What are some major differences between pre-mRNA and mature mRNA?
Pre-mRNA has its introns, no 5’ cap and no poly A tail
Name the 3 major ways in which all pre-mRNA’s are processed.
- 5’ - 7-methylguanosine cap with a 5’-5’ linkage
- cleavage / polyadenylation
Describe the addition of the 5’ cap to pre-mRNA
- Since the 5’ end has a triphosphate, the gamma phosphate is cleaved
- An “upside down” guanosine monophosphate (MGP) is added - 5’-5’ linkage
- Methylate the guanosine at the C7 position
The result is a triphosphate in the middle of a 5’-5’ linkage of the methylated guanosine and the 5’ residue of the new RNA
What are the conserved sequences of introns and where are they located in the intron?
- GU (GT) at the 5’ end
- A in the middle closer to the 3’ end
- AG at the 3’ end
What are the functions of the 5’ cap?
- Stabilize 5’ end. Protect from exonuclease activity.
- Recognition site for
i. CBC in the nucleus -
a. enhances splicing
b. enhances 3’ processing
c. enhances export from nucleus
ii. EIF4F in the cytoplasm (initiates translation by binding the cap and bringing the mRNA to the ribosome)
What is the conserved sequence at the 3’ end of the pre-mRNA and what is it used for?
AATAAA roughly 10-30 bp’s upstream from the cleavage point in the process of adding the poly A tail
List some genetic disorders caused by splicing defects.
- SMA (spinal muscular atrophy)
- CD44 - alt. splicing predicts metastasis in cancers
What is CD44?
Codes for a cell surface protein that moderates cell migration. Alt. splicing can predict cancer metastasis.
What is marfans syndrome?
splicing defect in the fibrolyn gene.
presents with tall, protruding chest, weak aorta
SMA (spinal muscular atrophy)?
name is very descriptive…
splicing defects causing mtr neuron degeneration
Name the factors that bind to the intron during splicing.
U2 associated factor protein
Where does U1 snRNA bind?
At the 5’ end of the intron on the GT (GU) site.
Where does the U2 snRNA bind?
at the A site in the “middle” of the intron
Where does the U2 associated factor protein bind?
at the 3’ end of the intron at the AG site
What is the A site in the intron also known as?
the branch point
Describe how the U1, U2 snRNA’s and the U2 assoc. factor bind to the intron vs. the exon…
From the A site in the first intron then to the GU site in the second intron, bridging an exon in between.
They “bridge” an exon because exons are shorter (easier to identify) than introns.
Describe the rxns in splicing once all the factors have bound.
- 2’-OH of the A site nuc. attack on the 5’ end’s phosphate
- 3’ -OH at the 5’ end of the intron nuc. attack on the phosphate at the 3’ end
- The two exons are joined by the usual phosphodiester bond.
Describe the two rxns that process the 3’ end of the pre-mRNA.
Add’n of poly A tail:
- cleavage about 20 bp downstream from the AATAAA sequence in the non-coding region
- Addn of 150 - 200 A’s
What enzyme does the poly A addition at the 3’ end?
Describe two instances of alternative poly adenylation.
- IgM heavy chain - stay on lymphocyte cell membrane vs. go into the serum
- Thalassemia - alpha-globin production altered with alternative poly A tail site (AATAAA corrupted to AATAAG)
What is the termination function of the 3’ end processing?
poly A tail signals termination of trxn
What are two major functions of the poly A tail?
- Stabilize the 3’ end. Protect from exonuclease activity.
2. Aid in translation later on in the ribosome.