8/21/14 - Post-Transcriptional Regulation

Question 1

What is constitutive splicing?

Answer 1

Splicing patterns that do not change.

Question 1

How might miRNAs promote tumor growth?

Answer 1

Overexpression of certain miRNAs could lead to increased degradation of tumor suppresor protein transcripts.

Question 2

Draw the pathway that shows the regulation of eIF2

Answer 2

Question 2

During apoptosis, genes encoding proteins which lead to cell death should be expressed, and all other genes should be silenced. What is one way a cell ensures only the appropriate genes are active?

Answer 2

Phosphorylation of eIF2 halts global protein translation, but IRES sites allow certain transcripts to bypass this initiation pathway. Normally, 5’ caps are needed to facilitate binding of small ribosomal subunit to the transcript, but IRES sites allow it to bind further down the transcript to initiate translation.

Question 3

What is the function of an IRES site?

Answer 3

Allows translation to begin downstream of the 5’ cap

Question 3

What is the main function of miRNA?

Answer 3

Degrade protein transcripts.

Question 4

Describe how alternative splicing generates different forms of immunoglobin proteins (draw)

Answer 4

-Immunoglobin has 2 splice donor sites -If cell uses first site, it will cleave and polyadenylate at the first stop codon, removing the downstream 3’ acceptor site, resulting in a shorter protein. Since 3’ site is missing, intron will not be removed and will be included in final transcript. This region has many hydrophilic amino acids, important for binding in secreted antibodies. -If cell uses 2nd site, it will cleave and polyadenylate at the 2nd stop codon, resulting in a longer transcript. Since the 3’ splice site is still present, the intron will be removed. The region downstream of the 3’ splice site contains many hydrophobic amino acids which facilitate membrane binding.

Question 4

How does eIF4E-BP block the recruitment of the small ribosomal unit to the capped end of the mRNA? (draw)

Answer 4

• eIF4G is a scaffold protein which binds eIF4E to form a complex that recruits small ribosomal subunit
• eIF4E-BP normally inhibits eIF4E
• When growth is signaled, kinases are released which phosphorylate eIF4E-BP so it can’t bind to eIF4E.
• With eIF4E free to bind with eIF4G, the small ribosomal subunit can be recruited.

Question 5

Describe the role of phosphatases in eIF2 function

Answer 5

Phosphatases cleave the phosphate group from eIF2, allowing it to bind normally to eIF2B and resume normal function.

Question 6

What does IRES stand for?

Answer 6

Internal ribosome entry site

Question 7

Draw the ubiquitin pathway

Answer 7

Question 9

How does the removal of 7-methylguanosine cap lead to mRNA degradation?

Answer 9

• Cap can no longer protect the 5’ end of the mRNA
• 5’ > 3’ exonuclease activity degrades mRNA
• This is a fast process

Question 11

What effect would phosphorylation of eIF4E-BP have on the translation of mRNAs containing an IRES site?

Answer 11

It would have little effect since eIF4E is not required to recruit the small ribosomal subunit in transcripts with an IRES site, only eIF4G.

Question 12

How does the RISC complex identify the correction mRNA location to silence?

Answer 12

It base pairs with the mRNA strand, forming a seed sequence that does not get translated.

Question 13

What is the role of eIF4E in translation initiation?

Answer 13

Binds to the 7-methylguanosine cap and facilitates the recruitment of the small ribosomal subunit

Question 13

What is the function of E3 in the recognition and ubiquination of protein substrates?

Answer 13

It catalyzes the transfer of ubiquitin to lysine residues on the target protein via isopeptide bonds

Question 14

What is the function of each main region on the proteasome?

Answer 14

1. Cap region recognizes ubiquitin chains and binds target proteins.
2. Cylindrical core region is where degradation occurs.

Question 15

Which 3 enzymes catalyze the activity of ubiquitin?

Answer 15

1. E1, ubiquitin activating enzyme
2. E2, ubiquitin conjugating enzyme
3. E3, ubiquitin ligase

Question 16

What are the two pathways by which a mRNA can be degraded?

Answer 16

1. Deadenylation of the polyA tail
2. Removal of the 5’ cap

Question 17

How do regulatory sequences in pre-mRNA act to regulate constitutive vs. alternative splicing?

Answer 17

Enhancer regions can bind SR/activator proteins to increase spliceosome activity. Silencing regions can bind repressors to decrease spliceosome activity

Question 18

In immunoglobin splicing, how does the cell decide which cleavage site to use?

Answer 18

The first cleavage site is suboptimal under normal conditions. When the cell is stimulated, TFs are expressed which aid in the recognition of the first cleavage site.

Question 19

What role does ubiquitin play within the cell?

Answer 19

Degrades both normal and abnormal proteins.

Question 20

Why is eiF4E thought to be overexpressed in some cancers?

Answer 20

• High [eIF4E] is needed to facilitate translation of difficult mRNAs
• Difficult mRNAs are made on purpose to more tightly regulate some cellular activities, such as cell proliferation
• Overexpression of eIF4E makes it easier for cell proliferation mRNAs to be translated, leading to more cell growth

Question 21

What circumstances might lead to the phosphorylation of eIF2?

Answer 21

If a cell is under stress, kinases are released which will phosphorylate eIF2.

Question 23

Describe the 6 major patterns of alternative RNA splicing.

Answer 23

1. Exon skipping 2. Alternative splice sites 3. Intron retention 4. Mutually exclusive exons 5. Alternative promoters 6. Alternative polyA sites

Question 24

What is the purpose of alternative splicing?

Answer 24

It allows for the production of different forms of a protein from a single gene

Question 25

What is the advantage to cells of unstable mRNAs and protein products?

Answer 25

It allows for greater regulation

Question 26

Alternative forms of immunoglobin proteins are often generated by which major pattern of RNA splicing?

Answer 26

Alternative polyadenylation sites

Question 28

What is the effect on translation when eIF2 becomes phosphorylated?

Answer 28

-It binds more tightly to eIF2B, forming an inactive complex -Since [eIF2] >> [eIF2B], all eIF2B will be bound in an inactive state -Without eIF2B, eIF2 can no longer be recycled back into eIF2-GTP and translation will not continue.

Question 29

Describe the role of ubiquination in the development of Parkinson’s Disease

Answer 29

A type of E3 ubiquitin ligase (known as Parkin) is unable to bind alpha-synuclein and mark it for degradation. Alpha-synuclein is then able to form insoluble protein lesions in the brain, leading to Parkinson’s Disease.

Question 30

What happens when RISC base pairing to mRNA is highly specific?

Answer 30

Argonaute nuclease within RISC complex is activated to quickly cleave the mRNA.

Question 31

What happens to ubiquitin chains following degradation of target protein?

Answer 31

They are recycled back into monomeric units

Question 32

The rate of degradation of a cellular consituent has what significance?

Answer 32

This rate determines how rapidly the consituent responds in response to changes within the cellular environment

Question 33

Give an example of how a protesasome inhibitor might be used to treat cancer (might want to draw)

Answer 33

1. NF-kappa-B activates cell proliferation genes
2. I-kappa-B inhibits NF-kappa-B, but is normally degraded by ubiquitin pathway.
3. Proteasome inhibitor would stabilize I-kappa-B, leading to inhibition of NF-kappa-B and cell proliferation.

Question 35

How does miRNA act to silence genes?

Answer 35

1. double strand miRNA interacts with proteins that discard one strand.
2. single strand miRNA associates with RISC complex to bind mRNA and silence that region

Question 36

What happens when RISC base pairing is not highly specific to the mRNA?

Answer 36

mRNA is transferred to a P-body and eventually degrades, but this is a slow process.

Question 37

How does deadenylation lead to degradation of a mRNA?

Answer 37

• PolyA tail loses ability to protect 3’ end of mRNA after ~30 nucleotides are lost
• Nucleases then begin to degrade in the 3’ to 5’ direction
• This is a slow process, and its rate depends on how quickly the polyA tail can be shortened