Test Flashcards
1
Q
What is a ribosome
A
- consist of rRNA core decorated with ribosomal proteins
- have a small and large subunit
- catalyse protein translation
2
Q
Ribosome biogenesis
A
- involves synthesis and assembly of rRNA and ribosomal proteins
- includes all 3 eukaryote RNA polymerase
- 2/3rd of total cellular energy budget is spent on ribosome biogenesis and translation
3
Q
Nucleolus
A
- sub-nuclear body
- DNA spare and protein dense
- site of rRNA transcription and ribosome pre-biogenesis
- contains rDNA
4
Q
What limits the number of ribosomes in a cell
A
rRNA transcription
5
Q
What is a limiting factor for protein translation
A
Ribosome number
6
Q
rDNA composition
A
- always present as repeats in eukaryotes
- a single rDNA repeat unit contains 18s, 5.8s and 28s rRNA subunits (transcribed via RNA Pol I)
- organised into arrays of head to tail tandem repeats
7
Q
3 levels of rRNA transcriptional regulation
A
- whole repeat array is activated or silenced
- individual repeats are activated or silenced
- transcriptional rate of active repeats modified
8
Q
How do different differentiated cells differ
A
epigenetically - each cell type has a unique profile of genes which are epigenetically turned off/on that determines its phenotype
9
Q
difference in gene expression amount in stem cells vs differentiated cells
A
stem cells have a greater amount of genes being expressed
10
Q
Genome organisation
A
- organisation may help determine gene expression profiles
- topologically associated domains = contain more than one gene with all genes having the same state (active or inactive)
- become more organised as they differentiate
11
Q
Yamanaka factors
A
- Oct3/4
- Sox2
- Klf4
- c-Myc
12
Q
The ribosome biogenesis paradox
A
- expected that reduced rRNA transcription would result in fewer ribosomes hence a reduced protein translation but this is not always observed
- differentiated cells have lower rRNA transcription but increased protein translation
13
Q
Hypothesis for paradox
A
- translation becomes more efficient in differentiated cells
- number of functional ribosomes in differentiated cells is increased despite ribosome biogenesis decreasing
- non-ribosomal translation
- paradox is an artefact
- translation-independent function for ribosomes in stem cells
- ribosome factory cells transferring ribosomes to other cells
14
Q
Translation becomes more efficient - supporting evidence
A
- Differentiated cells had extensive translatome remodelling and post transcriptional modifications (Baser et al., 2019) = modifications may be making it easier for ribosomes to translate leading to an increased efficiency
- Baser et al (2019) observed an increase in translational efficiency in neurones compared to NSCs
- Drosophila GSCs undergo splice changes when differentiating which alter the upregulation or downregulation of mRNA transcripts to be specific making translation more efficient (Samuels et al., 2024) - modifications to rRNA can make it more efficient such as the addition of a methyl group
- Differentiated cells become more organised possibly making translation more efficient
- Sabatier et al 2016 = higher amount fo stable ribosomes in differentiated = more efficient
15
Q
Translation becomes more efficient - opposing evidence
A
- Not all differentiated cells have a lower translation rate with decreased biogenesis, fetal HSCs have a higher protein synthesis than adult HSCs but the rate declines during differentiation (Magee & Signer, 2021)
- Sanchez et al (2016) showed that an increase in rRNA is correlated with an increase in translation rate
16
Q
Translation becomes more efficient - testing for hypothesis
A
- Ribosome sequencing = compare the mRNA covered by ribosomes during translation and compare to the total mRNA level
17
Q
Number of functional ribosomes in cell is increased - supporting evidence
A
- As ribosome biogenesis is extremely costly maybe stem cells are stock piling ribosomes prior to differentiation to save costs
- “priming differentiated cells with an abundance of ribosomes to allow for a sustained translation rate” - Ingolia et al., 2021
- ESCs have a high number of inactive ribosomes, though to be essential for priming gene expression for differentiation programmes (Gabut et al., 2020)
18
Q
Hayashi et al. 2014
A
- the inhibition of rRNA transcription via ActD or TF-IA KO in leukemia cells led to decreased rRNA levels
- decreased rRNA levels were accompanied by increased differentiation (detected via increased cell surface marker) and nucleolar shrinkage
- differentiation is independent of cell cycle as treatment with a CDK inhibitor did not see an increase in differentiation
- nucleolar size decrease could be due to movement of proteins out of the nucleolus into the nucleoplasm during differentiation
19
Q
Woolnough et al. 2016
A
- decreased rRNA transcription via via activin A or pol I inhibition leads to the exit of hESCs from pluripotency causing differentiation
- GRO-seq was used to measure rRNA levels
- Pol I inhibitor led to the pluripotency genes downregulation and cellular differentiated gene upregulation
20
Q
Signet et al. 2014
A
- HSCs have low protein synthesis with Rpl24+/- mutants impairing the ribosomal subunit
- Restricted progenitors had lowers OP-Puro incorporation thus less protein synthesis
21
Q
Sabatier et al. 2021
A
- iPSCs have high 60s but low 80s = large amount of immature non-functional ribosomes
- decreased polysomes in iPSCs = decreased translation
- SBDS involved in ribosome maturation
- SBDS is upregulated in differentiated cells while RiBi proteins are downregulated = decrease in the production of immature ribosomes and increase in maturation
22
Q
Stedman et al. 2015
A
- Notchless KO reduces ribosome biogenesis caused differentiation of intestinal stem cells
- Impaired biogenesis triggers differentiation even in the absence of p53 > shows importance of ribosome
23
Q
Jarzebowski et al. 2018
A
- HSCs have high pre-rRNA levels despite low protein synthesis = supports paradox
- Terminally differentiated cells showed decreased biogenesis
- used flow fish technique to quantify pre-rRNA levels
24
Q
Zismanov et al. 2016
A
- Satellite cells are quiescent for long periods and activate myogenic differentiation only in response to damage
- disruption of Eif2a phosphorylation causes exit from quiescence
- loss of pluripotency with eif2a phosphorylation inhibition (Amiri et al. 2024)
- P-Eif2a leads to global arrest of translation
- protective mechanism
25
Magee and signer 2021
- Protein synthesis rates decrease during HSC development
- both high and low rates of protein synthesis impair HSC self-renewal
- Fetal HSCs have higher protein synthesis than adult HSCs
but the rate declines during differentiation
- rate is tightly controlled throughout differentiation and development
26
Baser et al. 2019
- quiescent stem cells maintain low protein synthesis which increases during differentiation
- mTORC1 regulates translation, blocking pluripotency factors during differentiation
- Differentiated cells had extensive translatome remodelling and post transcriptional modifications = modifications may be making it easier for ribosomes to translate leading to an increased efficiency
- observed an increase in translational efficiency in neurones compared to NSCs
- used ribo sequencing
27
Samuels et al. 2024
- Drosophila GSCs have high ribosome biogenesis which decreases during differentiation
- translation levels increase during differentiation
- extensive translatome remodelling
- Indicates that reduced ribosome biogenesis promotes differentiation along with translation becoming more active.
- splicing changes which alter what genes are upregulated or downregulated
- Bru1 and Rbp9 upregulated during differentiation and are involved in splicing changes
- some genes are expressed but not translated during early stages of differentiation = storing mRNA for later = increased translation in differentiated cells
28
Gabut et al 2020
- observed that ESCs have increased levels of inactive ribosomes
- may be important for priming differentiated
29
Sampath et al 2008
- Used translational state array analysis to measure transcript abundance and ribosomal loading during ESC differentiation
- differentiated ESCs increased content of golgi and RER which is consistent with increased protein synthesis
- used spectro to measure polysome content
- increased absorbance in diff = increasec content = increased protein synthesis
- increased ribosomal loading in diff = increased efficiency
