Describe Crick’s central dogma with regards to the flow of genetic information:
Formed in 1958 by Crick
DNA –(transcription)–> mRNA –(translation)–>protein –(post-translational modifications)–> FUNCTIONAL PROTEIN
- transcription is carried out by RNA polymerase to make mRNA from a single strand of DNA
- mRNA is carried to the ribosome where tRNA brings over the appropriate amino acid by reading the mRNA in ‘codons’ (groups of 3 bases)
- there are 60 codons which all relate to ~20 amino acids
- chains of amino acids make up proteins
- proteins can undergo PMT to alter their function
- there are ~26, 000 genes, which produce ~300, 000 proteins
Describe transcription in detail:
- dsDNA –> ssDNA
- RNA polymerase uses ssDNA to make a strand of mRNA
- mRNA has uracil instead of thymine
- mRNA has a poly-A tail which allows it to be identified
- mRNA leaves the nucleus and enters the cytoplasm where it binds to ribosome
Describe translation in detail:
- ribosomes read the mRNA in groups of 3 bases called codons
- There are ~60 codons, which specify 1 out of 20 possible amino acids
- transferRNA molecules carry the appropriate amino acids to the ribosome and attach the correct amino acid to its corresponding codon
Why is the central dogma not true and what are its variations?
1) Alternate splicing: when mRNA is produced from DNA some EXONS can be spliced out, meaning different proteins can be coded for from the same strand of DNA (introns are always non-coding and so are spliced out)
2) MicroRNA’s: these are small non-coding molecules of RNA which bind to mRNA and either stop its translation or cause it to be degraded. MicroRNA recognise mRNA from its poly-AAA tail and bind to it this way.
3) Epigenetics: alter gene transcription so can cause up/downregulation of protein production
4) Post-translational modifications: methylation, ubiquitination, phosphorylation etc. mean that from 26, 000 genes we get ~300, 000 proteins! Often the reason why proteins fail to be identified on mass spectrometry
What techniques can be used in the lab to analyse RNA?
Northern blots
Reverse transcriptase PCR
Microarrays
In-situ hybridisation (ISH)
Describe Northern Blots:
- mRNA molecules separated out my size using electrophoresis
- molecules are blotted from electrophoresis gel onto a cellulose membrane
- cellulose membrane is exposed to probes which bind to complementary mRNA’s of interest
- probes are radioactively labelled -> so that when X-rays are applied they can be visualised
- the intensity of the bands produced indicates the level of mRNA present
Describe Reverse Transcriptase PCR:
Good for amplifying low levels of RNA and then analysing it
RNA is much more stable
Allows you to select mRNA molecules that you are only interested in rather than the whole genome of DNA
- use RNA as template for PCR instead of DNA
- incubate RNA with RT (reverse transcriptase) enzyme at 50oC
- This creates a strand of cDNA (complementary DNA)
- the cDNA and RNA are then split at 80oC
- cDNA then incubated with DNA polymerase at 72oC to make it into dsDNA and it is ready for PCR/cloning
Usually use 96 well plates and takes ~2hrs from start to finish
Describe RNA Microarrays:
- A tray with lots of wells containing mRNA is used
- Fragmented genetic material from a cancer cell and also a normal cell are added to the array tray
- if complementary mRNA to the contents of either cell type is present in the wells, they will bind and fluorescence will be released fluorescence detected by computer
- E.g. more red than green on the array tray means that the initial mRNA used in the wells is found more in cancer cells than normal cells
This process generates a SNAPSHOT OF GENE EXPRESSION levels at any one time and what mRNA’s are expressed by cells in a particular state
PAPER: Bullinger et al 2004 - used microarrays to create prognostically relevant sub groups of AML (formulated molecular subtypes based on gene expression) as currently AML is poorly classified disease
Describe micro-RNA’s and a use for them:
- small non-coding RNA molecules which have been evolutionarily conserved
- they are a RECENT discovery and give us insight into human variation in disease
PAPER: Vila-Navarro et al 2017 - using microRNA’s for the detection of pancreatic neoplasia
What are cDNA libraries and what can they be used for?
- cDNA is produced from fully transcribed mRNA it gives a SNAPSHOT of the genes that are expressed at one point in time in an organism
Process: mRNA collected -> undergoes reverse transcriptase to make into cDNA -> cDNA inserted into plasmids -> plasmids inserted into bacteria which grow and the cDNA is replicated -> plasmids then isolated and DNA purified
- tissue specific cDNA libraries can be produced
- cDNA is much shorter than the full DNA sequence so easier to work with and libraries are less resource-intensive to work with than having to screen a DNA library
Describe the biogenesis of micro-RNA:
1) Pri-miRNA is transcribed by RNA polymerase II from parent gene in the nucleus
2) DROSHA cleaves the pri-miRNA into pre-miRNA so that it is small enough to leave the nucleus
3) EXPORTIN transports the pre-miRNA out the nucleus and into the cytoplasm
4) DICER then cleaves the pre-miRNA into miRNA
5) miRNA associated with a RISC complex (RNA induced silencing complex) which guides the miRNA to specific gene
6) miRNA binds to mRNA (recognised by its poly-A tail) and either:
A) stops the mRNA being translated
b) degrades the mRNA
Gao et al 2018 showed that the miRNA 24 is pathologically elevated and causes cardiac hypertrophy and heart failure by reducing excitation-contraction coupling (therefore targeting miRNAs could be therapeutic)
Vila-Navarro et al 2017 -> showed that the miRNA landscape of PDAC and IPMN differ! (could be used as diagnostic biomarker)
What are the advantages of working with miRNA?
- miRNAs can regulate multiple components of the same pathway (therefore targeting one miRNA can be very effective)
- miRNA’s are long lived molecules with sustained effects
What are the disadvantages of working with miRNA?
- can have off-target/unintended effects
- a single miRNA can have beneficial AND toxic side effects
Describe ISH:
A labelled probe detects an mRNA target in situ (cells/tissue) allowing quantity and location to be identified
Describe key features of RNA and considerations to be thought of when working with it:
Features:
- single stranded (therefore not that stable)
- has no introns (these are spliced out)
- has a poly-AAA tail
- has uracil (not thymine)
Working considerations:
- work on ice
- good pipetting skills (small volumes)
- treat all equipment/surfaces with RNase ZAP (to remove any RNase enzymes which may degrade the RNA)
Key features of working with RNA:
- stabilise
- isolate
- quality check
- quantify
Why would we choose to use cDNA over RNA
- it is double stranded so can undergo further experimentation (PCR, plasmids, cloning)
- it is more stable
- it preserves the RNA sequence
- you can select certain mRNA molecules which you are interested in
