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

• gamma 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 intron

• 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

components of spliceosomes

• 5 snRNPS

• U1, U2, U4, U5, U6

what are snRNPs made of

1 functional RNA called small nuclear RNAs and multiple proteins

what is U6 SnRNP made of

• U6 snRNA

• many U6 associated proteins

snRNPs

sequentially interact with pre-mRNA to faciliate splicing 

spliceosome splicing process

• U1 snRNP binds to the 5’ splice site

• branch point binding protein (BBP) and U2 auxiliary factor (U2AF) binds to the branch point

• U2 snRNP binds to the branch point while displacing BBP and U2AF

• U4/U6 snRNP and U5 snRNP join the complex

• U6 binds to the 5’ splice site, displacing U1

• U4 leaves the complex

• displacement of U4 causes the first splicing reaction to occur

• 2nd splicing reaction occurs and the intron is removed

what is the basis of spicing

• RNA-RNA interactions

• snRNPs bind to mRNA and other snRNPs via RNA-RNA base pairings

• bring branch point adenine close to the 5’ splice site to facilitate first splicing reaction

example of RNA-RNA interaction

U2 snRNP holds the branch point using its U2snRNA

what are spliced sites bound by

exon junction complex (EJC)

what attracts snRNPS for splicing

phopsphorylation of Ser2 and Ser5 in the RNA poly II CTD

what does EJC do

• binds to the junction of 2 exons indicating proper removal of that intron

• multiple EJCs will bind if there were multiple introns

alternate splicing

• not all exons in the mRNA need to be kept in a mature mRNA

• can splice the same mRNA in different ways to make mature mRNA that code for the same versions of the protein

• different cells can trianscribe the same genes but splice the pre-mRNA differently to express different versions of the protein

alpha-tropomyosin

• regulate muscle contraction in muscle cells

• becomes the cytoskeleton in non-muscle cells

• alternate splicing of this gene produces different versions of its protein with functions tailored for each cell type

how are poly-A tail enzymes attracted

• RNA poly II is transcribing the 3’UTR 

• RNA poly II CTD is phosphorylated at Ser2 which attracts the enzymes

addition of poly A tail process

• 3’ UTR has a binding site for one of the poly A tail enzymes

• poly A tail enzymes bind to the binding site and cut the pre-mRNA about 30-40 nucleotides downstream

• other enzymes add about 200 adenines to the free 3’OH at the cut site

poly A tail binding site

AAUAAA

what happens after poly A tail binds

• poly A binding proteins bind to it

• mRNA is now mature and ready for export

qualities of a mature mRNA

• Cap binding complex

• exon junction complexes

• poly-A binding proteins

termination of elongation

• 5’ end of the RNA that is associated to RNA poly II is uncapped

• this RNA gets digested from the 5’ end

• makes the RNA dislodge from RNA poly II, facilitating elongation termination

where does the mature mRNA go

cytoplasm

nuclear pore complexes

gigantic, multi protein complex that forms a gate on the nuclear surface

tagged molecules in the nucleus

must be exported to cytoplasm

nuclear export receptors

bind to tagged nucleus molecules and help them travel through the NPC, into the cytoplasm