RNA-mediated gene regulation Flashcards
What is the central dogma ?
DNA-> Transcritpin -> mRNA/rRNA/tRNA-> ribosomes-> translation -> protein
What do you know about the human genome project ?
the human genome contains approximately 3 billion of these base pairs which reside in the 23 pairs of chromosomes within the nucleus of our cells.
- less than 2 % of the human genome encodes exonic mRNA that encodes proteins
- 25% of the human genome encodes introns
- it is estimated that 98% of the genome is transcribed into RNA
There are two types of non coding RNA?
- long non-coding RNA : >200bp
- short non coding RNA : siRNA , microRNA
What do we know about lond non-coding RNA?
- > 200bp
- have defined promoteers and chromatin marks as protein coding genes
- produced in many locations and can be sense or antisense to the coding genes
How do long non- coding RNA mediate gene regulation?
- since these are difficult to detect they are difficult to investigate
- they can function both in Cis ( at site of transcription) or in trans (away from transcription site )
IncRNAs - do not need to be translated or transported
and can bind to multiple proteins and act as a scafolld.
localised control of gene expression
a transcription of non- coding regions can lead to the recruitment of Chromatin mpdofoers to the area resulting in activation or repression of protein coding genes.
How do long non- coding RNA’s mediate gene regulation? -using the modulation of nuclear domains and sequestration
A-LncRNA can regulate paraspeckles ( can bind mRNAs and stabalise them!)
B-LncRNAs can regulate the polycomb body
C-LncRNAs can regulate splicing subnuclear domains
D-LncRNAs can regulate sequestrations of proteins in the nucleolus
LncRNAs can interact with chromatin and recruit the polycomb repressive complex - PRC2 to deposit H3K27me3 marks which is associated with transcriptional silencing
What is short non-coding RNA? and give some examples?
- RNAi ( RNA interference)is a sequence specific response to double stranded RNA charachterised by small RNAs
- siRNA , microRNA , piRNA
Why does the cell have siRNAs?
- double stranded RNA is a sign of danger to the cell
- produced during viral replication and at sites of inverted sequences
- RNAi is thought to be an ancient defence mechanism against virus and transposable elements !
How does siRNA work?
- the mechanism is highly conserved
- dicer proteins have typically two RNAse 3 domains and a PAZ domain
- the PAZ domain binds the end of the dsRNA
- the distance between the PAZ domain and the RNAse 3 domains determines the size of the product , working as a molecular ruler
- dicer products range between 20-25 nt long
- siRNA guide strand is bound by argonaute protein
- Argonaute proteins slice target transcripts
- Argonaute proteins have PAZ and PIWI domains
- Argonaute proteins form a family and not all habe slicder activity
How do microRNA work?
- the precursor is a single transcript that forms an imperfect hairpin structure.
Why is miRNA important?
it is important in development , controlling timing and organs
- it is important for housekeeping gene regulation
- important for apoptosis
- important for tumourogenesis , being both tumour supressor , oncogenes and disease specific signatures
- virus can encode miRNAs that change host gene expression.
What are piRNAs?
PIWI - interacting RNAs (piRNAS) are small non- coding RNAs that form the PiRISC complex (piRNA - induced silencing complex)
- they are not very well known
- piRNA physically associate with PIWI proteins and not AGO proteins
- piRNAs function to silence transposable elements in the germ line
What are riboswitches?
they are structured non- coding RNA domains that bind metabolites selectively and control gene expression
- nearly all riboswitches reside in non- coding regions of mRNAs
- derived from an ancient sensory and regulatory mechanism
- riboswitches work in Cis ( at the site of transcription)
What is CRISPR technology?
Clustered Regularly Interspaced Short Pallindromic Repeats ( CRISPR)
- derived from prokaryotic adaptiave immune system - offering bacteria and archaea protection from virus and conjugative plasmids
- has been used to delete, add, activate or suppress targetted genes in human cells , mice , rat etc
- quick , effective and mostly inexpensive way of producing KO or KI
How does CRISPR technology work?
- single guide RNA - consistent of a sequence complimentary to its target at its 5’ end and a cas9-recognisable structure at its 3’ end
- the target also needs a short sequence just outside the DNA- RNA hybridisation called the protospacer adjacent motif (PAM)
- most researchers have used the Cas9 protein that prefers NGG as PAM
