Eukaryotic Post Transcriptional Regulation

Question 1

What are the post transcriptional steps in gene expression that can be regulated.

Answer 1

1) Attentuation of mRNA
2) RNA processing control (alternative splicing, capping, cleavage/ polyadenylation RNA editing)
3) Nuclea export
4) translation control (localization control, translation initiation)
5) mRNA stability
6) Protein control (protein turnover, protein modification, protein inhibition)

Question 2

Describe the major patterns of alternative RNA splicing

Answer 2

1) Exon skipping
2) Alternative 3’ SS selection
3) Alternative 5’ SS selection
4) intron retention
5) mutually exclusive exons
6) Alternative promotors
7) Alternative poly(A)

Question 3

Exon skipping

Answer 3

When an exon is spliced out of the transcriopt with the flanking intron

Question 4

Alternative 3’ Splice Site selection

Alternative 5’ Splice site selection

Answer 4

Occur when two or more splice sites are recognized at one end of an exon.

Question 5

Intron retention

Answer 5

and intron remains in the mature mRNA transcript.

Question 6

mutually exclusive exons

Answer 6

Cell has to decide which exon to include between two exons. Cannot include both at the same time so it will alternate between which one is included.

Question 7

Why is alternative splicing important in fibronectin production?

Answer 7

Because there are two types of the fibronectin, membrane bound and soluble fibronectin. Both types are found on the same gene. Fibroblasts synthesize mRNA that included the exons EIIA and EIIB (these encode for the protein region responsible for binding to the cell membrane)

Through alternative splicing Hepatocytes synthesize an mRNA without these exons, resulting in fibronectin that circulates the blood.

Question 8

How do regulatory proteins aid in splice site selection?

Answer 8

Proteins will either promote the use of a splice site (positive) or block the use of another (negative)

Proteins that bind to a silencer sequences will prevent splicing of a specific region (negative control)

Proteins that bind to an enhancer sequence encourage the splicesome to use that specific site (positive control)

Question 9

What is the difference between constituitive and alternative splicing?

Answer 9

In alternative splicing levels of activator and repressor proteins vary between tissues

Question 10

If a gene doesn’t have an alternative splice site are splicesome activator proteins used

Answer 10

Yes, even though there are not other options for splicing, activator proteins are used to guarentee that the splicesome finds the correct splice site

Question 11

Explain how regulation of polyadenylation site selection during the processing of 3’ end of an immunoglobulin mRNA can result in thr formation of membrane bound and secreted forms of antibody from the same gene.

Answer 11

If a B-cell has not seen an antigen and been stimulated it will produce membrane bound antibody. The mRNA for the membrane bound antibody is long, and the intron sequence at the 3’ end is removed. If antigen stimulation occurs the RNA will be cleaved upstream from where the unstimulated splice site was. The product is shorter mRNA but the 3’ intron sequence is retained and remains as a coding sequence and results in a different polyadenylatin site. It codes for the hydrophillic c-terminal portion of the secreted antibody protein.

The regultion event is based on the cleavage and polyadenyltion site used.

Question 12

Describe the function eIF-2

Answer 12

eIF-2 forms a ternary complex with GTP and the initiator methionyl-tRNA. This complex binds to the 40S subunit and together they are recruited by the cap binding complex to the mRNA. The initiator tRNA bound to the 40S subunit interacts with the initiation codon in the mRNA. This triggers GTP hydrolysis, which leads to assembly of a functional ribosome on the mRNA.

Question 13

Describe the function of initiation factor eIF-2B in maintaining eIF-2 in its active state.

Answer 13

eIF-2B is a guanine nucleotide exchange factor that reactivates the inactive eUF2-GDP complex after is has been hydrolized.

Question 14

Exaplain the effect on eIF-2 phosphorylation on on mRNA translation

Answer 14

phosphorylation of eIF-2 inhibits protein synthesis because the phosphorylated eIF-2 binds to eIF-2B forming a stable complex that is inactive. There is more eIF-2 than eIF-2B so it doesn’t take much phosphorylated eIF-2 to inactivate the majority of eIF-2B.

Question 15

When does the phosphorylation of eIF-2 occur?

Answer 15

The phosphorylation of eIF-2 occurs in response to cellular stress: heat shock, glucose/amino acid deprivation. or in abscence of heme, growth factors or serum. also in response to interferon and double stranded RNA

Question 16

Describe the effect of phosphorylation of eIF-4E binding protein (eIF-4BP) on mRNA translatin.

Answer 16

eIF-4BP is an inhibitor of eIF-4E. eIF-4E is a cap binding protein that is activated by growth factors and hormones. Activation of eIF-4E leads to phosphorylation of eIF-4BP which allows a functional eIF-4E to complex with eIF-4G and recruit the small ribosomal subunit to mRNA.

Question 17

When does dephosphorylation of eIF-4BP occur.

Answer 17

it is triggered by serum deprivation, viral infection, heat shock, and the cell’s entrance into M phase of the cell cycle.

Question 18

Why is eIF-4G essential

Answer 18

It brings the 40S subunit to the mRNA. inactivation will lead to inhibition of potein synthesis.

Question 19

Why can dephosphorylated eIF-4BP block the recruitment of the small ribosomal subunit to the capped and of mRNA?

Answer 19

Because it binds to and inactivates eIF-4E which then cannot bind to eIF-4G in order to recruit th small ribosomal subunit to mRNA

Question 20

Describe the function of an internal ribosomal entry site (IRES) in mRNA translation

Answer 20

Allow the 40S subunit to bypass the cap when forming a functional translation initiation complex with the corresponding mRNA.

Question 21

Why is iEF-4E a protooncogene?

Answer 21

Increased expression of eIF-4E can lead to an increased translation of inefficient mRNAs that encode oncogenic proteins.

increased eIF-4E expression results from eIF4B phosphorylation

Question 22

Predict the effect of phosphorylation and de-phosphorylation of eIF-4BP on the translation of mRNAs that contain an IRES

Answer 22

dephosphorylation of iEF-4BP leads to inhibition of cap binding protein iEF-4E. This is thought to activate translation of mRNAs that contain IRES, while inhibiting cap dependent translation. Vice versa for phosphorylation of iEF-4BP

Question 23

compare the cap dependent mechanism of translation initiation to the IRES method of translation initiation

Answer 23

The cap dependent method requires a set of initiation factors whose assembly on the mRNA is stimulated by the presence of a 5’ cap and poly-A tail. needs eIF4E to bind to eIF4G.

The IRES dependent mechanism requires only a subset of the normal translation initiation factors (only eIF4G). These assemble directly on the folded IRES to recruit the 40S subunit to the mRNA.

Question 24

How do IRES allow eukaryotic cells to be similar to prokaryotic cells.

Answer 24

IRES allow for eukaryotic cells to have polycystronic messages.

Question 25

How are IRES involved in apoptosis?

Answer 25

When a cell sustains a lot of DNA damage the cell goes through apoptosis. Apoptosis is initiated by a family of proteases called caspases. Caspases cleave eIF4G which turns off the synthesis of cellular proteins and turns on the synthesis of proteins required for apoptosis. mRNA active during apoptosis contain an IRES

Question 26

What does the steady state of protein and mRNA synthesis depend on?

Answer 26

The rate of synthesis and degradation of protein or mRNA. Time required to achieve a new level of product depends soley on degradation.

Question 27

Explain the advantage of unstable intermediates or products to cells.

Answer 27

Unstable intermediates and products are degraded more easily. The rate of degradation of a cell constituent determines how quickly it responds to change.

Question 28

Describe two pathays by which mRNA can be degraded.

Answer 28

1) Deadenylation pathway. When a poly-A tail is de-adenyltated and shorted to less than 30 nucleotides a de-capping enzyme becomes active an removes the methylguanine cap. Exonucleases then degradethe mRNA from both ends.
2) micro RNAs bound to RISC bind to mRNAs they share complementarity with. If there is high complementarity between the miRNA and the mRNA, endonucleolytic cleavage of the mRNA occurs. If there is less complementarity the result is translational silencing.

Question 29

Describe the source of miRNAs

Answer 29

miRNA genes are located either withing introns of protein coding genes or in non-protein coding regions of DNA between genes (intergenic DNA)

Question 30

Describe miRNA assembly in an RNA induced silencing complex (RISC)

Answer 30

The double stranded mRNA precursor is cleaved in the nucleus by a nuclease (Drosher). The resulting pre-miRNA is transported into the cytosol where it is further cleaved by Dicer to form a small double stranded miRNA. This binds the protein Argonaut, which is present in a complex of proteins called RISC. One of the two strands is degraded and the left over strand is used by RISC to identify target mRNAs.

Question 31

Describe how risk identifies the mRNA it regulates

Answer 31

The mRNA target contains one or more seed sequences, 7 nucleotides long, complementary yo the miRNA.

Question 32

Describe and compare the two ways an miRNA can regulate the level or activity of an mRNA

Answer 32

1) If there is low complimentarity there will be translational silencing. Translation inhibited mRNAs accumulate at particular sites within the cytoplams called P-bodies where they are de-capped, deadenylated, and degraded.
2) if there is high complimentarity leads to endnucleolytic cleavage of the mRNA by argonaute.

Question 33

Describe nonsense mediated mRNA decay

Answer 33

When there is a successful exon linkage, exon junction complexes will bind marking the begining and end of the exon. The mRNA goes through a test run of translation. If the ribosome reaches a stop codon between two exon junction complexes it will recognize that this is a nonsense mutation because it hasn’t reached the 3’ exon junction complex that marks the end of the exon. The mRNA will be degraded.

Question 34

Describe the ubiquitin conjugation system

Answer 34

Ubiquitin tags proteins for destruction by an ATP dependent protease. The carboxyl-terminal glycine of ubiquitin becomes attached covalently to the episilon-amino groupo of lysine residues (iso-peptide bond). Ubiquitin molecules bind to each other and form a string of ubiquitin that hangs off the ysine chain. The protein will then go to a proteasome tobe degraded.

Question 35

What is ubiquitin

Answer 35

8.5 kilodalton protein found in all eukaryotic cells. has a hydrophobi globular core. It’s carboxyl-terminal glycine gets attached to epsilon-amino group lysines in order to mark proteins for degradation.

Question 36

E1

Answer 36

Ubiquitin activating enzyme

Question 37

E2

Answer 37

ubiqiutin conjugating enzyme

Question 38

E3

Answer 38

ubiquitin ligase

Question 39

Describe the ubiquitin pathway of protein turnover

Answer 39

Attachement of ubiquitin to proteins requires ATP and is catalyzed by 3 enzymes. Ubiquitin is bound to and activated by E1. E1 transfers the ubiquitin to E2. E2 then binds to E3 and the substrate to be degraded. The substrate is ubiquibated and the protein goes to the proteosome for degradation. The ubiquitin is recycled by E1.

Question 40

Describe the strucutre of the proteosome

Answer 40

It is a cylinder shape with two caps at each end. The central rings of the inside of the cylinder are proteases. whose active sites are only accessible from the interior of the cylinder. The caps are regulatory particles that recognize the protein substrate and remove the ubiquitin for reuse. The caps unfold the protein and thread it into the interior of the chamber.

Question 41

Describe the degradation of a protein by the proteosome.

Answer 41

The proteosome cap recognizes the ubiquidin tagged protein and unfold the protein using ATP. The protein is transported to the central core of the proteosome (also ATP dependent) where it is digested into peptide 7-12 amino acids in length. Actual peptide bond cleavage does not require ATP

Question 42

Describe the role of ubiquitin-protesome control of the cell cycle

Answer 42

E3 ligases APC, SCF, and SCF-3 recognize and ubiquinate cyclins. The presence or abscence of certain cyclins dictates where in the cell cycle a cell is.

Question 43

APC

Answer 43

Anaphase promoting complex. E3 ligase that triggers chromosome separation and the termination of mitosis. It recognizes an maino acid sequence called the mitotic cyclin destruction box found in M-phae cylcins.

Question 44

SCF

Answer 44

SCF promotes the transition from G1 phase to S phase and the progression through S phase

Question 45

SCF-3

Answer 45

E3 ubiquitin ligase that regulates levels of I-kapa beta. I-kapa beta inhibits NF-kapa beta. NFkb actiates genes that promote cell growth and survival. When IkB is phosphorylated it is inhibited, and then ubiquinated by SCF-3 + E2-ubiquitin. This allows NF-kappa B to be active

Question 46

What is the role of NFkB in cancer

Answer 46

it is often elevated in cancer cells because it promotes cell growth and survival. inhibition of IkB also leads to increased NFkB in cells. Inhibiting IkB degradation maintains NFkB in an inactive state which can lead to apoptosis in cancer cells

Question 47

What is the role of NFkB in inflammation

Answer 47

inhibiting IkB degradation leaves NFkB in an inactive state which can reduce inflamation and symptoms of autoimmune disorders

Question 48

Velcade and PS-519

Answer 48

Velcase- proteasome inhibitor approved for treatment of relapsing multiple myeloma, is being evaluated in clinical trials for treatment of many cancers including leukemia

PS-519 a proteasome inhibitor in phase II clinical trials for the treatment of reperfusion injuries in stroke.

PS-519 and Velcade were shown to reduce infarct size in animal models of reperfusion injury to the heart.

Question 49

Explain the function of the ubiquitin proteasome system on Parkinsons

Answer 49

Parkin is an E3 ubiquitin ligase. Alpha-synuclein a substrate of Parkin can produce insoluble aggreagate in regions of th brain if it is not degraded.

Question 50

Explain the function of the ubiquitin proteasome system on Alzheimers

Answer 50

beta-amyloid proteins form long fibrils in brain tissue. These amyloid fibers are resistant to degradation by proteolysis and accumulate in brain tissue.

Question 51

Explain the function of the ubiquitin proteasome system on hereditary tumors of retina, brain, kidney, and other tissues.

Answer 51

VHL (von hippel Lindau protein) a component of an E3 ligase targets HIF, a transcription factor that promotes angiogenesis.

Question 52

Explain the function of the ubiquitin proteasome system on hereditary breast and ovarian cancer

Answer 52

BRCA1 and BRCA2: components of an E3 ubiquitin ligase that activate proteins involved in DNA repair. (non-traditional role in ubiquination)

Question 53

Explain the function of the ubiquitin proteasome system on Angelman syndrome

Answer 53

UBE3A: deletion of this E3 ligase contributes to the mental retardation seen in Angelman syndrome