Post-translation Flashcards
How is the pioneering round of translation of an mRNA transcript different than the other one?
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)
Which RNA pol is responsible for synthesis of snRNA? tRNA? of 5S rRNA?
sn RNA = RNA pol II
tRNA = RNA pol III
5S rRNA (found in large subunit of Ribosome) = RNA pol III
What are the roles of SR proteins?
Serine-Arginine rich proteins
Bind exons on the pre-mRNA so introns are properly spliced, by binding to specific sequences of the RNA
What are different mechanisms of RNA surveillance and Quality control for translation of proper proteins?
- SR proteins define the exons so exons properly excised
- Polyadenylation of the pre-mRNA
- Export factors loaded onto the mRNA for it to get out of the nucleus
- NMD (nonsense mediated decay)
What is an important mechanism of quality control of the cell to prevent having truncated/ incomplete proteins?
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)
Is the stability of cytoplasmic mRNA similar across all organisms?
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
What is special the AUUUA sequence?
It is found as a repeat in many short-life mRNAs in eukaryotes in 3’ UTR → destabilizes mRNA
How/Where(on the mRNA) does RNA decay occur?
- 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
What is the structure of the exosome?
Responsible of the 3’ → 5’ decay of mRNA
- Regulatory lid → RNA helicase
- ATP base
- Exo-9
- RRP44
*3 and 4 are 2 exonuclease activities in channel - Endonuclease activity are exit of the channel in case exonucleases activities failed
What are 2 general ways by which protein expression can be regulated by RNA changes depending on cell environment?
- 3’ UTR region regulation stabilizes/destabilizes RNA (more or less degradation)
- 5’ UTR region regulation blocks translation before the coding region
How is stability of mammalian transferrin receptor TfR regulated in response to intracellular iron concentration?
What is TfR’s role?
*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
What can we deduce from a Western Blot and a Norther Blot that do not correlate?
(related to translational regulation)
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
In which direction do ribosomes read RNA?
5’ (cap) → 3’ (poly(A) tail)
How is synthesis of Ferritin regulated inside the cell depending on mRNA levels?
- Ferritin mRN has IREs (Iron Response Element / stem loops) in its 5’ UTR (before the coding region)
- Active IRE-BP bind to IREs → block ribosomes from passing → ribosomes don’t get to the coding region → protein not synthesized
- Inactive IRE-BP doesn’t bind → ferritin gets synthesized(want in high iron)
What is ferritin?
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
How does inhibition of mRNA translation has a pivotal role in drosophila embryo development?
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
Which C. elegan mutant goes through the 1st stage of dev. (on 4 stages) over and over? How?
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
What are the sequential steps through which biogenesis of microRNA proceeds? (in the nucleus)
- MicroRNA transcribed mostly by RNA pol II → capped and processed
- primary-miRNA folds up → dsRNA hairpin
- dsRNA hairpin digested by Drosha enzyme → pre-miRNA
- Exportin5 takes pre-miRNA (without Drosha)→ cytoplasm via NPC
What are the sequential steps through which biogenesis of microRNA proceeds? (in the cytoplasm)
- Dicer cleaves dsRNA into 21-23nt fragments
- Fragment are bound by Argonaute protein in an RNA-indued silencing complex (RISC) → miRISC
- ATP hydrolysis → unwinding/helicase activity of miRISC
- use miRNA product as gude to target complementary cellular RNA
- Associate by Watson-Crick IMPERFECT base pairing in the 3’ UTR → Block translation of destabilize mRNA target through de-adenylation
What is a dicer?
- RNAse II like enzyme
- Acts as a dimer to cleave dsRNA → 21-23nt fragments
How important is microRNA for gene control?
We estimate 60% of the predicted coding genes in our genom may be under microRNA-mediated control → VERY IMPORTANT
What is the process of RNAi?
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
What are the differences and similarities between RNAi and microRNA?
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
What are examples of non-coding RNAs nucleate complexes that trigger chromosomal silencing?
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
