Mature mRNA: fully processed (5’-cap, introns removed + exons joined, 3’ poly-A tail) and functional RNA
Pre-mRNA splicing: A process that removes introns from pre-mRNA and joins exons together to form mature mRNA
mRNA isoforms: a variant forms of mature mRNA (encoded by the same gene) formed as a result of differential
processing (such as due to alternative splicing) of pre-mRNA
Spliceosome: A large RNA-protein complex that catalyzes the removal of introns to generate mature mRNA
Splice sites: The sites at which exons are separated from their neighboring introns and at which two exons are subsequently
attached
- Sequences in pre-mRNAs are recognized by **
a) The 5’ splice site: Has a ** at the **’ end of the **. The arrow indicates the ** (also called the splice donor site)
b) The 3’ splice site: Has an ** at the **’end of the **. The arrow indicates the boundary of intron with the
downstream exon (also called the **).
c). A polypyrimidine tract: found **; usually contains a stretch of ** but can
contain **.
d). The branchpoint sequence (branch site): located **. ** is highly conserved
e) ** in the 5’ splice site, **, ** upstream of 3’ splice site and ** in the 3’ splice site
are highly conserved across mammals
f) In addition to these core splice sites, there are ‘’ and ‘**’ sequences within introns and exons that help to define **
- Sequences in pre-mRNAs are recognized by spliceosomes
a) The 5’ splice site: Has a GU dinucleotide at the 5’ end of the intron. The arrow indicates the exon-intron boundary (also called the splice donor site)
b) The 3’ splice site: Has an AG dinucleotide at the 3’end of the intron. The arrow indicates the boundary of intron with the
downstream exon (also called the splice acceptor site).
c). A polypyrimidine tract: found immediately upstream of the 3’ splice site; usually contains a stretch of Uracils but can
contain Cytosines.
d). The branchpoint sequence (branch site): located within the intron close to the 3’ splice site. Adenosine is highly conserved
e) GU in the 5’ splice site, branchpoint A, polypyrimidine tract upstream of 3’ splice site and AG in the 3’ splice site
are highly conserved across mammals
f) In addition to these core splice sites, there are ‘splicing enhancer’ and ‘splicing silencer’ sequences within introns and exons that help to define exon-intron junctions
- The spliceosome (splicing machinery)
a) The spliceosome (a complex of snRNPs) mediates **
b) snRNPs== a complex of **and **: **, **, **, **, **, and **.
c) Human spliceosome contains ~** proteins and ** separate snRNAs
i) snRNAs: The 5 ** rich RNA molecules are between ** nucleotides long termed **, **, **,
**, **.
ii) snRNAs have a distinct ** and form complexes with**
iii) snRNAs undergo ** during splicing
iv) ** catalyze the ** reactions - snRNAs display ** and ** **
- ** and ** snRNAs base pair with the 5’ splice site and **
a) Base pairing between U2snRNP and the branchpoint is not perfect and causes the **. The ** group of
Adenosine participates in the ** reaction. Binding of U2snRNP to the branchpoint is stabilized by **. ** binds to the ** and ** - The splicing reaction occurs through **
- The spliceosome (splicing machinery)
a) The spliceosome (a complex of snRNPs) mediates the transesterification reactions required for splicing
b) snRNPs=small nuclear ribonuclear proteins= a complex of snRNA (small nuclear RNA) and proteins: U1snRNP, U2snRNP, U3snRNP, U4snRNP, U5snRNP, and U6snRNP.
c) Human spliceosome contains ~300 proteins and 5 separate snRNAs
i) snRNAs: The 5 uridine rich RNA molecules are between 100-300 nucleotides long termed U1snRNA, U2snRNA, U4snRNA,
U5snRNA, U6snRNA.
ii) snRNAs have a distinct secondary structure and form complexes with a number of proteins
iii) snRNAs undergo structural rearrangements during splicing
iv) RNAs catalyze the transesterification reactions - snRNAs display intramolecular and intermolecular base pairing
- U1 and U2 snRNAs base pair with the 5’ splice site and branchpoint respectively
a) Base pairing between U2snRNP and the branchpoint is not perfect and causes the Adenosine to bulge out. The 2’-OH group of
Adenosine participates in the transesterification reaction. Binding of U2snRNP to the branchpoint is stabilized by U2AF. U2AF binds to the polypyrimidine tract and 3’ splice site - The splicing reaction occurs through assembly of spliceosome
SUMMARY OF THE SPLICING REACTION:
*The chemical reactions of splicing occur between ** at ** in the ** (the 5’ and
3’ splice sites) and the **
* Splicing occurs through two ** reactions, in which **
are broken and new ones are formed
* Catalytically active spliceosomes assemble **. The catalytic center is formed **
* The very early steps of spliceosome assembly involve binding of ** to the **ʹ **, binding of ** to
the ** and **, and binding of U2snRNP to **
* The rest of the splicing apparatus binds, sometimes displacing other factors.
* ** rearranges, first ** occurs.
* Other rearrangements then ** for the **second transesterification reaction
* ** is released. After splicing, **, and their components are **
SUMMARY OF THE SPLICING REACTION:
*The chemical reactions of splicing occur between nucleotides at exon-intron junctions in the pre-mRNA (the 5’ and
3’ splice sites) and the branchpoint inside the intron.
* Splicing occurs through two transesterifications reactions, in which phosphodiester bonds within the pre-mRNA
are broken and new ones are formed
* Catalytically active spliceosomes assemble stepwise on the pre-mRNA. The catalytic center is formed de novo on
the pre-mRNA.
* The very early steps of spliceosome assembly involve binding of U1snRNP to the 5ʹ splice site, binding of U2AF to
the polypyrimidine tract and 3’ splice site, and binding of U2snRNP to the branchpoint sequence (complex A).
* The rest of the splicing apparatus binds, sometimes displacing other factors.
* Pre-mRNA rearranges, first transesterification occurs.
* Other rearrangements then bring the two exons together for the second transesterification reaction
* Mature mRNA is released. After splicing, spliceosomes disassemble, and their components are recycled
- pre-mRNA splicing was revealed through in vitro splicing experiments
a) STEP 1:
* Synthesize
*
* Negative control:
b) STEP2:
.i)
ii).
iii).
iv).
- Negative control:
- Stop the reaction by
c) STEP3:
i).
ii) .
- pre-mRNA splicing was revealed through in vitro splicing experiments
a) STEP 1: Preparation of radiolabeled pre-mRNA
* Synthesize a mini-gene construct: genomic
fragment of a gene (exons and introns) is
cloned into a plasmid-containing a promoter
(e.g., T7, SP6)
* Mini-gene construct is incubated in a
‘transcription buffer’ containing T7 or SP6 RNA polymerase and nucleotides (NTPs) including 32 P-UTP (radiolabeled UTP)
* Negative control: Mini-gene construct + all
components of the transcription buffer except for T7 RNA polymerase
b) STEP2: Splicing reaction
.i) Radiolabeled pre-mRNA: generated from an in vitro transcription reaction
ii). Splicing reaction buffer that contains RNase inhibitor
iii). Splicing competent nuclear extract from HeLa cells
iv). Incubate the reaction at 30 degrees Celsius for T=0 to 120 minutes.
- Negative control: Reaction incubates at 30 degrees Celsius for T= 0 minutes.
- Stop the reaction by placing the tubes on dry ice
c) STEP3: Visualization of Splicing reaction
i). Run samples from the splicing reaction onto a denaturing polyacrylamide gel
ii) . Expose the gel to an X-ray film
- Spliceosomes can assemble through ** and **
- Intron definition: **
a) Intron definition: ** defines the intron that is to be removed.
b) Pairing of ** and ** + ** occurs across intron that is going to be excised, followed by **and **.
c) If introns are ** nucleo=des long, spliceosome will use **
d) Intron definition is predominantly used in **. Also, observed in ** and ** - Mammals use exon definition to **
a) Exon definition- **, rather than **, are recognized first by **: ** and
** + **. ** proteins bind to ** within exons and introns , recruit **and
** their interaction** with the . Exon definition is used when intron length exceeds, splicing
complexes first assemble across **- used to splice mammalian pre-mRNAs which typically
have large introns - ** stabilize the exon-defined complex
a) SR proteins recruit ** proteins to **and **
stabilize their interaction with the pre-mRNA.
b) SR proteins recruit ** to the ** and stabilize ** - As a general rule, if introns are shorter than about ** nucleotides the **. If
introns longer than ** nucleotides, the spliceosome **. - SR proteins **
- Exon definition is converted into ** since **
- Spliceosomes can assemble through intron and exon
- Intron definition: cross-intron assembly and splicing
a) Intron definition: The spliceosome defines the intron that is to be removed.
b) Pairing of U1 and U2 snRNPs + U2AF occurs across intron that is going to be excised, followed by full spliceosome assembly and splicing.
c) If introns are < 200-250 nucleo=des long, spliceosome will use intron defini=on
d) Intron definition is predominantly used in yeast. Also, observed in invertebrates and plants - Mammals use exon definition to define splice sites
a) Exon definition- exons, rather than introns, are recognized first by binding of early spliceosome factors: U1snRNP and
U2snRNP + U2AF. SR proteins bind to splicing enhancer sequences within exons and introns , recruit splicing factors and
stabilize their interaction with the pre-mRNA. Exon definition is used when intron length exceeds ~200-250 bp, splicing
complexes first assemble across an exon (~150 bp average)- used to splice mammalian pre-mRNAs which typically
have large introns - SR (serine rich) proteins stabilize the exon-defined complex
a) SR proteins recruit U2AF proteins to polypyrimdine tract and 3’ splice site
stabilize their interaction with the pre-mRNA.
b) SR proteins recruit U1 snRNP to the 5’ splice site and stabilize its interaction with the pre-mRNA - As a general rule, if introns are shorter than about 200-250 nucleotides the spliceosomes will use intron definition. If
introns longer than 250 nucleotides, the spliceosome will use exon definition. - SR proteins stabilize exon defined complex.
- Exon definition is converted into intron definition since splicing always takes place across introns.
- Constitutive versus alternative splicing
a) Constitutive exons: **
b) Alternative exons: ** - The splicing code: I**
a) Important nucleotide sequences that control splicing of exons
i) 1. **- **, **, **t, and ** (slide6)
ii). Splicing code: ** (** () , (i** **) and ** (r) , **
b) Splicing code is deciphered by **
i) SR proteins bind to ** ** and **. Splicing is posi=vely regulated by **
ii) ** bind to splicing silencer sequences- ** and **. Splicing is ** by splicing regulators**
- Constitutive versus alternative splicing
a) Constitutive exons: exons that are consistently conserved after splicing
b) Alternative exons: exons that are not consistently conserved after splicing - The splicing code: In addition to core splice sites, other sequences control splicing of exons
a) Important nucleotide sequences that control splicing of exons
i) 1. core splice site sequences- 5’ splice site, 3’ splice site, polypyrimidine tract, and branchpoint sequence (slide6)
ii). Splicing code: Splicing enhancer (ESE (exonic splicing enhancer) ,ISE (intronic splicing enhancer) and silencer sequences (ESS (exonic splicing silencer) , ISS (intronic splicing silencer))
b) Splicing code is deciphered by RNA binding proteins
i) SR proteins bind to splicing enhancer sequences- ESE and ISE. Splicing is posi=vely regulated by splicing regulators
ii) hnRNPs bind to splicing silencer sequences- ESS and ISS. Splicing is repressed by splicing regulators
- Splicing is positively regulated by **
a) SR proteins bind to ** **(/**), ** and **
b) SR proteins facilitate **
c) an exon will be included in the mature mRNA ONLY IF ** - Splicing is repressed by **(e.g., **)
a) hnRNPs block ** thus causing **
b) hnRNPs block **
- Splicing is positively regulated by splicing activators- SR proteins
a) SR proteins bind to splicing enhancer sequences (ESEs/ISEs), recruit splicing machinery and promote inclusion of exons
b) SR proteins facilitate exon inclusion
c) an exon will be included in the mature mRNA ONLY IF splicing machinery can bind to core splice sites around it. - Splicing is repressed by splicing repressors (e.g., hnRNPs)
a) hnRNPs block spliceosome assembly, thus causing exon exclusion from the mature mRNA
b) hnRNPs block exon inclusion.
Alternative splicing of the c-src pre-mRNA
1. Alternative splicing of the c-src mRNA in non-neuronal cells
a) Alternative splicing is found in , which encodes **
§ In non-neuronal cells ** binds to ** sites on ** of the ** exon, and it is **
2. Alternative splicing of the c-src mRNA in neuronal cells
a) In neuronal cells, ** gets degraded via ** Thus, PTB is unable to ** and, so ** is **. Note: miRNA function will be discussed in the next lecture**
Alternative splicing of the c-src pre-mRNA
1. Alternative splicing of the c-src mRNA in non-neuronal cells
a) Alternative splicing is found in c-src mRNA, which encodes SRC tyrosine kinase
§ In non-neuronal cells PTB binds to ISS sites on both sides of the N1 exon, and it is repressed from joining the final mature mRNA
2. Alternative splicing of the c-src mRNA in neuronal cells
a) In neuronal cells, PTB mRNA gets degraded via neuron-specific microRNA. Thus, PTB is unable to repress inclusion of N1 and, so N1 is included in the final mRNA. Note: miRNA function will be discussed in the next lecture
