Eukaryotic Genomes (4-7) Flashcards
(93 cards)
1
Q
How much human DNA is non-coding?
A
More than 50%
→ 1.5% is exons - coding part
→ as genomes get larger, an increasing proportion of the DNA is non-coding
2
Q
What makes human genomes so large?
A
- Gene duplication → families of genes and pseudogenes with often coordinated regulation
- Large introns → often containing retrotransposons
- Transposons → ability to change position in genome - LINES, SINES, retroviruses, retrotransposons
- Repetitive DNA → simple sequence repeats, segmental duplications
- Non-repetitive DNA
3
Q
What does gene duplication lead to?
A
Protein-coding genes have relative with which they share common ancestry
→ some exist in families and super-families
→ within a genome, families can be dispersed or clustered
→ maintenance of clusters implies functional co-ordination/regulation
→ expands the amount of DNA
4
Q
What is the globin gene family?
A
New genes formed by duplication during evolution
→ transcriptionally regulated gene cluster
→ duplicates can mutate individually - start getting divergent proteins
One ancestral globin → duplication → divergence → haemoglobin & myoglobin (different functions)
5
Q
How does haemoglobin gene expression change from foetal to adult?
A
Foetal → express alpha and gamma
Adult → changes to alpha and beta
Demonstrates one of the functions of expanding genome
→ gives the opportunity for developmental regulation - temporal transcription
6
Q
What are retrotransposons?
A
Type of genetic element found in genomes - capable of copying themselves into RNA then back to DNA which can be inserted at different locations within the genome
LINEs → long interspersed nuclear elements
SINEs → short interspersed nuclear elements
→ regulate gene expression by affecting chromatin structure, gene transcription and pre-mRNA processing
→ make up >40% of human genome
7
Q
What is the ‘beads on a string’ observation?
A
Chromatin first observed in 1974
→ visualised nucleosomes separated by linker DNA
→ with the removal of H1 by low salt conditions
8
Q
What is a nucleosome?
A
145-147 base pairs of DNA wrapped tightly ~1 2/3 turns around a globular protein complex
→ histone octamer: 2 copies of H2A, H2B, H3, H4
9
Q
What are the 5 major types of histone proteins
A
H2A, H2B, H3 and H4
H1 → linker histone
→ rich in +ve basic aa which interact with the -ve phosphate in DNA
10
Q
What did in vitro reconstruction of the nucleosome reveal?
A
H3 and H4 dimerise then form a tetramer
→ the stable tetramer wraps DNA in a soluble form leaving holes
→ enough space for H2A and H2B to dimerise and join nucleosome
→ N terminal tails protrude from the nucleosome
11
Q
How is DNA packed?
A
dsDNA
→ beads on a string
→ packed nucleosomes
→ extended nucleosomes
→ extended chromatin
→ compacted chromatin
→ metaphase chromosome
Each DNA molecule is packaged into a chromosomes 10,000-fold shorter than its extended length
12
Q
How can you transcribe DNA with the high levels of organisation and nucleosomes present?
A
Nucleosomes must be removed or remodelled to allow for transcription and replication
13
Q
What is DNA acetylation?
A
Reversible modification of histone tail lysine
→ +ve charge of lysine neutralised - proteins involved in transcription can access more easily
→ creates more open form of chromatin
14
Q
How does the cell choose which histone to be modified by acetylation to open the DNA for transcription?
A
Gene activator proteins bind chromatin remodelling complex
→ leads to remodelled nucleosomes, histone removal or histone replacement
Can also bind histone-modifying enzymes
→ read predominant chemical modification and copy it to other histones - spread of modification down the length of DNA
15
Q
What is DNA methylation?
A
1/2/3 methyl group added to aa
Specific lysine and arginine residues can be modified by methylation
→ can relax or compact chromatin depending on: which residue is methylated and the degree of methylation (context dependant)
16
Q
What are some other histone code modifications?
A
Phosphorylation → some serine and threonine resides - promotes transcription
Ubiquitylation → some lysine residues on H2A are recruitment sites for DNA repair, some are activating and on H2B are repressive
Citrullination → some arginine residues on H3 and H4
→ complex histone code determines gene expression - not fully understood
17
Q
How is the histone code maintained during transcription?
A
RNA Pol II stalls at nucleosome A
→ unwinds half of the DNA from nucleosome A
→ transcribed DNA is looped starts to wind around nucleosome A, while unwinding continues
Same nucleosome is transferred from in-front to behind the polymerase (from downstream to upstream as transcription proceeds)
18
Q
How is the pre-replicative complex formed?
A
The origins of replication are remarked with an ORC (origin recognition complex)
→ Cdc6 binds DNA at each side of ORC and recruits Cdt1
→ they recruit Mcm helices - opens strands of DNA
In G1 the pre-RC is formed but not active
19
Q
How is the pre-replicative complex activated in S phase?
A
Cdk2 phosphorylates Cdc6 → falls off and doesn’t recruit Cdt1
→ opens the origin for one round of replication
→ allows recruitment of primes clamp loader and DNA polymerases forming the active replication complex
20
Q
How is the histone code maintained during replication?
A
The replication machinery displaces H2A-H2B dimers
→ H3-H4 tetramers displaces and reused?
→ not clear but both new strands inherit them
Another set of histones is made to fill in the gaps
→ immediately acetylated
→ NAP-1 and GAF-1 chaperones guide histones into the available spaces
→ produces rebuilt histones - acetylated keeping structure ope
Acetylation is removed then histone-modifying enzyme/reader-writer complex copies epigenetic information
→ maintenance of histone code
→ transfer of epigenetic history
21
Q
What is epigenetics?
A
Heritable changes in gene expression that are not mediated at the DNA sequence level
Chromatin modifications
→ DNA methylation
→ histone modification
22
Q
How can SINEs regulated gene expression?
A
Non-epigenetic → act as enhancers or alternative promoters
→ recruit transcription factors and promote expression of nearby genes
Epigenetic → GC-rich so are hot spots for DNA methylation
→ can silence nearly genes stimulating chromatin condensation
23
Q
Why is transcriptional regulation needed?
A
Allows development of different tissues
Transition from childhood to adult
Deregulation can result in uncontrolled growth (cancers)
Allows reaction to environmental cues
24
Q
What 3 ways is transcription controlled?
A
- Chromatin structure
- RNA polymerase (and general TF) binding specificity
- Additional binding and activation factors
25
When is chromatin expressed/silenced?
Condensed chromatin → genes with highly packed heterochromatin are usually not expressed - silencing
De-condensed chromatin → expressed genes found in this 'open' formation
→ we can open/close chromatin shifting bias between expression and silencing
26
What is methylation of lysine at position 9 in the histone code associated with?
The formation of heterochromatin - gene silencing
→ H3K9me
→ type of heterochromatin depends on whether its tri- or uni- methylated
27
What is H327me associated with?
H3 → histone H3
K27me → lysine position 27 methylated
Associated with inactivation of HOX genes
→ developmental genes arrayed in the same order as the segments int he body - key role in organising body plan of embryo
28
What are some histone code modifications associated with gene expression?
H3S10p H3K14ac → gene expression
H3K4me H3K9ac → gene expression
→ relax DNA, open chromatin structure allowing for gene expression
29
What is the centromeric histone code modification?
H3K4me2 → double methylation of lysine at position 4
→ keeps structure open
→ important for centromere as must be open at all times
30
What is theorised about the scaffolding holding interphase chromosome in loops?
The scaffold is poorly understood but looped DNA assumed to be held by many scaffolding proteins
→ contains lots of topoisomerases for the winding and unwinding of DNA
Modification extends loops for high level of gene expression
31
Where is chromatin within the nucleus?
Heterochromatin → genes tuned off, close to the nuclear membrane associated iwht the lamina where its formed
Active regions → for genes that need to be turned on, the loop is opened up and relaxed
→ then moved to an area where all the requirements for transcription are concentrated
→ there is dynamic movements of genes within the nucleus
32
What are the two major types of heterochromatin?
Facultative heterochromatin → cell-type specific, can switch to euchromatin following developmental cues, characterised by a specific histone code mark
→ H3K27me3 that binds 'polycomb' proteins
Constitutive heterochromatin → regions that are consistently silenced in all cell type of an organism - centromeres, telomeres
→ H3K9me3 - carried out by histone methyltransferases (HMT)
33
How is the human centromere organised?
Centric chromatin flanked by peri centric chromatin
Centric chromatin → long highly repetitive chromatin structures
Double methylation of lysine 4 (H3K4me2) → gives centromere open structure allows for kinetochore attachment
→ important for function
34
What are telomeres?
Repetitive DNA sequence found at the end of each of our chromosomes
→ vary in length and repeated DNA sequence
→ vertebrate sequences at over several kb
→ yea several hundred bp
35
What is the end replication problem?
DNA is synthesised 5' to 3'
→ leading strand - continuous synthesis
→ lagging strand - Okazaki fragments
Gaps usually repaired by DNA ligase
→ but end gap on lagging strand can't be repaired because DNA synthesis required 3'-OH - there is no primer
→ means one of the strands made in shorter than the template
→ so telomeres get shorter over time and fray
36
What is the Hayflick limit?
The number of times a normal somatic, differentiated human cell population will divide before cell divisions stops
→ between 40 and 60 divisions
→ after the cell becomes senescent
→ in-built ageing mechanism
37
What is the compensatory mechanism for telomere shortening?
Telomerase binds the sing-stranded G-overhang
→ extends the 3' end of the parental strand using its own RNA subunit as a template (RNA directed DNA synthesis)
→ this provides more room for DNA primate to lay down a primer - which can be extended
(extends our telomeres, gives partial not full compensation for reduction in length of our telomeres)
38
Telomeres end up with a 3' unpaired end, why do they not fuse?
Exposed OH groups are reactive so the 3' end must hide somewhere
→ the 3' end hides from DNA repair mechanisms in sheltering complexes - to prevent fusion of telomeres
Shelterin complex
→ TRF 1 / 2 (telomeric repeat-binding factor 1 / 2)
→ RAP1 (repressor/activator protein)
and others
→ stimulates t-loop formation that displaces the d-loop - results in base paining at the 3' end
39
What is Werner syndrome?
Accelerated ageing caused by mutation in in the WRN gene
→ WRN helices protein important for DNA repair and telomeric DNA replication
→ telomeres that are usually normally replicated by lagging strand synthesis are not replicated efficiently in Werner cells
(inheritance: autosomal recessive)
40
What do human Pol II promoters typically look like?
Contain multiple *cis*-activing elements that bind proteins
BRE^u → upstream B recognition element - binds TFIIB
TATA box → binds TATA- binding protein (TBP)
BRE^d → downstream B recognition element - binds TFIIB
Inr → initiator element binds TFIID
Individual elements not always present → Pol II promoters are very variable
41
What is the basal transcription apparatus?
TFIID complex binds TATA box via TBP, aided by TFIIA
TBP recruits TFIIB which recognises BRE^u and BRE^d and accurately positions RNA Pol II at the start site of transcription
TFIIE, RNA Pol II?TFIIF are recruited
TFIIF → stabilised RNA Pol II interactions with TFIIE and TFIIH
TFIIH is recruited by TFIIE
→ won't move until further signals
42
How is elongation initiated?
TFIIH complex → helicase opens DNA double helix
→ kinase phosphorylates the C' - terminal domain of the RNA Pol II L' subunit
RNA polymerases don't require a primer - elongation starts
RNA Pol II now disengages from the cluster of general transcription factors - undergoes conformational change that tightens its interaction with DNA
→ phosphorylation of the CTD marks the transitions from initiation to elongation
43
How can polymerases binding to the TATA box regulate transcription?
Different TATA boxes are recognised with different affinities
→ some are more efficient at stimulating transcription can others
44
What is the G-less cassette transcription assay?
A promoter is cloned upstream of a G-less cassette (made from A, C, T bases only)
→ purified TES, RNA Pol II, ATP, CTP and [alpha^32P]-UTP added
Result → radioactive RNA transcript of a defined size
→ can be electrophoreses through polyacrylamide gels and quantified following autoradiography
No GTP is supplied the RNA is truncated at the point which a 'G' should be inserted
45
What are some additional binding factors that can control transcription?
Activator/repressor proteins
Mediator proteins
Chromatin-modifying proteins
46
What are homeodomains?
Type of DNA-binding domain that can help regulate gene expression
→ Helix 3 binds the major groove of DNA making specific interactions between aa and nucleotides
→ shape and DNA binding is conserved
47
What are zinc finger motifs?
A small structural motif with key Cys and His residues that coordinate a zinc ion stabilising the fold
→ 2 beta strands - 2 Sys residues which binds one zinc molecules
→ 2 alpha strands - 2 His residues that bind same zinc molecule
→ interacts with major groove of DNA
Key charged molecules Arg and His make specific contact with G residues - often cluster in DNA
→ GC rich areas prime binding sites for zinc finger motifs
→ demonstrates sequence specificity
48
What are leucine zippers?
Type of DNA-binding domain
→ cross-shaped binding to specific sequences
49
What is multimerisation and combinatorial control?
Combinatorial regulation is a powerful mechanism that enables tight control of gene expression
→ via integration of multiple signalling pathways that induce different transcription factors required for enhanceosome assembly
→ all 3 pathways activated - high levels of expression
→ depending on stresses cell is responding to
50
What is an enhanceosome?
Complex formed by the binding of multiple transcription factors to specific DNA sequences - enhancers
→ cooperative binding of different proteins to forma fully active enhanceosome
→ enhancer recruits high-mobility group (HMG) proteins which regulate chromatin structure - ensure the target gene can be accessed by the transcription factors
→ make the promote accessible
→ tight regulation of gene expression
51
What is the Philadelphia chromosome?
Gene abnormality found in some individuals with certain types of leukaemia
→ results from a gene translocation creating a fusion gene called BCR-ABL1 has abnormal kinase activity so is permanently switched on
→ leads to unregulated cell proliferation
52
What is Burkitt's lymphoma?
A type of non-Hodgkin lymphoma caused by inappropriate regulation of an oncogene by a very strong enhancer
53
What are some eukaryotic Pol II post-transcriptional events?
RNA Pol II transcription
→ possible attenuation (RNA transcript aborts)
→ 5' capping
→ splicing
→ 3' capping
if these fail leads to non-functional RNA sequences - degraded in the nucleus
→ possible RNA editing
→ nuclear export
not all events occur for each transcript
→ they require factors that bind the phosphorylated C-terminal domain (CTD) of RNA Pol II
54
What marks the transition from transcriptional initiation to elongation?
Phosphorylation of the seryls 2 and 5 on the CTD
→ acts as recruitment sites for enzymes that modify the transcript
→ the CTD of Pol II comprises multiple repeat regions of a 7aa sequence YSPTSPS
55
How is the 5' cap added to RNA transcripts?
RNA triphosphatase removes one phosphate from the nascent RNA transcript
→ leaves site for guanylyl transferase to cleave a diphosphate off a GTP and attach it to the remaining P pair
→ this is then modified by methyltransferases
→ forms an unusual 5'to5' triphosphate bridge
Result: cap put on backwards so 5' end can mascaraed as th 3' end → no longer vulnerable to degradation by 5' exonuclease
56
What are the capping enzymes?
RNA triphosphatase
Guanylyl transferase
Methyltransferases
→ recruited by the phosphorylated CTD of Pol II
→ 5' capping is co-ordinated with transcription
57
What is the purpose of a 5' cap?
Characterises Pol II transcripts from other RNA molecules → no other transcripts are modified like this
Stabilised the RNA → there is no 5' phosphate so its resistant to 5' exonuclease
Aids in further processing and export to the cytosol
Required for efficient translation of mRNAs
58
How are capped mRNAs regulated?
Decapping
→ 5' caps protect mRNAs from exonuclease activity
→ regulation depends on (in part) decaying pathways
Multi subunit cytosolic ATP-dependant decapping enzyme complex
→ removes the 5' cap
→ restores the 5' phosphate
→ venerable to 5' exonuclease
→ mRNA can no longer interact with ribosome - stops protein synthesis
Regulated decapping controls the half-life of a mRNA
59
How was it determined that RNA itself is catalytic?
Firstly an intron was identified, then it was found to be excised from the primary transcript
→ wanted to see what enzyme did this
Cech and his team
→ added phenol - denature protein
→ and boiled
intron still accumulated
→ allowed them to come to the conclusion that RNA could catalyse functions previously thought to be enzyme functions - intron described as a ribozyme
60
How was the splicing mechanism discovered?
Cech and his team used 'Tetrahymena' rRNA transcripts
61
How do human mRNA transcripts undergo splicing?
Use an inbuilt co-factor (branch site adenosine)
1. The RNA folds by base pairing → creates a complex structure placing the branch site hydroxyl in a position where it can attack the phosphate at the 5' splice junction
2. This adenosine now has 3 phosphodiester bonds → one is a 2', 5' phosphodiester bonds - circularises the intro into lariat structure
→ the 3' OH of the upstream exon (nucleophilic) attacks the phosphate at the 3' splice site
3. Exons are fused and the intron is removed as a lariat
62
What protein complex controls splicing?
By the spliceosome: an RNA and protein complex
→ 5 snRNPs (small nuclear ribonucleoproteins) U1, U2, U4, U5, U6 - small nuclear RNA complex with at least 7 protein subunits
Splicing must be precise so spliceosome recognises specific sequences at:
→ the 5' splice site
→ the branch site
→ the poly pyrimidine tract (poly Y)
Base-pairing specificity → allows assembly of spliceosome
Other proteins → stabilise, provide other functions
→ both allows precise splicing as splice sites and branch site are bought close together
63
How does the spliceosome work?
1. U1 snRNP binds the 5' splice site → base pairing provides specificity
2. Branch binding protein (BBP) and the protein U2 auxiliary factor (U2AF) bind the are that encodes branch site adenosine and associated pyrimidine tract
3. U2 is recruited → displaces BBP
4. A pre-formed trimer of U4, U5 and U6 associates
5. A molecular rearrangement (loss of U1 and U4) activates the complex → 5' splice site is exposed and can be attacked by the branch site adenosine
6. The 2'-OH attacks the 5' splice site phosphate
64
Which sequences are recognised by the spliceosome?
U1 only recognises 9 bases → only 3 of these are highly conserved
→ lots of different strengths of sequences identifying the 5' splice site
Splicing signals are consensus sequences → individual motifs have different affinities for spliceosome components
→ some are more efficient at stimulating splicing than others
65
How are the U1, U2, U4 AND U5, snRNPs made?
1. Transcription by Pol II, capped and shipped to cytosol
2. In cytosol 7 Sm proteins are loaded in a ring onto the snRNA by SMN protein
→ cap hypermethylated - signal to return to nucleus
3. Transported to nucleus and maturation into e.g. U1 snRNP by association with other proteins
(U6 lacks Sm proteins and remains nuclear)
SMN → Survival Motor Neuron
66
What is the 'exon definition' hypothesis?
Exons are defined by the binding of proteins during transcription → allows U1 to find the 5' splice site - how it recognises just a few bases
→ if you have a defines end of the first exon by the binding of SR protein as the U1 is scanning its only got to find a suitable site next to a SR protein
→ provides the added specificity that allows U1 to bind
Long exons have hnRNP to inhibit splicing
67
What is the role of the poly Y tract in alternative splicing?
A strong consensus poly Y tract → 3' end of intron strongly defined - strong defining of where U2 binds
→ normal splicing
A weak consensus poly Y tract → sometimes U2AF binds, BBP binds, U2 recruited, 3' end defined - normal splicing
→ sometimes U2AF disengages and U2 not bound, 3' end not defined - alternative splicing/exon skipping g
→ alternative splicing is regulated by the strength of the poly Y tract
68
How can alternative splicing be controlled in a cell-type specific manner?
U2AF is a family of proteins whose members have different abilities to find poly Y tracts
→ generates protein control which can be controlled developmentally
Different cells express different versions → example of a need for weak ineractions
e.g. mRNA in thyroid cells produces calcitonin but in neuronal cells produces calcitonin gene related peptide - one gene: more than one protein
69
What is haemophilia?
A blood clotting disorder caused by mis-splicing
→ mutation causes A to G transition in intron 3 of the F9 gene
→ creates a false 3'- splice site
→ leads to mis-splicing producing a defective protein
70
What is spinal muscular atrophy (SMA)?
During the biogenesis of snRNPs Sm proteins are loaded onto the snRNA by SMN (survival motor neuron) protein
→ there are 2 versions of SMN (1 does 90% and 2 does 10% of loading)
→ most of the SMN2 protein made is non-functional because the mRNA was mis-spliced
→ SMN deficiency caused by *SMN1* mutation leads to widespread splicing deficiency - especially in motor neurons
71
What happens at the 3' end of a pre-mRNA?
Addition of a poly (A) tail
72
How is the poly(A) tail added to pre-mRNA?
1. Recruitment of the cleavage factors → poly(A) site bound by CPSF, cleave site CF, G/U rich region CStF
2. Interactions between CStF and CPSF provide a recruitment site for the polymerase (PAP)
3. Conformational change exposes cleave site → RNA cleaved by CF
→ provides a 3'-OH for PAP
4. PAP adds adenine residues → slow adenylation until ~12 added
5. Poly(A) binding protein binds → switch from slow to fast adenylation for ~200, then PAP disengages
73
What are the functions of polyadenylation?
Nuclear export
Translation
Stability of mRNA
74
What is RNA editing?
Many RNAs need to have their bases changed
→ occurs in eukaryotic tRNA, rRNA, mRNA and miRNA, archaea and bacteria (universal)
→ in trypanosomes there is extensive RNA editing of some kinetoplast genes
→ in vertebrates RNA editing occurs in the nucleus
75
In what species does the most dramatic gene editing occur?
*Trypansoma brucei*
→ the mitochondrial DNA (kinetoplast DNA) contains a mixture of maxicircles and minicircles interlocked
→ the maxicircle kDNA encodes two rRNAs, one ribosomal protein and seventeen protein-coding sequences - 12 of the ORFs require RNA editing in order to be converted into translatable mRNAs
→ some maxicircle mRNAs are extensively edited - many Us added
→ some maxicircle genes are encrypted - transcripts must be decrypted before being read
76
How do trypanosomes modify their RNA transcripts?
1. GuideRNA 1 encoded on a minicircle binds the target RNA by pairing at its 5' anchor and 3' tail sequences
2. Specialised proteins recognise the addition/deletion sites and add/remove Us → copying the information from the gRNA - resulting in an extended mRNA
3. The editing provides an anchor site for gRNA 2 - encoded on a different minicircle → same process repeats
→ every loop on the gRNA has an A - determines where Us will be inserted
77
Why do mitochondria of trypanosomes and plants utilise extensive RNA editing?
Its speculated that different guideRNAs are expressed in different environments
→ is RNA editing a primitive way to change the expression of genes?
78
What is mammalian nuclear APOBEC-mediated mRNA editing?
Cytosine deaminated to generated uracil
→ change of amine to oxygen
→ involves an editosome
79
What does an editosome consist of?
APOBEC (ApoB mRNA editinf enzyme catalytic subunit) - 2 domains - catalytic / pseudo catalytic
→ pseudo catalytic - turns itself off until its associated at the site of deamination along with an auxiliary complex (ACF - APOBEC complementation factor)
→ the combination (editosome) recognises RNA sequences around the editing site then specifically edits
80
What is the function of the APOBEC editosome?
RNA editing of Apo-B gene - C to U editing on exon 26
(ApoB required for uptake and transport of cholesterol)
Liver → no editing - ApoB-100, lipid-associated, binds LDL receptors
Intestine → strong expression of APOBEC thus there is C to U editing - creates stop codon creating shorter version of ApoB
→ ApoB-48 - lipid-associated, doesn't bind LDL receptors
→ one gene, two proteins/functions - separates by cell-specific expression
81
What is mammalian nuclear ADAR-mediated mRNA editing?
Adenosine deaminated to inosine by ADAR (adenosine deaminase acting on RNA)
→ amine to oxygen
→ iodine is a mimic guanosine
ADAR requires dsRNA → binds a fold back i.e. stem loop structure
Exon → I is translated as G - can change protein sequence
Intron → I mimics G - can create new splice sites
→ ADAR driven editing can alter splice sites - regulate alternative splicing and protein function
82
Why would there be inverted repeats in our mRNA?
Imagine 2 *Alu* elements inserted into opposite strands of DNA → when mRNA is made complementary base pairing causes foldbacks (allows ADAR editing)
*Alu* elements → a class of primate-specific retrotransposons ~300 bases long - sub-group of SINEs
There are more than one million *Alu* elements dispersed in out genomes → provides plenty of opportunity to derive dsRNA
83
What is the value of ADAR-mediated RNA editing?
May have evolved as a defence system to inactivate retroviruses/retrotransposons → protects our genome
Enhances genome plasticity → generate new protein functions
ADAR can recognise DNA:RNA hybrids → role in DNA repair
Neurobiology link to epilepsy
84
How is mRNA edited for nuclear transport?
Export factors are loaded on to a mRNA during transcription by the C-terminal tail of one of the large subunits of RNA Pol II
→ nuclear export is fully integrated into mRNA maturation
At a late stage in splicing a nuclear export receptor complex is transferred from the CTD of Pol II
→ provides a nuclear export signal (NES) allowing message to be exported to the cytoplasm
mRNP - complex compacted particle of mRNA and proteins → mature enough to be exported
85
How does nuclear export of mRNA occur?
1. The nuclear export signal (NER) binds nuclear cage around a nuclear pore
→ mRNP is threaded through 5' end first, SR proteins removed and recycled
2. The cap binding protein (CBC) is removed → replaced by eukaryotic initiation factor 4E (elF4E) and circularised with elF4G (NER returned to nucleus)
3. The mRNP is now ready for translation
86
Why is miRNA of high importance?
microRNA action in cytosol still being fully understood
In 2020 → ~500 genes controlled by miRNAs
In 2024 → 1/3 eukaryotic genes regulated by miRNA
Role of miRNAs in regulating gene expression might be as important as that of transcription factors
87
What are miRNAs?
MicroRNA → small non-coding regulatory RNAs processed from dsRNA precursors
→ interfere with expression of other RNAs
→ typically stem-loop ds structure
→ 2-base overhang at the 3' end
→ GC-rich - stable structure
88
What is involved in the biogenesis of miRNA?
Canonical pathway → miRNA precursors can be found inter genetically (sometimes in clusters)
→ primary miRNAs formed
→ Drosha in the nucleus makes specific cuts at cleavage sites forming pre-miRNA
Non-canonical pathway → encoded in an intron (a mitron)
→ mitron lariat formed - debranching and base-pairing forms pre-miRNA
89
How are pre-miRNAs exported from the nucleus?
Processed pre-miRNAs don't have a 5' cap nor a poly-A tail → required exporting 5/Ran GTP
Exportin 5 binds the pre-miRNA (required the 2bp overhang)
→ associates with Ran GTP - allows export through the nucleus
→ complex falls apart in the cytosol
→ miRNA stays in cytosol - other components recycled
90
How is RISC (RNA-induced silencing complex) formed?
Cytosolic pre-miRNA binds Dicer ( a cytosolic RNAse III family member) → forms a mature duplex: guide strand and passenger strand
Cytosolic chaperones (Hsp90/70) bind argonaute → change conformation to open - can accept miRNA
Mature duplex loaded onto AGO, passenger strand degraded → left with RISC - a regulatory complex that can regulate expression of other genes
91
What is the role of RISC?
If the guide RNA of RISC has strong homology to the target gene → will bind strongly to the mRNA
→ argonaute slices it in 2 - leaves 2 sites for nucleases to attack
→ also recruits a deadenylase
→ degradation of mRNA
If the guide RNA has weak homology to target mRNA
→ translation inhibition
Shuts down gene expression of target gene
92
What are some examples of miRNA action?
miR-184 inhibits *Numbl*
→ controls neural stem cell differentiation - miR-184 antagonises and stops differentiation
miR7 / miR-206 inhibit *Pax7*
→ muscle cells
93
What can happen when miRNAs go wrong?
Dysregulation of protein expression
Severe developmental defects e.g. in limb formation, heart defects
Cancer
Neurological defects, behavioural effects
Infertility
Dicer is inactivated in fertilised eggs, blocking the generation of all miRNAs at this developmental stage
