CLASS: 11-12:00
DATE: Sept 15, 2010
PROFESSOR: CHEN
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II.
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RNA and Post Transcriptional Regulation
Scribe: Christine Sirna
Proof: Louisa Warren
Page 1 of 5
CONSTITUTIVE SPLICING V. ALTERNATIVE SPLICING [S76]
a. For the second part of post transcriptional regulation we will be continuing what we were doing yesterday
b. We talked about splicing yesterday and there are a few more things to emphasize about splicing
c. There are 2 modes
i. Constitutive and alternative splicing
1. Constitutive: every intron is removed and every exon is incorporated into the mRNA
a. No exceptions to this
b. This produces only a single from of mature mRNA because all introns are incorporated
d. In eukaryotic genes, this can give rise to multiple forms of mature RNA
e. This can occur through 4 mechanisms
i. Use of different promotors
1. In one gene 2 promoters can be used
ii. Use of different polyadenylalation sites
iii. Alternatively splicing the exons found in the primary transcript
iv. Combination of the three mechanisms
f. We focus on aternative splicing: exons can be incorporated in an alternative way
i. One contains a certain number of exons and another mRNA contains a different amount of exons
ii. There are some exons which are different
CONSTITUTIVE mRNA SPLICING [S77]
a. I’m going to use a diagram to show you and give you a better idea
b. Here is consititutive mRNA splicing
c. This gene (A) contains three exons and 2 introns
i. Will be constitutively spliced
ii. All 3 exons will be found in mature RNA so there are no exceptions
d. Also you compare cell types (cell A and cell B)
i. Have same gene but they splice the pre RNA identically so they are identical
ii. No difference between A cell and B cell
iii. All exons are incorporated into the mRNA
iv. This is constitutive splicing
ALTERNATIVE mRNA SPLCING I [S78]
a. What is alternative mRNA splicing?
i. One exon can be there or not there in mature RNA
b. First we look at A cell as example
i. Same gene
ii. Exon 2 can be alternative spliced
iii. Can be in mature RNA or not in mature RNA
iv. Produce 2 mature RNA
1. One contains all three exons and the other mature RNA contains only exon 1 and exon 3 in the
absence of exon 2
a. This is alternative splicing
c. Also in B cell splicing pattern is identical to A cell
i. No difference in splicing pattern in A and B cell
ALTNERNATIVE mRNA SPLICING II [S79]
a. In many cases there will be a differential in mature RNA in different cell types
b. This is another alternative splicing example
c. The pattern depends on the cell types
d. Exon II can be there or not there
e. In A cell there is a mechanism to include the exon 2
i. Produce RNA with 3 exons
f. Same gene and same primary RNA in C Cell
g. C cell has a mechanism to exclude exon 2
i. So you produce only mRNA with exon 1 and 3
h. This is alternative splicing but it also depends on cell type, where the gene is expressed in the different cell
types
i. In the B cell type both isoforms can be produced
i. There is a regulation on alternative splicing and which exons depend on cell types
ALTERNATIVE mRNA SPLCING CREATES PROTEIN ISOFORMS [S80]
a. One of the reasons to have alternative splicing RNA is to increase coding potential of a gene
CLASS: 11-12:00
Scribe: Christine Sirna
DATE: Sept 15, 2010
Proof: Louisa Warren
PROFESSOR: CHEN
RNA and Post Transcriptional Regulation
Page 2 of 5
b. If you have one gene that is expressed that has many exons and the presence of exons is a combination
(some exons are there and in another mature RNA you are coding for other exons) you encode different
protein isoforms even though they are from the same gene so you increase the coding potential
i. One gene makes different protein isoforms through alternative splicing
c. Example is Skeletal muscle troponin T gene
i. Has 18 exons and 11 out of 18 are found in all mature mRNA even though it can be alternatively spliced
ii. Many different mature RNA but 11 out of 18 are always there
iii. 5 of the exons (4-8) can be combinatorial,
1. one exon can be there, two can be there or three can be there
2. These five exons can all be together or there can be only 3 of the 5 in mature RNA
iv. 16 and 17 are mutually exclusive: only one will be there
1. they will never both be present
d. If you do the math, or calculation, and all possibilities of splicing can occur, how many mature RNA can be
produced?
i. 64 different mature RNA from one gene
1. These 64 mature RNA that encode protein, some regions are identical and some are not identical
2. Produce protein isoforms that have similar function but not completely identical function
ii. This example is to increase coding potential of one gene
VI. ALTERNATIVE SPLICING EXPANDS THE CODING POTENTIAL OF THE GENOME [S81]
a. Next example is also alternative splicing but depends on cell type, depends on where gene is expressed on
what cell type
b. This is an alpha tropomyosin gene
i. It can be alternatively spliced depending on cell type
c. If this gene is expressed in striated muscle
i. It will be spliced according to this pattern (points to slide) but same gene
d. If expressed in smooth muscle, it will be spliced according to this pattern (points to slide)
e. There are minor differences between these two proteins
i. One of them contains the exon but in smooth muscle, it does not contain this exon
ii. It is alternatively spliced and the pattern depends on the cell type
f. They do a similar function but there are some minor differences in the function of this protein
VII. mRNA EXPORT [S82]
a. That concludes nuclear events of mRNA processing
i. capping, splicing, and polyadenylation
b. Now mRNA is processed
i. Contains cap and Poly A at C terminal end
c. Introns are removed and so now RNA is ready for export to cytoplasm
VIII. DIAGRAM [S83]
a. There is regulation but he will give us simplified model here
b. This is mature RNA
i. Contains 5’ cap structure with CBC protein
ii. Contains poly A tail with poly A binding protein
iii. There are other proteins that are recruited to the RNA
c. RNA will be exported 5’ end first
d. At some point the complex bound to the cap structure is repressed by another translation initiation factor
which is important for translation initiation
e. Some proteins will stay together with RNA after mRNA is exported
f. Some RNA binding proteins will be restricted to the nucleus
IX. POST TRANSCIPTIONAL REGULATION IN THE CYTOPLASM [S84]
a. There is another post transcriptional regulation that occurs in the cytoplasm in addition to the nucleus
b. 3 things can happen to mRNA in the cytoplasm
i. First thing is translation: translate mRNA to protein
ii. Second is degradation:
1. mRNA is not as stable as DNA so once it is translated it needs to be degraded to shut down
expression of gene
2. Degradation rate varies from one RNA to another
a. Some are very unstable and will take an hour and some mRNA are more stable and they stay
in the cell for days
iii. RNA is localized to a specific compartment before it can be translated
1. You make RNA but cannot translate it
CLASS: 11-12:00
Scribe: Christine Sirna
DATE: Sept 15, 2010
Proof: Louisa Warren
PROFESSOR: CHEN
RNA and Post Transcriptional Regulation
Page 3 of 5
2. RNA needs to be moved to specific compartments to undergo translation
3. This is mRNA localization
X. THE COMPETITION BETWEEN mRNA TRANSLATION AND mRNA DECAY [S85]
a. We know that translation and mRNA degradation compete with each other
b. If mRNA is under translation it will not be degraded
i. If you inhibit mRNA translation you push mRNA in the direction to be degraded
ii. Reason for this is because 5’ cap structure is recognized by a protein complex and poly A tail is recognized
by poly A binding protein
iii. There is a protein interaction between the protein complex
iv. Now RNA is circularized within cell
c. Circularization serves 2 purposes:
i. Stimulate translation
ii. Protect mRNA from degradation because both ends are protected
d. Enzyme that degrades RNA won’t get access to RNA (because it is circularized)
i. If you interrupt interaction, both ends will then be exposed
e. Enzyme Poly A nuclease that removes poly A tail will then get access to mRNA and now the mRNA can be
degraded
f. There is competition between translation and degradation
XI. TWO MECHANISMS OF EUKARYOTIC mRNA DECAY [S86]
a. Generally speaking there are 2 degradation pathways in mRNA cell
i. One is the poly A shortening dependent
1. Poly A tail has to be removed first
2. Poly A tail binds with poly A binding proteins which protects the 3’ end
3. If enzyme removes poly A tail, then mRNA becomes unstable
b. So the first step is to remove the Poly A tail
c. Then the RNA can be degraded from 5’ to 3’ or 3’ to 5’
i. This is happening to majority of mRNA, most mRNA is degraded by this pathway
d. There are a few exceptions that only occur on a few of the mRNA
e. There is a second pathway
i. This occurs when the enzyme can cleave RNA in the middle
ii. The Endonuclease cleaves the RNA in the middle
iii. Then RNA can be degraded by similar mechanism
f. These are 2 methods of degradation in eukaryotic cells
g. The pathway on the left side occurs most frequently
XII. mRNA LOCALIZATION [S87]
a. Another regulation is called mRNA localization but it doesn’t happen to many RNA only a few RNA are under
this type of regulation
b. Generally speaking, protein is translated from mRNA and then protein product will be directed to membrane,
within the cytoplasm or the mitochondria
c. Generally speaking we make a protein and then direct the protein to specific compartment
d. If you want to make a membrane bound protein, you direct RNA to the ER
i. Then translate RNA inside the ER and then the protein can direct to the membrane
ii. That is the difference between membrane bound protein and cytoplasmic protein
e. Our subject here is that we made an mRNA
i. RNA will now be translated in the beginning
ii. There is a mechanism to repress translation
iii. Now we direct RNA to a certain area
1. Happens most often in neurons, we direct to the axon and then translate a protein and this is called
mRNA localization
2. This RNA has to be properly localized first before translation
f. 3 features required for mRNA localization
i. One is specific RNA sequence found in RNA and mostly in 3’ translated region
ii. Second it requires protein to recognize specific sequence
iii. Third you require some kind of cytoskeleton protein so now the RNA protein complex can follow the
cytoskeleton and direct to the specific compartment
XIII. THE IMPORTANCE OF 3’ UTR IN mRNA LOCALIZATION [S88]
a. This is an example to show that the sequence within RNA is important for localization
b. At a certain stage there are many nuclei in apical part of cell
i. Apical part is at the top of the picture
CLASS: 11-12:00
Scribe: Christine Sirna
DATE: Sept 15, 2010
Proof: Louisa Warren
PROFESSOR: CHEN
RNA and Post Transcriptional Regulation
Page 4 of 5
c. There is RNA called hairy RNA which will normally be localized to apical region
d. People want to demonstrate that a specific sequence is required for localization of this RNA in this region
i. They can synthesize RNA in the lab
ii. One RNA contains specific sequence and other is RNA that lacks specific sequence
iii. Then label RNA with different colors
1. One with specific localization sequence label with red
2. Other one without specific sequence you label with green
iv. Now inject both RNA into the cell and then wait about 30 minutes
e. Then go under microscope to know where RNA is localized
f. One without specific sequence in 3’ UTR region is not properly localized because we know this RNA is not
localized within the cytoplasm
g. One with specific sequence is properly localized in apical part of cell
h. Specific sequence in RNA is required for localization
i. Of course we require the protein to bind to this specific sequence
XIV.
RNA EDITING: ANOTHER WAY TO INCREASE THE DIVERSITY OF GENETIC INFORMATION [S89]
a. There is one more thing: RNA editing
b. What is RNA editing?
i. It is the change of one or two nucleotides on RNA
1. Change nucleotide to another nucleotide
ii. Then after the change you do not find the nucleotide on DNA anymore
iii. You edit or change the sequence after RNA is synthesized
c. It is very rare in mammalian cells and there are only 2 examples
i. A to I editing (I looks like G) so basically A is changed to G
ii. Other one is change C to U
d. After the change don’t find U in DNA instead you will find C
e. When you say how come it is not found in DNA? Then you know RNA is edited
f. Why do we need RNA editing? There are 3 reasons
i. Alter AA coding possibilities
1. Change A to I
2. I looks likes G
3. So normally A will base pair to U
4. I looks like G and G base pairs with C on slide you should change to C (where it says I will pair
with…)
ii. Introduce pre mature stop codon
1. Can change codon that normally coded for an amino acid so now translation will stop there and you
make a truncated protein
iii. Change splice site
iv. Change conserved splicing site and then somehow inhibit splicing
XV. RNA EDITING- A to I EDITING [S90]
a. Two examples
i. One is A to I editing
1. Doesn’t occur spontaneously
a. Requires protein complex
b. Requires enzyme
2. Here A needs to be added to I
3. So now RNA forms specialized secondary structure so now base pairs are together
4. Enzyme which converts A to I will bind to secondary structure
5. So enzyme has enzyme editing activity will convert A to I
a. Called adenosine deaminase acting on RNA (ADAR)
b. So now deaminate adenosine and also it will bind to RNA
c. This enzyme is not only an enzyme it will also second double stranded RNA and will bind to
double strand RNA and find the A and convert to I
d. I will base pair with C
e. So now you change the amino acid codon
XVI.
EDITING OF APOLIPOPROTEIN B (apoB) mRNA – C to U [S91]
a. Another example that occurs on gene Apolipoprotein B
i. It is expressed in liver or intestine
b. Depending on where it is expressed
i. In the liver the mRNA is not edited
CLASS: 11-12:00
Scribe: Christine Sirna
DATE: Sept 15, 2010
Proof: Louisa Warren
PROFESSOR: CHEN
RNA and Post Transcriptional Regulation
Page 5 of 5
ii. In the intestine it will be edited
c. Here is the total content CAA (total gene shown in picture)
i. If expressed In liver, because there is no editing on RNA you will make a longer protein
ii. If this RNA is expressed in intestine, there will be RNA editing
d. Machinery that recognizes the sequence is called an editosome
i. This is a protein complex that recognizes sequence and converts C to U
ii. So this changes the CAA codon to UAA and the UAA is a stop codon
1. Translation will now stop and you will make truncated protein that is shorter than the one made in the
liver
e. Depending on where RNA is expressed and whether it is edited or not you produce different protein isoforms
from one gene
f. Also increases the coding potential
XVII. UNIFIED THEORY OF GENE EXPRESSION [S92]
a. Read at home
XVIII. DIAGRAM [S93]
a. This is a contemporary view of gene expression
b. Already mentioned that mRNA processing occurs right after RNA is synthesized but before synthesis is
complete
c. Every step is a subdivision of entire step
i. One step can occur before the previous one is complete
d. Here is an example
i. The promoter sequence (on bottom left in blue circle) on RNA polymerase used to transcribe mRNA as
soon as 5’ region is transcribed, the capping occurs on RNA
1. Cap structure will be recognized by cap binding protein
ii. RNA polymerase II synthesizes introns
iii. Splicing machinery will assemble on introns
iv. This means the introns has been matched to tell the machinery that this is the intron
v. Doesn’t mean that intron has to be removed. It is just recognized but not removed
vi. Then you can see the 3’ end of mRNA will be cleaved
vii. 5’ end is already in cytoplasm
viii. Before RNA synthesis is complete 5’ end of RNA is already exported into cytoplasm
ix. Continuous steps, one step has occurred before the previous one is complete
x. That is the contemporary view of gene expression
e. In lecture, he had to explain using a linear method
f. But in terms of timing, it occurs in short time and can occur simultaneously
g. That concludes lecture
[End 31:50 mins]