Transcriptome Flashcards
Define: transcriptome
Sum total of RNA present in the cell
Why is defining the transcriptome important from an academic and a translational viewpoint?
Academic: knowledge of all active genes and how they are regulated
Translational: disease progression - transcription perturbed
How many protein coding genes in the human genome?
19,716 (around 20,000)
How many annotated transcripts are there in TOTAL in the genome?
150,352 (around 150,000)
How many transcripts are produced for each protein-coding gene?
7.6 (between 7 and 8)
Of active genes, what percentage are ‘house-keeping’, and what percentage are tissue-specific?
70% are house-keeping
30% are tissue-specific
Of the 20,000 protein coding genes, how many are ON and how many are OFF in active in any given cell type?
Half: so 10,000 are on, 10,000 are off
Name 5 experimental techniques that can be used to determine which genes in a cell are active
1) Northern blot
2) RT-PCR
3) qRT-PCR
4) Microarray
5) RNA-seq
Give one disadvantage of using mRNA to monitor gene expression
It is easily degraded
Give two advantages of using mRNA to monitor gene expression
1) can be probed with high specificity due to base-pairing of nucleic acids - HYBRIDISATION
2) many transcripts can be probed simultaneously from the same sample (e.g. Microarray)
Give 4 advantages of the Northern Blot technique for monitoring gene expression
1) definitive way to measure mRNA levels
2) can measure SIZE of transcript and compare to ORF
3) can detect multiple RNA species - alternative splicing
4) cheap
Why is Northern Blotting now essentially ‘phased out’?
It is very labour intensive - would need 47,000 to measure the transcriptome of a particular cell type
Which technique has now essentially replaced Northern blotting?
Reverse transcriptase PCR (RT-PCR)
What are the two stages seen in a graph of PCR (Fluorescence against Cycle)?
Exponential phase
Plateau phase
What is qRT-PCR?
Quantitative RT-PCR
What does qRT-PCR involve?
1) short nucleotide probe with Reporter-Quencher bound to the cDNA
2) reporter cleaved from quencher by nuclease activity of Taq polymerase
3) PCR machine measures fluorescence intensity EVERY cycle
1st Generation Microarrays
?
In 2nd Generation Microarrays, how many samples can be tested per chip?
12
In 2nd Gen Microarrays, how many transcripts can be measured?
> 47,000
What are 2 disadvantages of 2nd Generation Microarrays?
1) Expensive - £600 per chip
2) Lots of data analysis
What are the 5 main steps in RNA-seq?
1) long RNAs covered into cDNA library through either RNA fragmentation or DNA fragmentation
2) sequencing adapters are added to each cDNA fragment
3) short sequence obtained from each cDNA using HTS technology
4) sequence reads aligned with reference genome/transcriptome and classified as three types: 1) exonic reads, 2) junction reads, 3) poly(A) end-reads
5) reads used to generate a base-resolution expression profile for each gene
What are the ‘spots’ on a 1st gen microarray?
Spots = oligo (60 nts)
What is cRNA?
Complement of cDNA
Which three main breakthroughs have been made using RNA-seq?
1) divergent transcription
2) polymerase pausing
3) enhancer transcription
What is an advantage of RNA-seq over microarrays?
RNA-seq doesn’t involve using a chip - you sequence everything and anything
What technique would you use to determine whether Gene A is downregulated by treatment B?
RT-PCR (single gene analysis, which has replaced Northern Blotting)
How would you determine which genes are overexpressed in cells?
Screening - 1000s of transcripts in dozens of samples
Microarray or RNA seq
What underlies cancer cells?
Changes in gene expression
How can gene expression relate to cancer?
It can enhance or repress the hallmark capabilities
In normal cells, 70% of the active genes are house-keeping genes and 30% are tissue-specific. What happens in cancer cells?
There is a lack of differentiation (ANAPLASIA) - two different tissue-specific cells will becomes more similar as the cells ‘forget’ cellular identity
What is EZH2?
Enhancer of Zeste Homologue 2
What is the clinical significance of EZH2?
It is over-expressed in metastatic prostate cancer and in B-cell lymphoma
How can the overexpression of EZH2 in metastatic cells be proven?
Microarray (RNA)
RT-PCR (RNA)
Western Blot (protein) (compare against housekeeping genes e.g. Beta-tubulin)
What type of protein is EZH2?
Histone methyltransferase - methylates H3K27me3, a REPRESSIVE mark. Part of the polycomb complex, PRC2
How does methylation of H3K27 affect transcription?
H3K27me3 is repressive
Which complex is EZH2 a part of?
PRC2
Which 3 proteins make up the core of the PRC2 complex?
EED, SUZ12 and EZH2
How can EZH2 affect cancer progression?
Its overexpression can promote tumorigenesis via epigenetic silencing of tumour suppression genes (e.g. ANXA6 in gastric cancer)
What is anaplasia?
Change in transcriptional profile seen in cancer cells that results in a loss of a cell’s identity
Give an example of a tumour suppression gene that is epigenetically silenced by EZH2
ANXA6 in gastric cancer
How may EZH2 be targeted to treat cancer?
Its inhibition may be therapeutic strategy for lymphoma with EZH2-activating mutations
What does EZH2 do?
It is a histone methyltransferase. It adds three methyl groups to Histone 3 Lysine 27 (H3K27) to turn off transcription
Why does EZH2 make a good drug target?
It is an enzyme so its active site can be targeted (the SET domain of its active site)
What is the function of EZH2?
It is the catalytic subunit of PRC2.
It transfers methyl groups from the co-factor SAM to H3K37.
Has also been found to methylate non-histone proteins.
Why can overexpression of EZH2 be used as a diagnostic marker of cancer?
Because it is typically not expressed in healthy adult cells: it is only found in actively dividing cells
Give some examples of drugs that inhibit EZH2?
GSK126, DZNep
Which particular domain of EZH2 has been targeted for therapeutic treatment?
The SET domain active site
