Lecture 19 Flashcards
What are introns?
Non-coding regions
What are exons?
Coding regions
Are introns or exons transcribed into pre-mRNA?
Both
How to form mature mRNA?
Need to splice: Introns are excised and exons are covalently linked to form mature mRNA
What procedure first detected splicing?
R-looping
What happened during R-looping?
- ssDNA was hybridized to its corresponding mRNA and imaged to electron microscopy
- large loops of DNA observed that didn’t anneal
- these loops encode introns that are excised out of the pre-mRNA to make the mRNA molecule
What two ways to detect splicing?
- R-looping
- Comparing sequence of a gene to that of its corresponding complementary DNA (cDNA), which is synthesized from the mRNA. Introns in the gene are present in the ssDNA template strand but have been excised from the processed mRNA
Typically DNA has more introns or exons
Introns
What are the three types of introns?
Spliceosome “spliceosomal” introns, Group I introns, Group II introns
What are spliceosome “spliceosomal” introns
- The largest class of introns, inc those found in nuclear mRNA primary transcripts
- non-self-splicing introns
What are Group I introns
- Found in some nuclear, mitochondrial and chloroplast genes encoding for rRNAs, mRNA and tRNAs
- self-splicing
What are group II introns
- found among rare examples of introns in bacteria
- self-splicing
What is used to remove spliceosome introns from mRNA
- use a large RNA-protein complex called a spliceosome to catalyze splicing
How do self-splicing introns differ from spliceosomal introns?
- have very specific RNA sequences that catalyze their own excision and joining
What are catalytic RNA molecules are called
ribozymes
In eukaryotes, spliceosome introns have what common motif?
The intron begins with GU and ends with AG
In spliceosome introns: What is the consensus sequence at the 5’ splice site in vertebrates?
AG/GUAAGU
In spliceosome introns: What is the consensus sequence at the 3’ splice site?
a stretch of 10 pyrimidines, (Py)n, U or C, followed by any base (N) and then often by C, and always ending with AG
In spliceosome introns: Where is the branch site
20-50 nt upstream of (i.e., 5’ to) the 3’ splice site
In spliceosome introns: What is always located at the branch site
An adenosine
In spliceosome introns: What are the conserved residues critical for spliceosome-mediated splicing
- 5’ splice site AGGUAAGU
- 3’ splice site AG
- Branch site A
What happens during the first trans-esterfication reaction in splicing?
The 2-OH of branch site A
nucleophilically attacks the 5’ phosphate of the 1st nucleotide of the intron. This cleaves the
phosphodiester bond between the intron and the upstream exon and forms a new 2’-5’ phosphodiester
bond between A and the intron
What happens during the first trans-esterfication reaction in splicing?
The free 3’-OH of G on the upstream exon performs a nucleophilic attack of the 5’ phosphate of the 1st nucleotide in the downstream exon, joining the two and releasing the looped “lariat” structure.
How is the lariat structure formed?
nucleophilic attack by 2’-OH on branch site A – on 5’ phosphate on 1st G in intron – breaks the GG bond between the upstream exon and the intron, forms a new AG phosphodiester bond
How are the exons covalently linked?
nucleophilic attack by newly liberated 3’-OH on upstream exon G to 5’ phosphate on 1st nt of
downstream exon, breaking phosphodiester bond between this nt and intron G. This splices the axons together and releases the intron in the form of a lariat structure
How is the lariat structure degraded?
By nucleases in the nucleus
What is the spliceosome complex made up of?
highly conserved
structure made up of the mRNA to be spliced
plus ~300 proteins and 5 small nuclear RNAs
What makes the 5 snRNAs in the spliceosome complex
- 100-200 nucleotides long
- made by RNAP II
How are snRNPs (small nuclear ribonucleoproteins) made
When the snRNAs associate with spliceosome proteins
What are the 5 snRNPs?
U1, U2, U4, U5, U6
Fxn of the 5 snRNPs?
involved in splicing reactions. These bind
critical sites on pre-mRNA by base pairing
Role of snRNP U1?
Binds the 5’ splice site and the 3’ splice site
Role of snRNP U2?
Binds the branch site and forms part of the catalytic center
Role of snRNP U5?
Binds the 5’ splice site
Role of snRNP U4?
Masks the catalytic activity of U6
Role of snRNP U6?
Catalyzes splicing
How does U1 bind to 5’ splice site?
- splicing begins with binding of the U1 snRNP to the 5’ splice site via
complementary base pairing btwn the pre-mRNA and the snRNA of the snRNP - U1 snRNA (cyan)contains a highly conserved six-nucleotide sequence that base pairs to the intron at the 5’ splice site - this binding initiates spliceosome assembly
Addition of what else besides U1 snRNP leads to formation of the spliceosome?
addition of U2 (binds to branch site), U4, U5 and U6 snRNPs lead to formation of the spliceosome
What causes the branch-site adenylate to bulge?
- imperfect base-pairing of U2 at the branch site causes the branch-site
adenylate to bulge, which helps activate it for nucleophilic attack via its 2’ OH, at the 5’ splice site
What requires ATP hydrolysis, assembly or spliceosome or splicing?
assembly of the spliceosome requires ATP hydrolysis
Where does assembly of the spliceosome occur?
Assembly of the splicesome occurs on the CTD of RNAP II – snRNPs associate with the CTD and bind the splice sites as they emerge from the polymerase
What forms when U2 base-pairs with U6?
catalytic splicing center
When does splicing occur?
DURING transcription
What is the proposed mechanism for bringing the 5’ and 3’ splice site together?
- as the first splice junction (the 5’ splice site) is synthesized, it is bound by a tethered spliceosome component (U1?)
- the second (3’) splice site on the other side of the intron is then captured by the spliceosome complex as it passes, bringing
the splice sites together - after splicing, the intron is degraded in the nucleus by nucleases
What does self-splicing require?
- pre-mRNA (containing self-splicing introns)
- Mg2+
- free G (GDP, GMP, GTP, guanosine) – originally included in the rxn mixture because it was thought that ATP or GTP might be needed as an energy source but instead was found to be the required co-factor
Because no spliceosome is involved in self-splicing Group I introns what is used?
intron itself serves as the enzyme that catalyzes splicing - a ribozyme. Folding of the intron brings the exon ends together for splicing
What is an enzyme that catalyzes splicing called
Ribozyme
3 steps in self-splicing by Group I introns?
- the 3’-OH of a free GMP
(guanosine monophosphate)
attacks the phosphate in the first nucleotide of the intron (an A), breaking the phosphodiester bond
at the splice site and establishing a new phosphodiester bond between G and the intron - a transesterification
reaction
• as in spliceosome-mediated splicing, the new 3’-OH of the upstream exon (5’ exon/exon 1) then nucleophilically attacks the first nucleotide (U) in the downstream exon (3’ exon/exon 2), releasing the intron and linking the
two exons together
• no lariat structure is formed, intron is degraded by nucleases
What do Group I introns use GMP for
attack the phosphate of the first A of the intron
In the Group I trans-esterification reaction, the phosphodiester bond is first between where and then between where after trans-esterfication?
U-A then G-A.
Does Group I intron splicing require ATP?
No
2 steps in self-splicing by Group II introns?
2’-OH of branch-site A within the intron attacks the phosphate of the first G in the intron, transferring the
phosphodiester bond from the U to the A and forming a branched lariat structure
• the 3’-OH of the upstream exon then attacks the first U in the
downstream exon, transferring the phosphodiester bond from the lariat structure to the upstream exon and releasing the lariat structure, which gets
degraded by nucleases
In self-splicing by Group II introns how many phosphodiester bonds does adenosine have
3
Benefit of alternative splicing?
- provides a powerful mechanism for expanding the diversity of genomic sequences – means that one gene can produce multiple gene products
- Generate diversity
- splicing at alternate splice sites to include or exclude different exons can lead to the production of several gene products from one gene
How can alternative splicing be regulated?
alternative splicing can be regulated to give differential splicing at different times during development and in different cell types
What are alternative splice products called
Isoforms
What dictates which splice sites are chosen?
Cell-specific proteins interact with spliceosomes and dictate which splice sites are chosen.
