Week 6 - RNA Splicing and Processing Flashcards
Pre-mRNA
the nuclear transcript that is processed by modification and splicing to give an mRNA
RNA splicing
the process of excising the introns from RNA and connecting the exons into an continuous mRNA
RNA is modified
in the nucleus by
- additions to the 5’ and 4’ ends
- by splicing to remove the introns
RNA splicing and modification
The 5 end of eukaryotic mRNA is
capped
The 5’ end of eukaryotic mRNA is capped
- a 5’ cap is formed by adding a G to the terminal base of the trancript via a 5’-5’ link
- 5’ 7-methylguanosine cap
- the cap structure is recognized by protein factors to influence mRNA stability, splicing, export, and translation
- major function is to protect the mRNA from degradation
- cap is recognized by cap binding protein heterodimer (CBP20/80) to facilitate export from the nucleus
5’ cap
7-methylguanosine
- add CH3 at C7 (methylated guanine)
- 5’ to 5’ phosphotriester linkage
- protects unstable RNA (stops degradation)
The 5’ capping process takes place
during trancription
• may be important for release from pausing of transcription
3 forms of (5’) capping
- all contain cap 0
- the 5’ cap of most mRNA is monomethylated, but some small noncoding RNAs are trimethylated
- 50% of mRNA is capped at 20 nucleotides
- at 30 nucleotides almost all mRNA is capped
3 forms of capping
… enzymes work together to add the cap
- 3 enzymes work together to add the cap
- RNA triphosphatatse and guanylytransferase activities are present in the same protein and called the capping enzyme
Capping enzyme
RNA triphosphatase and guanylytransferase activities are present in the same protein
Addition of the 5’ cap
PICTURE
Addition of the 5’ cap
basic steps
- remove 1 P (from the 3 at the 5’ end)
- transfers guanosne to 5’ of RNA, 5’-5’ phosphodiester link
(^both in capping enzyme)
- add methyl to guanosine
Capping enzyme is recruited by
the CTD of RNA polymerase II
Capping enzyme is recruited by the CTD of RNA polymerase II
- RNA pol II CTD must be phosphorylated on Ser-5 to target a transcript for capping
- CE interacts with Ser-5 phosphorylated pol II
- capping must happen in transcription
Capping enzyme is recruited by the CTD of RNA polymerase II
PICTURE
RNA splicing occurs
during and/or after transcription
Splicing makes
transcript diversity
diversity in protein function in splicing the transcript
Nuclear splice sites are
short sequences
Nuclear splice sites are short sequences
- splice sites - sequences immediately surrounding the exon-_intron_ boundaries
- the 5’ splice site at the 5’ (left) end of the intron includes the consensus sequence GU
- the 3’ splice site at the 3’ (right) end of the intron includes the consensus sequence AG
- GU-AG or U2 type introns (98% of human introns)
Splice sites
sequences immediately surrounding the exon-intron boundaries
sequence motifs, consensus in intron that allows splicing
U2-type introns (98% of human introns)
The 5’ splice site at the 5’ (left) end of the intron includes the consensus sequence
GU
U2-type introns (98% of human introns)
The 3’ splice site at the 3’ (right) end of the intron includes the consensus sequence
AG
GU-AG
U2-type introns
98% of human introns
Exon-intron boundaries
U12-type introns (0.1% of human introns)
- the 5’ splice site at the 5’ end (left) end of the intron includes the consensus sequence AU
- the 3’ splice site at the 3’ (right) end of the intron includes the consensus sequence AC
•
Splice sites are read in
pairs
Splice sites are read in pairs
- splicing depends only on recognition of pairs of splice sites
- all 5’ splice sites are functionally equivalent, and all 3’ splice sites are functionally equivalent
- splicing junctions are recognized only in the correct pairwise combinations
- need to known 5’ splice site in same intron as 3’ splice site
Pre-mRNA splicing proceeds through a
lariat
Pre-mRNA splicing proceeds through a lariat
- splicing requires the 5’ and 3’ splice sites and a branch site just upstream of the 3’ splice site
- a lariat is formed when the intron is cleaved at the 5’ (GU) splice site, and the 5’ end is joined to a 2’ position at the A at the branch site in the intron (covalent bond)
- 2 transesterification reactions - a reaction that breaks and makes chemical bonds in a coordinated transfer so that no energy is required
- the intron is released as a lariat when it is cleaved at the 3’ (AG) splice site and the left and right exons are then ligated together
Pre-mRNA proceeds through a lariat
PICTURE
Pre-mRNA splicing proceeds through a lariat
2’
- 2’-hydroxyl on branching nucleotide initiates nucleophilic attack on 5’ splice site
- 3’-hydroxyl generated at the 3’ end of the exon during the first transesterification initiates a nucleophilic attack on the phosphodiester bond at the 3’-splice site
Lariat formation
2’
PICTURE
% compositioin
- only 25% of genome is exon+intron
- 1% exon
- 24% intron
- ~25% transcribed
… are required for splicing
snRNAs
snRNAs are required for splicing
- small cytoplasmic RNAs - scRNA, scyrps
- small nuclear RNA - snRNA, snurps
- small nucleolar RNA - snoRNA
Small cytoplasmic RA
(scRNA, scyrps)
RNAs that are present in the cytoplasm
(sometimes found in the nucleus also)
Small nuclear RNA
(snRNA, snurps)
- one of many small RNA species confined to the nucleus
- several of them are invovled in splicing or other RNA processing reactions
- snRNAs exist as ribonucleoprotein particles
(snRNA + several proteins) = snRNP or snurp
• binds RNA not DNA because single-stranded –> hyrbidize
Small nucleolar RNA
(snoRNA)
a small nuclear RNA that is localized in the nucleolus
splice tRNAs, other rRNA
Each snRNA is present in its own
small riboucleoprotein particle
The 5 snRNPs involved in splicing are
- U1
- U2
- U5
- U4
- U6
Together with some additional proteins the snRNPs form
the spliceosome
Spliceosome
weight, snRNPs
- ~12 MDa
- 5 snRNPs account for almost half of the mass
- 141 proteins + 5 RNAs - sequentially
Splicing factor
a protein component of the spliceosome that is not part of one of the snRNPs