L12: pre-mRNA modification during eukaryotic transcription

what is the timing of the modifications controlled by

phosphorylation state of the RNA poly II CTD tail

what does Ser5 phosphorylation do

• attracts enzymes for 5’ capping close to RNA poly II

• 5’ end of the new pre-mRNA is just coming out of RNA poly II

• 5’ capping enzymes are recruited at a specific time to cap this end

Ser2 and Ser5 phosphorylation

• attracts spliceosomes close to RNA poly II

• protein CDS with introns and exons are being produced during this time

• spliceosomes recruited at perfect timing

what happens at the end of elongation

• phosphatases dephosphorylate ser5

• only ser2 is phosphorylated now

• this attracts poly-A enzymes close RNA poly II

• 3’ UTR being coded at this time

what happens to the other side chains in the CTD during elongation

also get phosphorylated to provide further regulation

5’ cap structure

guanosine attached to phosphates by a 5’-5’ triphosphate bridge

how does 5’ cap form

• beta phosphate at the 5’ end of the pre-mRNA gets removed

• beta and gamma phosphates of the GTP gets removed

• remaining phosphates connect the molecules in a 5’-5’ config

• N7 of the cap and 2’ OH of the first couple nucleotides get methylated

what happens after the 5’ cap attaches

• CAP binding complex binds to it

• signals that the pre-mRNA has been processed fully at the 5’ end

CAP binding complex structure

heterodimer

ssRNA

• linear chain of nucleic acids

• can have another branch added to its middle via 2’ OH

where do introns exist

between 2 exons

intron structure

• 5’ splice site

• 3’ splice site

• branch point adenine- in the middle of the intron

how does splicing occur

• the 2’OH of branch point adenine attaches to the 5’ end of the intron

• leaves the 3’ end of the exon 1 with a free 3’ OH

• the 3’ OH of exon 1 gets attached to the 5’ end of exon 2

• this detaches the 3’ end of the intron from pre-mRNA

what happens to the intron after being spliced

forms a Q shaped structure

what is the 5’ C in the branch point adenine connected to

3’ carbon of the nucleotide immediately upstream

what is the 3’ C in the branch point adenine connected to

5’ carbon of the nucleotide immediately downstream

what is the 2’ C in the branch point adenine connected to

• 5’ carbon of what used to be the first nucleotide of the intro

• always G

conserved sequences in introns

• ‘GU’ at the 5’ end

• ‘AG’ at the 3’ end

• branch point adenine

• spliceosome recognize and bind to these sequences