Lecture 11: mRNA Processing and Export

What is involved in mRNA processing?

• 5’ capping

• cleavage at poly(A) site

• polyadenylation

• RNA splicing

What are pre-mRNAs and mRNAs bound with?

mRNPs

What type of reactions are involved in pre-mRNA splicing?

transesterification reactions

What are the 5’ cap functions?

• stabilizes the mRNA

• facilitates the export of mRNP out of the nucleus

• aids in translatability

True or False: Introns (3000 nucleotides) are usually longer than exons (150-200 nucleotides).

true

What are sequence features required for splicing?

• 5’ splice site

• 3’ splice site

• branch point A

• polypyrimidine tract

True or False: 5’ splice and 3’ splice sites are conserved sites.

true

Where are the 5’ and 3’ splice sites?

5’ end of the intron and 3’ end of the intron

What is branch point A?

13 nucleotides upstream from the 3’ splice sites

What are some characteristics of branch point A?

• necessary for splicing to occur

• conserved

• adenosine nucleotide

Where is the polypyrimidine tract located?

between branch point A and the 3’ splice site

What are some characteristics of the polypyrimidine tract?

• 10-12 base pairs long

• splicing factor binds here

What is the complex that carries out pre-mRNA splicing?

the spliceosome

What does the spliceosome contain?

the splicing snRNPs and at least 150-200 proteins

What do usnRNAs (U1, U2, U4, U5, and U6) help with?

the splicing reaction

True or False: The spliceosome can theoretically splice by itself; the complex doesn’t require ribozymes.

true

What may the spliceosomes be evolved from?

self-splicing enzymes

What structure is formed during the first nucleophilic attack?

a lariat/lasso structure

Where is there base pairing between in the early splicing process?

pre-mRNA, U1 snRNA, and U2 snRNA

What is the splicing process?

1. Branch A undergo the first nucleophilic attack

2. U1 snRNA binds to the 5’ splice site and forms hydrogen bonds

3. U2 snRNA binds close to branch point A and forms hydrogen bonds

Why do the snRNAs need to form a hydrogen bond?

In order for splicing to happen, the U1 and 2 snRNAs have to get closer together

What conserved sequence is at the 5’ end and at the 3’ end?

GU at the 5’ end and AG at the 3’ end

What happens if there’s a mutation in pre-mRNA’s 5’ splice site?

splicing is blocked

What happens if there’s a compensatory mutation in U1?

splicing is restored

What does ESE stand for?

exonic splicing enhancer

What are some characteristics of the ESE?

• 6-7 nucleotides long 

• SR proteins bind here 

• Consensus sequence

What do SR proteins contribute to splicing?

SR proteins contribute specificity to direct splicing

What does U2AF65 recognize?

the polypyrimidine tract

What does the U2AF35 recognize?

the splice sites

What do SR proteins recognize, and what does it result in?

the ESE, and it defines the junction of intron and exon and recruits proteins to this junction

What do SR proteins recruit to the ESE?

recruits U2 factors

What is the cross-exon recognition complex?

A protein complex that forms a pre-mRNA strand at the junction of two exons that have been joined together during RNA splicing

How does the RNA Pol II CTD coordinate transcription?

the CTD of RNA Pol II gets phosphorylated and acts as a landing pad for other factors

Phosphorylation of what helps with transcription initiation and elongation?

serine 2 and serine 5, respectively

How does CTD function as a master regulator?

it recruits…

• capping enzymes

• splicing proteins to facilitate splicing

• polyadenylation factors

• certain factors depending on where RNA Pol II is located on the gene

What is alternative splicing?

a process by which different combinations of exons (the coding regions) are joined together to produce multiple mRNA variants from a single pre-mRNA transcript

What does alternative splicing form?

isoforms

What is important to know about the sxl gene?

• Expressed only in females

• Activated by a promoter only active in females

• Pioneer TF is only active in females

• Binds to sxl pre-mRNA and prevents splicing

Which exon is removed out of the transcript in a female embryo?

exon 3

What is sxl?

a splicing repressor

What exon do males contain that females don’t?

exon 3

What does the female sxl isoform contain?

a stop codon (UAA)

What does the male sxl isoform result in?

• degradation of sxl mRNA

• nonfunctional mRNAs

In females, what happens to the tra gene?

• splicing is prevented

• exon 2 is removed

In males, what happens to the tra gene?

• exon 2 is present

• no tra protein

What does the tra protein bind to?

dsx

What is the tra protein?

a splicing activator

In females, what does the tra protein form?

a functional complex with SRSFs (Rbp1 and Tra2)

What does the tra protein and SRSFs bind to?

exon 4

What does SRSF recruit?

splicing factors

What does the tra protein and SRSFs complex facilitate?

the splicing of the intron

True or False: Exon 4 is removed from the tra gene.

true

What is important to know about dsx in males and females?

• There’s no stop codons present

• Males have a longer dsx protein compared to females

• Dsx proteins target different genes

• Female-specific TF targets different genes (and so do male-specific TFs)

In order for a Poll II transcript to be polyadenylated, what must the sequence contain?

a polyadenylation sequence (AAUAAA) and a G/U rich region near the 3’ end

What does the Pol II CTD help with?

the recruitment of proteins needed for polyadenulation

What does AAAUAA recruit?

a CPSE (cleavage and polyadenylation specificity enzyme)

What does the GU region downstream of AAUAAA recruit?

CSTFs (cleavage stimulation factors)

What does PAP do?

it catalyzes poly(A) synthesis

Where does cleavage of the mRNA occur?

between the G/U-rich region and the AAUAAA sequence

True or False: mRNA processing can be core and post-transcriptional.

true

Splicing begins core-transcriptionally but ends what?

post-transcriptionally

What are the only genes that don’t undergo polyadenylation?

histone genes

What does poly(A) help with?

• translatability

• export of RNA

• mRNA stability

True or False: Cancer cells use polyadenylation to make different isoforms of proteins.

true

What are some important reminders about pre-mRNA?

• RNA is never present in the naked form; there’s always proteins around it!

• All transcriptions start with an exon

• Splicesome complex is involved with splicing

• As soon as splicing is finished, the intron forms a lariat structure

  • Enzymes will linearize the intron and be degraded by ribonucleases

As soon as ligation occurs, what happens?

the EJC (20 nucleotides upstream) will be loaded

What binds to the poly(A) tail every 12 adenosine resiudes?

PABP

What does PABPII do?

prevents degradation of mRNA and endonuclease activity

What does the capping structure prevent?

prevents the 5’ ribonuclease from degrading the 5’ end

What does the poly(A) tail prevent?

the degradation of the 3’ end

How is the mRNA structure transported out of the nucleus?

through the NPC

What is export of the RNP facilitated by?

Tap and P15 (NXF1 and NXT1, respectively)

What do NXTs and NXFs do?

recognize the EJCs and interact with the nucleoporin complexes like an exportin would

True or False: The cell must eject proteins from the mRNP in order to get through the NPC.

true

What does the CBC recruit when in the cytoplasm?

the eiFAE

In the cytoplasm, what exchanges with what?

PABPII exchanges with PABPI

True or False: Cell controls the translatability of the transcript through the exchange of CBC and eiFAE; eiFAE can interact with PABPI.

true

What type of RNA loads ribosomes more efficiently?

circularized RNA

What happens in nonsense mediated decay?

• Ribosomes remove EJC

• Removal EJCs will result in splicing junctions

• Pioneer ribosomes dislocates the EJCs

When are ribosomes dislodged from the transcript?

when ribosomes reach the stop codon/nonsense codons

Introns tend to have a high number of what?

stop codons

When a stop codon is present, what happens?

• the ribosome will get dislodged from an EJC

• EJCs will recruit deadenylase to short the poly(A) tail from 150-250 residues to around 30 residues—exposing the 3’ end to degrade the exosome

What type of activity does the exosome contain?

3’ exoribonuclease

True or False: The stop codon must be upstream of the last EJC to prevent nonsense mediated decay.

true