Post-Transcriptional & Translational Regulation

Question 1

What specific elements in the 3’ UTR of cells is responsible for regulating synthesis of the transferrin receptor?

Answer 1

Iron Responsive Elements (IRE)

Question 2

Describe the mechanism of miRNA.

Answer 2

Pre-miRNA bases pairs with itself in the nucleus before moving to the cytosol. A Dicer RNase processes the pre-miRNA to produce double-stranded miRNA. One strand binds with an argonaute protein and forms a mature RNA-induced silencing complex (RISC). The miRNA acts as a guide that brings the nuclease into contact with the target mRNA by complementary base pairing. mRNA is degraded and protein synthesis limited. Inhibition of protein synthesis often requires 2 miRNAs binding.

Question 3

What splice sites conform less to consensus sequences and are often not bound unless sites with greater affinity to consensus sequences cannot be found?

Answer 3

Cryptic Spice Sites

Question 4

Describe the default mechanism for mRNA degradation, when there are no regulatory sequences present.

Answer 4

mRNA degradation begins with slow deadenylation of the poly-A tail by a deadenylase. When the poly-A tail becomes very short, deadenylase is replaced by an exosome, which rapidly degrades the poly-A tail. From the 5’ end, a decapping enzyme and nuclease degrade the mRNA.

Question 5

Describe the mechanism of global translational inhibition.

Answer 5

eIF2+GTP is required to initiate translation. eIF2B is a mediator that replaces GDP with GTP. When eIF2 is phosphorylated, eIF2B binds but is unable to unbind thus stopping most translation.

Question 6

How does translational regulation differ from transcriptional regulation?

Answer 6

Translational regulation effects protein levels much more quickly.

Question 7

Where does translation take place?

Answer 7

Cytoplasm

Question 8

What enzyme is responsible for breaking down the poly-A tail?

Answer 8

Deadenylase

Question 9

Describe the mechanism of RNAi.

Answer 9

A dicer cuts the RNA into small interfering RNAs (siRNA). A siRNA strand combines with argonaute proteins to form a RISC. The siRNA acts as a guide to locate and destroy foreign RNA.

Question 10

What are the types of alternative splicing?

Answer 10

Constitutive & Regulated

Question 11

Describe negative and positive control of regulated alternative splicing.

Answer 11

Negative control - Involves a repressor, which prevents recognition of splicing sites by snRNPs

Positive control - Involves an activator, which activates recognition of a splicing site so that it is removed by a spliceosome

Question 12

What protein recognizes splice sites in pre-mRNA?

Answer 12

snRNP (small nuclear ribonucleoproteins)

Question 13

Where does RNA editing take place?

Answer 13

Nucleus

Note, this process is very rare

Question 14

What is alternative polyadenylation?

Answer 14

Many transcripts have alternative cleavage and polyadenylation sites that can generate different mRNA isoforms with different 3’ UTRs (untranslated regions). These different UTR sequences allow for inclusion or exclusion of regulatory elements, without changing the amino acid sequence.

Question 15

What is the relationship between mRNA stability and protein synthesis?

Answer 15

The more stable the mRNA, the longer it takes to be degraded, and the more protein is translated. mRNA degradation is often regulated by sequences in the 3’ UTR.

Question 16

How do viruses inhibit cap-dependent translation of host mRNAs?

Answer 16

Decrease availability of eIF4E, which binds the 5’ cap to initiate translation

Question 17

What dictates translational regulation?

Answer 17

• Sequences in the 3’ UTR
• Accessibility of the 5’ cap

Question 18

What is the function of MicroRNAs (miRNAs)?

Answer 18

To fine-tune protein synthesis by reducing protein that is made

Question 19

What are the features of constitutive alternative splicing?

Answer 19

Constitutive spicing is relatively unregulated due to intron sequence ambiguity (consensus binding sites). Splicing factors first bind to mRNAs that have a higher binding affinity for the consensus site. If none exist, splicing sites then bind mRNA with lower binding affinity. This form of splicing often makes defective mRNAs that are quickly degraded.

Question 20

What enzyme synthesizes miRNA?

Answer 20

RNA Polymerase II

Question 21

What is an exon junction complex?

Answer 21

EJCs mark the location of removed introns and play a role in regulation.

Question 22

What splicing sites conform well to consensus sequences of splicing factors?

Answer 22

Canonical Splice Sites

Question 23

What post-transcriptional modifications mark a successful mRNA?

Answer 23

5’ Cap

3’ Poly-A Tail

Exon Junction Complexes

Question 24

What post-transcriptional modification process is responsible for the production of both ApoB100 and ApoB48 from the same mRNA?

Answer 24

RNA Editing

Question 25

Describe the mechanism of alternative mRNA degradation.

Answer 25

Some mRNA contain recognition sites for an endonuclease in the 3' UTR. The endonuclease cleaves the poly-A tail, allowing the nuclease to degrade mRNA from the 5' end and deacetylase from the 3' end. This process is more rapid.

Question 26

How does stabilization of IREs in the 5' UTR of ferritin affect protein synthesis?

Answer 26

When IREs are destabilized, the 5' cap is accessible, translation occurs, and protein is synthesized. When iron levels are low, a protein binds and stabilizes IRE, but blocks access to the 5' cap, thus preventing translation. This process is the opposite of IRE binding in the 3' UTR and translation of the transferrin receptor.

Question 27

What is nonsense-mediated mRNA decay?

Answer 27

mRNA is “tested” in a pioneer round of translation immediately outside the nucleus. If there is an error in the mRNA and a stop codon is reached before all exon junction complexes are reached, this indicates a nonsense mutation and triggers mRNA degradation.

Question 28

How are splice sites identified?

Answer 28

Splice sites are recognized by snRNPs through complementary base-pairing with consensus sequences in the pre-mRNA.

Question 29

What nucleotide changes occur during RNA editing?

Answer 29

C to U - creates a stop codon A to Inosine - changes the amino acid (very important in the brain) *Note, this process is rare and only takes place in the nucleus*

Question 30

What cell states activate kinases that cause global inhibition of translation of most mRNAs?

Answer 30

1. Amino acid starvation 2. Heme deficiency 3. Accumulation of misfolded proteins in the RER 4. dsRNA

Question 31

What targets long, double-stranded RNA molecules, which are sometimes viral intermediates?

Answer 31

RNA interference (RNAi)

Question 32

What in the 3' UTR are recognized by proteins that decrease or increase the rate of deadenylation?

Answer 32

AUUUA Elements (AREs)

Question 33

What is the unfolded protein response (UPR, aka ER stress response)?

Answer 33

Protein misfolding in the ER activated UPR. Translation is inhibited through phosphorylation of eIF2 and synthesis of chaperone proteins is increased. If misfolds cannot be resolved, the cell undergoes apoptosis.

Question 34

What is the effect of iron levels on synthesis of the transferrin receptor?

Answer 34

When iron concentration is high in the cell, IREs are unbound by proteins and results in mRNA degradation. When iron concentrations are low, proteins bind IREs to stabilize the mRNA and promote translation of the transferrin receptor.

Question 35

What enzyme is responsible for changes in the nucleotide sequence during RNA editing?

Answer 35

Deaminases, which are expressed in a cell/tissue- or development phase-specific manner.

Question 36

What are the features of regulated alternative splicing?

Answer 36

Regulated splicing is more intentional and involves addition or removal of functional domains with assistance from activators or repressors. Regulated splicing is tissue- or development stage-specific, with only one version of the protein expressed in the cell.

Question 37

What are the characteristics of translational regulation?

Answer 37

* Quick effects * Important for developmental regulation * Important in enucleated cells * Most regulated stage or protein synthesis

Question 38

Describe a spliceosome, including major features.

Answer 38

Spliceosomes are large molecules of RNA and protein. They are complex and dynamic, thus flexible, and require extensive ATP hydrolysis for assembly.

Question 39

What enzyme splices and removes introns from pre-mRNA?

Answer 39

Spiceosome