Post-transcriptional Gene Regulation

Question 1

Describe the different forms of alternative splicing that are regulated events following transcription.

Answer 1

Alternative splicing can lead to:

exon skipping

Alternative 3’ or 5’ splice site selection

Intron retention (i.e. not splicing)

Mutually exclusive exons (i.e. one or the other is included)

Question 2

How can alternative splicing impact post-transcriptional processes including translation and mRNA packaging?

Answer 2

Alternative splicing can determine which promoter is included in the mRNA transcript

It can also dictate which cleavage and polyA site is included

Both of these splicing events are not the regulated event

Question 3

How can 1 mRNA code for a family of proteins with different functions?

Answer 3

Alternative splicing of an mRNA can determine which exons are included in the transcript.

Example: Fibronectin can exist in the extracellular matrix of fibroblasts or in hepatocytes. These fibronectin forms differ by two integrin binding domains that are crucial for adhesion in the basement membrane, but are spliced out of the hepatocyte form.

Question 4

Describe how DSCAM can have more than 38,000 different splicing patterns from 4 exons.

Answer 4

Splicing of this gene choses 1 base pair from each of 4 exons (A1-12, B1-48, C1-33 and D1-2)

12*48*33*2 = 38,016 possible splicing patterns

Question 5

How can an mRNA with a strong splice site be prevented from splicing?

Answer 5

Negative control

A repressor protein can bind to a silencer sequence to inhibit splicing

Because of the strong interaction (near perfect base pairing), even in the absence of an SR protein, the spliceosome would be able to initiate splicing.

The repressor protein prevents this splicing from occuring

Question 6

How can a weak splice site be recognized by a spliceosome?

Answer 6

Usually, the spliceosome will pass over weak splice sites

In the presence of an activator protein (i.e. SR proteins), the spliceosome will recognize the weak splice site and initiate splicing

Question 7

What is the difference between constitutive splicing and alternative splicing?

Answer 7

In alternative splicing, the levels of the activator and repressor proteins vary between tissues leading to different mRNAs in different tissues

Question 8

What regulatory proteins aid in the selection of splice sites?

Answer 8

SR proteins (serine, arginine rich proteins) bind to ESEs (exon splicing enhancers) along with the complex of U proteins that make up the spliceosome

Question 9

Describe the differences in splicing between the membrane-bound antibody and the secreted antibody.

Answer 9

The antibody gene contains 2 potential polyA sites and 2 stop codons

The membrane-bound form of antibodies is produced when the second cleavage/polyA site is used (this is the optimal site). The intron contains the first stop codon, but is spliced out to form the antibody with a terminal hydrophobic peptide that can insert into the membrane.

The secreted antibody is produced when the first cleavage/polyA site is used. This causes part of the intron to be incorporated into the mRNA because the 3’ splice site is cleaved away. The final protein has a terminal hydrophobic peptide from the intron region.

Question 10

What are the roles of eIF2, eIF4E and eIF4G?

Answer 10

These are involved in the initiation of translation

eIF2 bound with a GTP is attached to the initiator tRNA that is attached to the P-site of the small ribosomal subunit

eIF4E is a cap binding protein

eIF4G is a scaffold protein that binds to the ribosomal subunit to allow it to scan along the mRNA searching for the start codon

Question 11

When the small ribosomal subunit reaches the AUG start codon, what happens?

Answer 11

GTP is hydrolyzed causing eIF2 and the other initiation factors to dissociate from the complex.

The large ribosomal subunit can then bind to the Methionine tRNA at the P-site.

Question 12

How is eIF2 recycled and how does this regulate translation?

Answer 12

Following hydrolysis of GTP, eIF2 is bound to GDP

When there is enough energy in the cell, eIF2B (a guanine nucleotide exchange factor) removes the GDP and replaces it with a GTP to reform an active eIF2

If the cell is stressed, a protein kinase phosphorylates eIF2-GDP which keeps eIF2 in inactive form and dramatically slows protein synthesis

Question 13

Describe the steps in the regulation of cap-binding proteins.

Answer 13

The cap binding protein eIF-4E is bound to inhibitor 4E-BP. It can still bind to the mRNA, but cannot interact with eIF-4G which prevents the ribosome from attaching

4E-BP is inactivated by phosphorylation which frees up the eIF-4E

Question 14

How are inefficient transcripts translated properly?

Answer 14

eIF4E is able to process mRNA that have difficult-to-read conformations

These mRNAs are not properly translated when active eIF4E is present at low levels

Question 15

What is an IRES?

Answer 15

Internal ribosome entry site

This allows for cap independent translation

Translation can be initiated without eIF4E by using an IRES. Still need the eIF4G to bind to the small ribosomal subunit

This is a way the cell can turn off general translation, but still keep a subset of proteins activated (for example, this occurs in apoptosis)

Question 16

True or false: the time it takes to achieve the steady state level of mRNA depends on the rate of mRNA synthesis and the rate of mRNA degradation

Answer 16

FALSE

The time it takes to reach steady state is only affected by the rate of degradation

Question 17

A cell with a degradation rate of 10 minutes will respond to changes in rate of synthesis ________ than a cell with a degradation rate of 1 minute

(Faster or Slower)

Answer 17

Slower

The rate of degradation of a cellular constituent determines how rapidly it responds to changes in synthesis rate

Question 18

How are old mRNAs degraded?

Answer 18

Over time, the length of the poly-A tail decreases (deadenylation)

When the tail is <30 base pairs, the mRNA will be degraded from both ends (3’–>5’ by an exonuclease, and then 5’—>3’ after the cap is removed)

Question 19

What determines how stable and mRNA molecule is?

Answer 19

How fast the poly-A tail is degraded

Question 20

What process competes with de-adenylation?

Answer 20

Translaton

When an mRNA is being translated, the poly-A tail is protected from deadenylation by poly-A-binding proteins involved with the translation machinery

Question 21

Describe how miRNAs regulate gene expression

Answer 21

miRNAs are short RNAs that form hairpins by folding on themselves

The hairpin is cleaved by Dicer

Argonaute discards one of the 2 strands leaving the RISC complex and one strand to basepair with mRNA

Depending on the extent of the basepair matching, the mRNA will either be rapidly degraded (extensive matching) or translation will be reduced (less extensive mathing)

Question 22

What is a seed sequence?

Answer 22

The bound portion between miRNA and mRNA, which only needs to be 7 base pairs long

Question 23

True or false: A single miRNA can regulate many genes

Answer 23

True.

Because only 7 base pairs need to match and they do not need to be perfect or sequential, one miRNA can regulate many genes

Question 24

How are broken/unuseful RNA sequences degraded?

Answer 24

Nonsense-mediated mRNA decay

Splice errors often produce premature stop codons in the middle of coding regions due to frameshifts

Exon-junction complexes (EJC) bind following successful splicing

Upon entering the cytosol, a test translation is conducted

If no errors exist, then all the EJCs should be pushed off the mRNA

EJCs present on the mRNA following test translation is a red flag because a stop codon must have been reached, the mRNA will be rapidly degraded

Question 25

What role does ubiquitin play in protein degradation?

Answer 25

Ubiquitin is the tag that marks proteins for degradation by the proteasome

Question 26

Describe the ubiquitin pathway of protein turnover

Answer 26

E1 (ubiquitin activating enzyme) is ubiquitinated

The ubiquitin is transferred from E1 to E2 (ubiquitin conjugating enzyme)

E2 complexes with E3 (ubiquitin protein ligase) and binds to the substrate to ubiquitinate

The substrate is then polyubiquitinated

The proteosome cap binds to the ubiquitinated tail and the substrate is degraded within the cylinder

The ubiquitins are then recycled back to E1

Question 27

Describe the structure of the proteasome

Answer 27

The proteasome is composed of a central 20S cylinder supplemented with two 19S caps

Question 28

How are cell cycle regulators degraded?

Answer 28

The ubiquitin system is used to rapidly degrade cell cycle regulators.

Specific regulators are present at different stages of the cell cycle, and should not be present for the other cell cycle stages, so rapid degradation is necessary

E3 is expressed at the end of each stage, which causes ubiquitination of proteins marking them for degradation

Question 29

How could proteasome inhibition be a target for cancer treatment?

Answer 29

NF-kappa B is normally contained within the cell as an inactive complex with its inhibitor I-kappa B

When I-kappa B is phosphorylated, it dissociates from NFkB allowing NFkB to enter the nucleus and cause cell growth and survival.

I-kappa B is normally degraded by the proteasome

By blocking proteasome activity, we may be able to keep the concentration of IkB higher leading to less gene activation by NFkB