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BIS477 World of RNA > Ribosome profiling > Flashcards

Ribosome profiling Flashcards

1
Q

Why doesn’t RNA/protein levels within a cell tell you what is being actively translated?

A

Analysis of total RNA/total protein present in a cell is performed under steady state conditions so doesn’t tell you which proteins are currently being actively translated

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2
Q

Why doesn’t mRNA/protein level necessarily correlate with amount of currently translated product? (4)

A
  • Low abundance mRNAs can be highly translated
  • High abundance mRNAs can be poorly translated
  • Stable proteins with slow turnover rate might not be being actively translated
  • Annotated ORFs aren’t the only RNA species being translated e.g. uORFs and smORFS
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3
Q

What are smORFs? (2)

A
  • Small ORFs
  • uORFs are a type of smORFs
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4
Q

What are polysomes?

A

mRNAs being translated have multiple ribosomes associated which form polysome structures

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5
Q

What is ribosome profiling? (2)

A
  • In vivo approach to determine which RNAs are actively being translated
  • Can isolate the polysomes, digest the RNA and sequence the regions that are protected by the ribosome to see exactly what is being translated
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6
Q

What are the steps of ribosome profiling? (8)

A
  • Prepare any type of cell culture
  • Rapidly inhibit translation to freeze ribosomes in position via antibiotics/flash freezing
  • Prepare cell extract and isolate polysomes by centrifugation (mRNA with bound ribosomes)
  • RNase treatment digests all RNA not protected by ribosomes, protected fragments are 28-32nt (organism dependent) consistently sized
  • Isolate ribosomes and associated RNA fragments by density gradient centrifugation
  • Library preparation of the isolated RNA fragments by adding linkers and RT and PCR similar to CRAC or iCLIP
  • High throughput sequencing and alignment of the results
  • Do RNAseq in parallel
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7
Q

What antibiotic is commonly used to inhibit translation? (2)

A
  • Cycloheximide
  • Inhibitor of translation elongation
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8
Q

Why do you do RNAseq in parallel to ribosome profiling?

A

Need to know the total amount of mRNA in the cell because the amount of translation from ribosome profiling is relative to total mRNA present

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9
Q

What are the strengths of ribosome profiling? (5)

A
  • No genetic manipulation of cells needed
  • Quantifiable
  • Sensitive (rare translation events can be identified)
  • Precise positional information (footprint size)
  • Detects changes in protein expression directly (in contrast to analysis of steady state RNA/protein levels)
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10
Q

What are the limitations of ribosome profiling? (5)

A
  • Difficult to efficiently freeze ribosomes in place during sampling
  • Artefacts arise from antibiotics as each antibiotic only inhibits specific steps in translation e.g. cycloheximide only stops elongating ribosomes so no view of translational start sites
  • Short length of fragments can lead to ambiguity in identification of similar sequences when mapping
  • The assumption that each ribosome associated with the RNA leads to the production of the protein
  • The assumption that elongation rate is the same globally
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11
Q

What are the features of ribosome profiling data? (4)

A
  • Ribosome footprint reads which can be mapped to the transcriptome
  • Very sharp boundaries at the 5’ and 3’ ends indicating start and stop sites
  • Should see sequence coverage over the entire ORF
  • Evidence of codon periodicity (more frequent reads at every 3rd nucleotide) which is indicative of active translation and can infer frame of translation from this pattern (required for quality control of the data)
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12
Q

Why is codon periodicity evident from RNA profiling? (3)

A
  • Ribosome reads in triplets not base by base
  • RNase treatment generates RNA footprints of the same size (28-32nt depending on organism)
  • Can map reads to single point in ribosome (e.g. P site) and if you map all reads to the same point, every 3rd nucleotide will map more often to this point
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13
Q

What are the E, P and A sites?

A

3 binding sites for tRNA on the ribosome

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14
Q

What are the 3 main types of data you get from ribosome profiling?

A
  • How is the ribosome translating
  • How much translating is taking place
  • What RNAs are being translated
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15
Q

What information can you get about how the ribosome is translating? (2)

A
  • Can identify defects e.g. pausing, stop codon read through
  • A peak in the sequence reads indicates ribosome pausing which may be caused by a structural element impeding elongation
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16
Q

What was discovered about Dom34 from ribosome profiling? (5)

A
  • Dom34 is a factor that rescues stalled ribosomes in cytoplasmic surveillance pathways
  • Did ribosome profiling across all genes of a wild-type cells and Dom34 deletion cells
  • Deletion of Dom34 showed peak of ribosome footprints in the 3’ UTR of a subset of genes
  • The 3’ UTR footprints lacked codon periodicity which indicates not being actively translated
  • These footprints mean that the ribosome is there but not translating but also not being released or recycled following termination showing that Dom34 is involved in removal of the ribosome for the surveillance pathway
17
Q

What information can you get about how much translation is taking place? (3)

A
  • mRNA levels don’t necessarily correlate with amount of translation and protein produced
  • The density of hits provides evidence of how much translation of a given RNA is taking place
  • Compare amount of translation with the levels of mRNA in the cell to calculate translation efficiency for each mRNA
18
Q

What was discovered about the E.coli FoF1-ATP synthase operon from ribosome profiling? (3)

A
  • E.coli FoF1-ATP synthase operon is transcribed together so levels of mRNA should be the same for each component
  • Ribosome footprint shows that ribosome occupancy is not equal across each component so the synthesis rate of some components is greater than others
  • Good correlation between synthesis rates and stoichiometry of the protein components in the complex
19
Q

What is an operon?

A

Group of genes that are controlled by a single promoter and transcribed into a single mRNA transcript in prokaryotes

20
Q

What is a polycistronic transcript?

A

mRNA transcript containing multiple genes

21
Q

What is an example of information about what RNAs are being translated from ribosome profiling? (2)

A
  • The position of the hits provides information about what RNA is being translated
  • Translation not just of protein coding RNA (mRNA) but also smORFs including uORFs
22
Q

Why is it difficult to identify and characterise smORFS? (3)

A
  • Loads of short putative ORFs can be identified in the genome (10-100 codons) but challenging to validate experimentally
  • Very small sequences so might only make a protein 10-15 amino acids long but computational methods of predicting protein coding ORFs requires 100 amino acids
  • Conservation and homology are difficult to determine for short sequences which are used to predict protein coding capacity
23
Q

How are new areas of translation identified?

A

Ribosome profiling has identified many ribosome footprints outside of canonical coding regions that coincide with predicted smORFS

24
Q

What kinds of smORFs have been identified from ribosome profiling? (6)

A
  • uORFS: purely 5’ UTR or 5’ UTR with exonic overlap
  • Exonic
  • Exonic alternatively spliced
  • Intronic: purely intronic or intronic with exonic overlap
  • 3’ UTR
  • Intergenic smoRF located between 2 genes
25
Q

What is the criteria for identification of a smORF from ribosome profiling data? (4)

A
  • Sharp borders outside of start and stop sites
  • Codon periodicity
  • Sensitive to inhibitors of translation (same as for normal ORFs)
  • Encoded peptides from some smORFS identified by ribosome profiling have been shown experimentally to have biological function but the majority await identification
26
Q

What is the main limitation of ribosome profiling in terms of identifying novel open reading frames?

A

Difficulty in identifying uORFs and smORFs which overlap with canonical ORFs because don’t get info about start sites when using cycloheximide for ribosome profiling

27
Q

What is GTI-seq? (5)

A
  • Global translation initiation sequencing
  • Variation of ribosome profiling which identifies global translation start sites
  • Involves a drug which inhibits the transition between initiation and elongation
  • All other ribosomes complete their translation cycle so only ribosomes at the start site are left
  • Can identify uORFs and smORFs by comparing ribosome profiling and GTI-seq data
28
Q

Which drugs are used for GTI-seq? (2)

A
  • Lactimidomycin
  • Harringtonine
29
Q

What was surprising from looking at GTI-seq data? (2)

A
  • Was thought that all translation started at AUG codon but the majority of global translation starts at near cognate start sites e.g. CUG, GUG etc.
  • Initiation seems to be more flexible and low-fidelity than initially believed
30
Q

What is the function of smORF translation? (2)

A
  • A few smORF encoded peptides have been shown to have biological function
  • Majority await identification
31
Q

What is the function of uORF translation? (2)

A
  • The process of translation of the uORF rather than the resultant peptide itself appear to mediate the regulatory effects on the downstream coding sequence
  • No indication that peptides from uORFs act in trans or that they are stable (thought to be regulatory rather than protein encoding)

BIS477 World of RNA (14 decks)

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