Post-translation

Question 1

How is the pioneering round of translation of an mRNA transcript different than the other one?

Answer 1

RNA helicase in Nuclear Pore Complex is not 100% efficient, leaves some nuclear proteins bound to DNA → Ribosomes knock off protein on its way (nuclear and cytoplasmic)

Question 2

Which RNA pol is responsible for synthesis of snRNA? tRNA? of 5S rRNA?

Answer 2

sn RNA = RNA pol II
tRNA = RNA pol III
5S rRNA (found in large subunit of Ribosome) = RNA pol III

Question 3

What are the roles of SR proteins?

Answer 3

Serine-Arginine rich proteins
Bind exons on the pre-mRNA so introns are properly spliced, by binding to specific sequences of the RNA

Question 4

What are different mechanisms of RNA surveillance and Quality control for translation of proper proteins?

Answer 4

1. SR proteins define the exons so exons properly excised
2. Polyadenylation of the pre-mRNA
3. Export factors loaded onto the mRNA for it to get out of the nucleus
4. NMD (nonsense mediated decay)

Question 5

What is an important mechanism of quality control of the cell to prevent having truncated/ incomplete proteins?

Answer 5

NMD
Nonsense mediated decay = cellular quality control
- Degrades mRNA with premature termination codon
- Nonsense mutations
- Presence of proteins still on mRNA after pioneering translation
By identifying mRNA with proteins still bound to it after the pioneering round of translation

Prevents synthesis of truncated or incomplete proteins to would get in the way of proper function of the cells (ex: by competing for specific hormone receptors)

Question 6

Is the stability of cytoplasmic mRNA similar across all organisms?

Answer 6

No, varies a lot
E.coli = 3-5 min half-life → not long because environment changes rapidly so need to change the proteins that are synthesized to adapt rapidly
Yeast = 22 min half-life
Humans = 10h → sticks around longer because more stable environment
*Not all mRNAs last 10h, some are shorter, ex: immune cells

Question 7

What is special the AUUUA sequence?

Answer 7

It is found as a repeat in many short-life mRNAs in eukaryotes in 3’ UTR → destabilizes mRNA

Question 8

How/Where(on the mRNA) does RNA decay occur?

Answer 8

• It occurs at both ends of the protein
  *Sometime RNA can even be cleaved → double attack!!! → both ends of both segments

5’ → 3’ decay: in P-bodies (liquid-liquid condensate)
- decapping by decapping enzyme
- decay by XRN1

3’ → 5’ decay:
- Deadenylase complex
- Exosome

Question 9

What is the structure of the exosome?
Responsible of the 3’ → 5’ decay of mRNA

Answer 9

1. Regulatory lid → RNA helicase
2. ATP base
3. Exo-9
4. RRP44
   *3 and 4 are 2 exonuclease activities in channel
5. Endonuclease activity are exit of the channel in case exonucleases activities failed

Question 10

What are 2 general ways by which protein expression can be regulated by RNA changes depending on cell environment?

Answer 10

1. 3’ UTR region regulation stabilizes/destabilizes RNA (more or less degradation)
2. 5’ UTR region regulation blocks translation before the coding region

Question 11

How is stability of mammalian transferrin receptor TfR regulated in response to intracellular iron concentration?
What is TfR’s role?

Answer 11

*TfR is needed for import of iron into the cell
1. 3’ UTR of TfR mRNA has IREs (Iron Response Element) = AU rich-elements
Recall AU-rich sequences destabilize mRNA
2. IRE-BP (IRE-binding proteins) have 2 iron concentration-dependent conformations: active (low iron) and inactive (high iron)
3. In low iron, active IRE-BP binds to IRE in 3’ UTR to prevent degradation of mRNA → more TfR proteins produced → more iron import

Question 12

What can we deduce from a Western Blot and a Norther Blot that do not correlate?
(related to translational regulation)

Answer 12

Western = proteins
Northern = RNA
Translation is affected, but levels of RNA stay constant, RNA is not degraded(5’ UTR block)
*Usually linear relation, more mRNA = more protein, when not the case, translation if affected

Question 13

In which direction do ribosomes read RNA?

Answer 13

5’ (cap) → 3’ (poly(A) tail)

Question 14

How is synthesis of Ferritin regulated inside the cell depending on mRNA levels?

Answer 14

1. Ferritin mRN has IREs (Iron Response Element / stem loops) in its 5’ UTR (before the coding region)
2. Active IRE-BP bind to IREs → block ribosomes from passing → ribosomes don’t get to the coding region → protein not synthesized
3. Inactive IRE-BP doesn’t bind → ferritin gets synthesized(want in high iron)

Question 15

What is ferritin?

Answer 15

Ferritin = intracellular protein that binds iron ions, preventing accumulation of toxic levels of free iron ions
When high iron, want more ferritin to bind to it
When low iron, want less ferritin to have more free iron in cell

Question 16

How does inhibition of mRNA translation has a pivotal role in drosophila embryo development?

Answer 16

Uniform hunchback mRNA distribution across dorsophila larva but not uniform protein expression
Nanos RNA found only in posterior
- Hunchback protein necessary only for anterior end
- Nanos = zinc finger RNA binding protein → interacts with 3’ UTR of hunchback mRNA → no translation in the posterior even though hunchbach mRNA is there

Question 17

Which C. elegan mutant goes through the 1st stage of dev. (on 4 stages) over and over? How?

Answer 17

lin-14 gf mutant
lin-14 is important gene which regulates timing of developmental effects

lin-4 also involved in development but mutation in lin-4 would cause larva to skip stages of development
encodes a small RNA that has considerable homolgy to 3’ UTR of lin-14 → will bind to it and affect tranlation of lin-14 mRNA
Regulation of translation trough miRNA concept with Argonaute protein in RISC and Dicer

Question 18

What are the sequential steps through which biogenesis of microRNA proceeds? (in the nucleus)

Answer 18

1. MicroRNA transcribed mostly by RNA pol II → capped and processed
2. primary-miRNA folds up → dsRNA hairpin
3. dsRNA hairpin digested by Drosha enzyme → pre-miRNA
4. Exportin5 takes pre-miRNA (without Drosha)→ cytoplasm via NPC

Question 19

What are the sequential steps through which biogenesis of microRNA proceeds? (in the cytoplasm)

Answer 19

5. Dicer cleaves dsRNA into 21-23nt fragments
6. Fragment are bound by Argonaute protein in an RNA-indued silencing complex (RISC) → miRISC
7. ATP hydrolysis → unwinding/helicase activity of miRISC
8. use miRNA product as gude to target complementary cellular RNA
9. Associate by Watson-Crick IMPERFECT base pairing in the 3’ UTR → Block translation of destabilize mRNA target through de-adenylation

Question 20

What is a dicer?

Answer 20

• RNAse II like enzyme
• Acts as a dimer to cleave dsRNA → 21-23nt fragments

Question 21

How important is microRNA for gene control?

Answer 21

We estimate 60% of the predicted coding genes in our genom may be under microRNA-mediated control → VERY IMPORTANT

Question 22

What is the process of RNAi?

Answer 22

Same process as miRNA but bind with 100% homology to target region → cleavage of the target mRNA by cytoplasmic ribonucleases

We know cleavage of RNA = kiss of death → attack from both ends x2

Question 22

What are the differences and similarities between RNAi and microRNA?

Answer 22

Both are trigered by dsRNA molecules
Both natural processes part of endogenous regulatory mechanisms for gene expression

microRNA = imperfect base-pairing → translation inhibition, mRNA destabilization

RNAi = 100% base-pairing → mRNA degradation (dead bye bye)

*Good example of other function for RNAs, that coding for proteins

Question 23

What are examples of non-coding RNAs nucleate complexes that trigger chromosomal silencing?

Answer 23

In yeast:
Small RNAs required for silencing mechanism of centromeric heterochromatin → Centromic regions associated with kinetocor-a structure must be silenced, important for accurate cell division

dsRNA recruits proteins → generating H3K9me3

*Similar mechanisms in vertebrates

Question 24

What are piRNAs and PIWI proteins? How do they function?

Answer 24

Role: protect germ lines of most animals

piRNAs transcribed from a DNA cluster of disabled transposable elements in flies
piRNA precursor (long RNA) → piRNA intermeditate (does NOT require dicer)
PIWI = Argonaute protein required for
piRNA + PIWI → complex → piRNA guides → bind to target through antisense complementarity → PIWI cuts transposon RNA → on of the sections reused as template for more piRNAs protecting germ cells from transposable elements

Question 25

Why are piRNAs and PIWI proteins important for the germ lines of most animals?

Answer 25

They cut RNA transposons which are sequences of RNA that can jump and create mutations or disrupt specific important sequences

Question 26

By which mechanism do viruses defy miRNA degradation?

Answer 26

miRNA response elements = where miRNA binds on the 3’UTR. By reducing them, less place to binds?

By forming circle RNA that doesn’t have a 3’UTR

ex: Viral sponge RNAs soak up miRNAs so they don’t bind to target site

Use circular RNA or linear look it up

Question 27

What are Barr Bodies?

Answer 27

Inactive X chromosome in females (inactivated during embryonic development)

Question 28

What can we tell about the patched nature of the fur of calico cats?

Answer 28

• They are all females
• See mosaic because of the different regions where different X chromosomes have been inactivated

Question 29

What is the n-1 rule?

Answer 29

There can only be 1 active X chromosome so n-1 X chromosomes have to be inactivated
DOSAGE COMPENSATION

Question 30

By what process is chromosome X silenced in females?

Answer 30

1. Decision of which X chromosome to inactivate = random process ocurring early in cell division (embryogenesis)
2. XIST gene on chosen X chromosome becomes activated
3. XIST → long non-coding RNA that coats entire length of chosen X chromosome in cis (same from which its transcribed)
4. XIST RNA recruits chromatin modifying complexes → compaction of chromatin + silencing → H3K27me and H4 anti-acetylates
5. TSIX (antisens to XIST) transcribed from opposit stran of XIST gene → inhibits spread of XIST RNA on the other X chromosome (that should remain active)

*both XIST and TSIX are long non-coding RNAs
*Example of how RNA can regulte gene expression

Question 31

What is involved in the recruitment of a chromatin modifyin complex by XIST?

Answer 31

SHARP protein recognizes XIST, forms a condensate around the X chromosome that should be inactivated
- XIST RNA acts as glue for formation of the condensate
- Have histone deacetylase → compaction
- H3K27me
-Genes from inactivated X chromosome not 100% extinguished but like 99%

Question 32

What are different reasons why the de-adenylation of the 3’ end of mRNA causes mRNA degradation?

Answer 32

1. facilitate 3’ → 5’ degradation by exosome
2. Exosome can chew up all the way to the cap
3. mRNA can’t forms loops with the PABPC interacting with the cap to protect from degradation

Question 33

What is Drosha?

Answer 33

an enzyme that digests primary miRNA → pre-miRNA

Exportin5 takes pre-miRNA → cytoplasm

Question 34

To what region of the target mRNA does miRNA usually bind?

Answer 34

To the 3’ UTR, binds imperfeclty
Binding → block translation or destabilize the deadenylation

Question 35

How is dsRNA involved in silencing mechanism?

Answer 35

by miRNA, siRNA
Also by nucleating a complex that involved several proteins that are involved in generating H3K8me3

Question 36

What is the effect of DNA methylation on chromatin states?

Answer 36

Histone code (sequences and placement of histone methylations) read by epigenic readers and then used to modify histones in proximity through mSin3 (inactivator)

HMT make sure histone code is kept from cell to cell