RNA biology ALL Flashcards
1
Q
Transcription initiation
A
- RNA pol = recruited to Pol promoter w/ TF
- RNA pol II TF assoc at TATA promoter via TBP, then TFIIA/B bind, Pol recruited w/ TFIIF/H
- RNA pol transcribes gens in 5’-3’ direction to form pre-mRNA
2
Q
Prokaryotic vs eukaryotic transcription
A
- Eukaryotic transcrip/translat occur in different compartments
- Unlike bacteria x have co-transcription
- Means RNA potentially exposed to exo-ribonucl.
- Recruitment of ribosomes in prokaryotes = controlled by 5’ SD
- Eukaryotes x have SD
- Eukaryotic genes = interrupted coding sequences
3
Q
Pre-mRNA processing
A
- co-transcriptional
- Processing largely complete b4 termination, tight interconnection
- Different processing linked to different stages of transcription (capping early, splicing early + late, 3’ end late)
4
Q
Evidence - pre-mRNA processing
A
- All 3 occur in vitro w/o transcription
- Some components of processing interact w/ general TF, recruited to PIC
- Transcription activators indirectly influence efficiency of processing reactions
- Some factors disengage w/ TF + attach to polymerase
- So some factors recruited early
- Promoter swap experiment (GOI engineered so promoter specificity for recruitment of Pol replaced by sequence that recruits RNA Pol I) → pre-mRNA that x be processed
- CTD of polII links mRNA processing to transcription, deletion has ↑ effect on pre-mRNA processing
5
Q
CTD of RNA polII
A
- Composed of random heptad repeated motifs: YSPT SPS
- Ser2,5,7 = substrate for phosph by RNAPII
- Typically unphosph, Ser 5 phosph by Cdk7 of TFIIH
- Later in elongation, serine 2 phosph
- Phosph of CTD = dynamic
- Kinases (2 main kinases = cdk7 (phosph ser 5) + cdk9 (phosph ser 2)
- CTD phosphatases (reverse phosph in position dependent manner, Fcp1 targets Ser5/2, SCP dephosph Ser5)
6
Q
Phosph status of CTD changes along gene
A
- Follow Pol w/ ChIP, use Ab that recognise Ser2/5
- Phosph at Ser5 at start, peaks early in transcription cycle then ↓
- Opposite of Ser2 (↓ Ser5 corresponds ↑ Ser2 which peaks at end of transcription then ↓)
- At TSS, low phosph, at end FCP-1 dephosph
- CTD provides code
- Dynamics ↑ complex, Ser5 phosph linked to splicing, kinases/phosphatases x known
7
Q
Capping overview
A
- Occurs very early elongation just after RNA Pol dis-engaged from promoter
- Cdk7 phosph Pol II Ser 5, Pol II disengages from PIC
- Transcription machinery reconfigured
8
Q
Stage II - arrest of RNAPII
A
- RNAPII stops synthesis after 20-40 bp of RNA
- RNA exits Pol II via exit channel close to CTD
- RNA Pol assoc w. processing factors, DSIF engages Pol by Spt5 interacting w/ Pol near exit channel
- Complex recognised by NELF → RNA pol arrest
- Capping E recruited to CTD
9
Q
Capping
A
- Starts after 20-30 nucleotides
- Involves 3 reactions, finalised by assoc. of modified cap nucleotide
- (RNA triphosphatase carries out 5’ triphosphate → diphosphate)
- (guanyltransferase transfers GTP to GMP w/ diphosphate, GMP attached to 1st nucleotide)
- (methytransferase uses 5-adenosylmethionie to methylate G at pos 7)
10
Q
Capping control
A
- DXO recognises incompletely capped transcript, removes cap + destroys transcript
- Also if methyltransferase x recognise G = substrate for DXO
- If mRNA x bind cap binding complex, exposed ends recognised by decapping E Dep1.2
- Competition btw cap binding complex + DXO or Xin2 binding to cap
11
Q
Capping function
A
- Protects mRNA from degradation by 5’-3’ exonuc
- Stimulates splicing
- Nuclear-cytoplasmic export
- Translation initiation
12
Q
After capping
A
- RNA pol = arrested state, needs to be released
- Cdk9 phosph ser 2 of CTD + subunits DSIF + NELF → rearrangement of transcription complex, TF recruited, RNAP released
13
Q
Promoter proximal stalling importance
A
- Important for capping, allows gene regulation
- Regulation relies on PTEfb, recruited after pausing, causes phosph of Ser CTD
- Chance to activate genes ↑ rapidly
14
Q
pTEfb activation ↑ regulated
A
- Promoter proximal release integrated into signalling pathway
- Recruitment of pTEfb to Pol = RLS
- Activation of pTEFb = subject to ↑ controls
- Most pTEFb = initially inactive, associate w/ HEX1M1, 7SK associates + traps pTEFb inactive
- pTEFb = released due to signals like HIFa, TF, mediator
15
Q
Regulation of gene expression during drosophila development
A
- Embryogenesis, pol II recruited + subsequently paused, genes in permissive state
- Poised Pol II = present across ↑ tissues during pattern formation
- Pause Pol allows activation e.g. upon exposure of cells to gradient of morphogens
16
Q
pre-mRNA splicing overview
A
- In more than 95% of genes, involves transcription of exon/intron structure, resolved w/ splicing
- Coding info = arranged in exons separated by introns
- Need to remove introns
17
Q
Removal of introns
A
- Splicing = mRNA-mediated trans-esterification → 2 sequential breakages = rejoining of sugar phosphate backbone
- Mediated by SNURPs in spliceosome
- Adenosine in intron carries nucleophilic attack w/ 2’OH onto phosph of 1st nuc in exon
- Exon has free 3’OH attacks exon/intron border → intron released as Lariat
18
Q
Protein encoding genes vary in size
A
- B-globin gene = 146aa, 1.6kb
- Titin = 34,350 aa, 283 Kb
- Dystrophin = 3,685 aa but 2.4Kb (30,770 introns)
19
Q
Cis-elements in pre-mRNA
A
- Specific sequences surrounding exon/intron structure at start or end (5’SS or 3’SS gives directionality)
- 5’SS = characterised by sequences in exon + intron, around 10 conserved nucleotides
- 3’SS = us 3 nucleotides of splice site, pyrimidine trap followed by branch point
20
Q
Spliceosome
A
- Has over 200 proteins, 5 RNA players: U1,2,4,5,6snRNA
- snRNA = SM bs, 200 nt in length, conserved 2o structure, have ds region of RNA, ss region e.g. 5’ end, important to recognise specific 5’/3’ sequences
- U4+6 interact together via 2p structures → catalytic centre
- 2 types of protein the snRNAs associate w/ : RNA specific e.g. 70K vs common e.g. SM proteins
21
Q
Assembly of spliceosome on pre-mRNA
A
- 3’ + 5’ SS = recognised by biding of SF2
- U1 snRNA recognises 5’ SS by GU at starts of most introns
- 5’ part of U1 = ssRNA + bp w/ exon/intron
- U2AF recognises pyrimidine track + AG at 3’ SS
- U2 snRNA helps other proteins assoc. w/ branch point, A at branch point = bulged out
- U4,5,6 snRNA appear as a 3, U4+6 join
- Causes ↑ rearrangement, U1 + 4 ejected
- U6 snRNA replaces U1 at 5’SS, U6 + 2 interact
- Complex RNA-RNA interaction, 2’OH + exon 1 brought close
- 2’OH carries out nucleophilic attack on exon-intron border
- After 1st translocation, have 2nd major 2nd rearragement → 2nd transest.
- 3’ SS executes 2nd nuc attack
22
Q
Co-transcriptional splicing
A
- Splicing = post-translational in complex w/ RNA Pol II(phosph at Ser5)
- Free 5’SS remains assoc w/ complex until polymerase transcribes sequences ds of exon
- Co-transcription = important
23
Q
Exon junction complex
A
- Splicing also marks mRNA
- Co-immunoprecip shows EJC assoc. w/ spliced mRNA
- Proteins deposited 20-40 us of exon-intron junction
- In cytoplasm, some factors release, others join
24
Q
Catalysis of RNA splicing
A
- Discovery of self-splicing RNA = importance of snRNA
- Self splicing introns = group I/II, fold + self-cleave, similar reaction to spliceosome but w/o protein
- Suggests some catalytic activity in residues in RNA not protein
- E.g. group II RNA
- rRNA have introns that need to be excised, intronic regions fold into structures, 2’OH carries out nuc. attack, leaves 3’OH w/ 2’5’ linkage of intron, 3’OH nuc. attack on intron border → 2 exons fuse
25
Circular RNAs
- Involved in gene regulation + disease
- Results from backspacing (ds 5'SS attacks 3'SS of previous exon → circular RNA, bp btw repeat sequences in introns, circ RNAs can be exported-
- E.g. = role as sponge for miRNA + RNA binding protein
26
Transcription + processing
- 3' end of pre-mRNA needs to be processed
- Occurs by cleavage + polyadenylation
- 2 outcomes = matures 3' end, instigates transcription termination
27
End of protein coding gene
- Poly A signal in pre-mRNA defines end
- Recognised by components cleavage + polyadenylation complex
- Recognition of polyAsite = co-transcriptional
- Some factors like CPSF = recruited to Pol at promoter via TFIID
- Several factors of complex interact w/ RNAPol II or directly w/ CTD
- Deletion of CTD impairs 3' end termination
28
What signals direct 3' end formation
- When RNA pol reaches end of transcription unit, transcribe regions surrounding Poly A site
- Cis elements that direct cleavage = bipartite, AT rich
- 2nd signal ds of cleave site= GT rich
- 2 steps: cleavage + polyadenylation of 5' product
- Once cis-elements recognised + polyad machinery assembled, cleavage occurs
- 3'OH on maturing mRNA created + 5' phosphate at pre-mRNA
29
Recognition of poly A site
- CPSF + CTSF
- Once both = assembled, CF1 + polyA polymerase recruited → 3' end formation complex
- Endonuclease activity in CPSF 73kDa subunit cleaves btw 2 sequence elements
30
5' 1/2 of cleaved RNA = polyadenylated
- 3'OH = substrate for polyadenylation
- PAP adds 200-250 A, tail covered w/ PABP
- Creates uniform 3' end, critical for nuclear cytoplasmic export
- PolyA tail assoc w/ PABP w/ CAP binding complex
31
Most but not all mRNAs are polyadenylated
- Replication dependent histone genes x polyadenyl
- x have introns, expression = cell cycle regulated
- Instead of polyA signals have SLBP
- Histone ds element = recognised by U7snRNP through bs in 5' of U7snRNA
- Leads to cleavage reaction, release mature RNA
- CPSF + Cstf needed
32
Regulation of replication dependent histone gene expression
- Linked to DNA synthesis + cell cycle regulation
- u7snRNA = expressed in cell cycle
- But SLBP = regulated in cell cycle
- SLBP means mRNA is stable in cytoplasm + circularises mRNA by providing interaction point btw CAP + SLBP
- End of mRAN synthesis activates pathway that phosph SLBP → degraded
33
3' end formation + termination
- Function = no. of polymerases limited, termination means can be regulated, failure to terminate risks affecting ds processes)
- Some cleavage + polyadenyl dissoc from Pol → pre-mRNA
- Cleavage means 1/2 pre-mRNA still attached to Pol which keeps elongating
- Cleavage provides 5' end that x have cap, degraded
34
Effects of processing on transcription
- pre-mRNA processing can feedback = influence transcription
- Other e.g. of feedback loop e.g. capping = dependent on transcription apparatus but incomplete capping terminates transcription
- Splicing feedback as splicing 1st intron can enhance initiation via TFIIH + splicing U2 ↑ local conc of PTeFb
35
Increasing complexity
- Processing reactions interconnected
- e.g. pre-mRNA not just linked w/ transcription, also communicate w/ each other e.g. CAP binding complex stimulates splicing of 1st intron + 3' end formation
- e.g. splicing of introns enhances splicing of others
36
Expanding the size of the proteome
- ↑ no. of available components = key for evolution of more complex organisms
- ↑ proteins by ↑ no. of genes or alternative splicing
- Alternative processing reactions inc/ alternative pre-mRNA splicing, alternative polyadenylation + pre-mRNA editing
37
Expansion of protein by alternative splicing
- Allows permutation of exons allowing cells to make new proteins from a single gene
- Normally exons = next to each other
- Certain exons can be present or absent in some isoforms
- ↑ diversity w/o uparrow DNA contect
38
Common alternative splicing mechanism
1. Exon skipping (certain exons skipped or included)
2. Intron retention (mRNA differ depending on whether or not they have introns in mature mRNA, creates mRNA w/ diff coding capacity)
3. Alternative 5' donor sites of 3' acceptor site (additional sites activated in certain circumstances → cystic exons, creates variety of transcripts)
4. Mutually exclusive exons (inclusion/ exclusion of exons → mRNA that differ in coding capacity)
5. Alternative promoters (makes greater variety of possible transcripts)
39
Alternative splicing leads to diversity
- e.g. Dsipshilia Dscam gene → more than 38,000 isoforms
- Creates plasticity needed to govern complex exon connections
- Relies on arrangement of certain exon clusters that define certain exons
- 12 alternative variants of exon 4, 48 of exon 6
40
Trans factor
- In addition to spliceosome, additional sequence elements are present in pre-mRNA + additional trans-factors
- 2 groups of trans-factor: +ve splicing factors (SR proteins) -ve factors = hnRNPs
41
Recognition of splice sites in introns alone x explain alternative splicing
- If was only dependent on intronic sequences, alternative splicing = hard to explain
- Some introns = huge
- Exon definition model: recognition of splice sites = initiated from exon-centric view
- Exon bridge linking 3' + 5' splice sites of different exons so pan-exon association
42
Regulating splice site recognition w/ SR proteins
- Within exon sequences, have sequences that modulate recognition of a splice site
- Could be 3'/5' weak splice site
- Exonic splicing enhancer = recognised by SR proteins, +ve effect by facilitating interaction w/ U2AF + U1snRNA
- This is converted to a cross intron assembly
43
Initiation of exon regulation via silencer sequences
- Exons also have exon splicer silencer (ESS)
- -ve factors bind these sequences, prevent recognition of 5'/3'SS → exon exclusion
- 3'/5' SS x defined but exon sites for other exons either side may be defined → cross exon boundary
44
Splicing of individual exons
- Competition btw +ve and -ve factors at overlapping enhancer/silencer sequences → skipping/exclusion
- Binding affinity + conc. of factors = key
- Silencing factors can bind intronic splicing silencers
- Regulation = through exon definition, presence/absence of exon enhancer seq + assoc of proteins like SR + hnRNP
- IF [hnRNP] ↑, can nucleate from ESS + create steric hindrance over 3'SS, if [SR] ↑, prevents hnRNP blocking 3'SS
- ↑ complicated
45
Example - sex determination in Drosphilia
- Depends on ratio btw X chromosome + autosome
- Regulator promoter only active in female early embryos → Sx1 gene transcription → functional protein that regulates expression from a diff promoter
- In males, lack of Sx1 → activation from 2nd promoter → pre-mRNA that x spliced, exon included that has stop codon → non-functional protein
- Sx1 regulates other pre-mRNA that regulate other splice factors e.g. Tra
- Tra pre-mRNA excluding exon → functional Tra by Sx1 preventing recognition of 3'SS → stop spliced out
46
DNA methylation
- Regulation of alternative splicing = linked to transcription + pol speed
- Achieved via coupling DNA meth. to Pol speed
- Alternative splicing of exon 4-6 = lymphocyte development, weak 3'SS
- If pol transcribes slowly btw 5+6, weak 3'SS engages w/ spliceosome + assembles spliceosome, if fast x
- CTCF btw exon 5+6 forms obstruction on DNA, if DNA x methylated CTCF forms block, slows elongation
- Splicing patterns can be inherited by epigenetic modification
- Chromatin modifications can direct inclusion/exclusion of exons e.g. =ve regulator PTB
- Chromatin adaptors assoc. w/ certain chromatin modifications
47
Alternative polyadenylation
- ↑ polyA sites in pre-mRNA, usage can be regulated
- polyA site in pre-mRNA us of final stop, alternative usage = coding region APA
- If polyA site = ds of stop codon in UTRs, UTRAPA used
- Coding APA → production of mRNA w/ different coding potential, UTRAPA → transcript diversity w/ mRNA isoforms
- E.g. Hlgm heavy chain : alternative polyA + splicing, dependent on 2 polyA sites that create alternately cleaved poly-A, recognition of polyA site, in immature B cells, ↓ levels of cleavage, us polyA site = suppressed
48
Alternative cleavage + polyadenylation
- 70% of human genes undergo alternatively
- Alternative 3' end processing → mRNA w/ different 3' UTRs
- This x Δ protein coding info but effects expression + mRNA localisation
- e.g. cancer mRNA has shorter UTRs, avoiding potential miRNA targets, achieved w/ different polyA sites ds of coding
- UTR has sites e.g. mRNA target sites, dstab elements
49
RNA editing
1. substituting base within mRNA
- E.g. ADAR Δ A→I
- During translation I is read as G → sub of aa in final product
- Seratonin receptor e.g. 5HT2c receptor Δ 5 codons by A→I
- E.g. C→U by APOBEC1
- C-U in CAA → UAA (stop codon), happens in apolipoproteoin, makes shortened 2153 aa
- In liver, CAA x recognised by comp factors → whole protein 5463 aa
2. Epitranscriptome
- All 4 nucleotides can be modified by methylation, ↑ variety, both coding + nc transcripts
- Most common = MGA methylation of A w/ METTL4 + WTAP methyltransferase + erasers like FTO
- Read by readers like hnRNPC, stimulate transcription/alternative splicing
- Pseudouridyl of nucleotides through snoRNA guided mechanism or through PUS
- Pseudouridyl at UGA stop → 'read through' by ribosome
50
Cap snatching by influenza
- Capping confers stability + ensures export from nucleus
- Depends on RNAPII
- Creates issue for virus which transcribe host DNA w/ own polymerase as lack CTD
- RNA virus often replicate in cytoplasm, use cells capping machinery
- Influenza virus = in nucleus, x have CTD, steals cap form emerging pre-mRNA w/ endoribonuc acclivity
- Cap is used as a primer to initiate transcription of its own RNA
- Viral RNA then capped so protected from 5'-3' degradation + exported to cytoplasm
- Host RNA x have cap, vulnerable to nucleases
51
Capping, early checkpoint release + disease
- Protein encoded by herpes simplex virus inhibits action of CDK9, prevents phosph of Ser 2
- Pol stalls at early checkpoint + x released, host transcripts suppressed
52
Mutations that affect pre-mRNA processing
- Mutations often found in regions needed for processing machinery to assoc w/ pre-mRNA e.g. 5'SS, ESE, 3'SS
- Mutation affecting splice site → ↓ splicing of exons + faulty mRNA
- Mutation in polyA signal ↓ or ↑ cleavage + poly adenyl
- 2 types of mutation: 1. splicing mutation that directly effect consensus signal cause exon skipping, 2. mutation that affect trans-factor so impair splicing machinery
- E.g. fontrotemporal dementia has mutations in MAPT genes
E,g, Tau enriched in axons, Tau3/4 ration is highly regulated, deviation → accumulation of Tau → neuroses.
- E.g. DMD caused by deletions + mutations e.g. T→A sub in exon 31 that creates an ESS forces exon31 skipping
- Mutation in cis-elements: can promote cancer initiation + progression e.g. KIT oncogene
- Mutation in splicing factors:
- E.g. PWS = ↑ symptoms like restricted growth
- Deletion of paternal SNURF-SnRNP causes PWS by loss of snoRNA, SnoRNA HB11=S2 comp to 5HT2CR pre-mRNA
- PWS = mutation in 5HT2CR, deletion in chromosome 15 that encodes snoRNA inc SNORD115
- SNORD115 assoc w/ exon 5 + suppresses activation of 5a splice site
53
Mis-splicing in cancer
- No. of mutations that inactivate splice sites either directly or indirectly by affecting splice regulatory sequencing
- Mutations to spliceosome e.g. U2AF
54
3' end formation + disease
- Largely occur in essential cis-elements, leads to Low or Gof
- E.g. thalassemia due to mutation in AATAAA hexamer (recognition signal needed for interaction of CPSF w/ pre-mRNA)
- Mutation in B globin gene Δ hexamer so CPSF x recognise → ↓ B globin protein → aggregation
- Mutation in coagulation factor III where weak consensus site
55
Influenza A virus
- Virus transcribes template, poly-adenyl occurs by transcribing U-rich region in template strand + repeatedly copying U-stretch → export
- mRNA translated to make NS1 which assoc. w/ CPSF1
- AAUAAA in pre-mRNA comprises host mRNA
- NS1 also inhibits splicing by binding + inactivating u6 snRNP
56
Nuclear-cytoplasmic transport
- 5' cap, 3' polyA tail + PABP both protect + involved in export
- Next to have 3' + 5' UTR
- Export = interactions btw adaptors on RNA or sequence motifs on RNA cargo recognised by receptor molecule
- Nuclear pore complex forms basket structure in nuc, protruding filaments in cytoskeleton
- Porin proteins comprise nuclear pore, region that lack 2o form channel
- Pore allows small molecules to pass, carrier proteins needed for larger
- E needed to assemble diffusion competent complex
57
Karyopherin family
- Traffic = dependent on these, act as exportins + importing
- E.g. cargo w/ export sequence recognised by exporting, assoc. w/ Ran-GTP, complex diffuses through pore → cyt
- In cyt, GAP binds Ran-GTP + hydrolyses GTP → disengages complex
- Ran-GEF imports Ran-GDP into nucleus + converted to Ran-GTP
- Ran-GTP used to release cargo imported to nucleus via importing
58
Import/export of non-coding RNAs
- Exportins specific for ncRNA, also use karyopherin
- E.g. tRNA assoc. w/ exportin-t
- SnRNA exported by Phax/CRM1
59
Export of mRNA/mRNPs
- mRNA exported from nuc→cytoplasm in Ran-GTP independent pathway
- Instead, use tap + p15 (TAP/Mex in yeast)
- Like Ran-GTP they associate w/ cargo
- Aly (part of EJC) = adaptor for export receptor TAP15
- TAP15 assoc w/ nuclear pore by interacting w/ component of nuclear pore complex
- SR/Aly-TAP15 translocate mRNA to nuclear pore
60
Link between transcription, exosfome + mRNA transport
- Transcription apparatus has factors like SPT5/6 that deal w/ chromatin + exo factors involved in quality control
- In yeast, export complex is assoc w/ RNA pol II + may be deposited on mRNA co-transcriptionally
- Interaction btw TAP15 + Sac 3 = protein-protein interaction
- Gating effect by pulling out DNA of a particular gene toward nuclear pore
- E.g. SAGA recruited to promoter far from nuclear periphery
61
RNA localisation
- Some RNAs localised to specific regions where needed
E.g. = neurons (mRNA transported to axon into synapses, mRNA localis → polarisation)
E.g. = migrating fibroblasts (localisation of B-actin mRNA to focal adhesion plaque, fibroblasts migrate along trajectory)
E.g. epithelia (E-cadherin + b-actin mRNA localised)
62
Transport of RNA
- Require specific 3'UTR sequences
- Proteins like ZBP-1 B-actin + MAP2 = adaptor that allow mRNP to assoc. w/ proteins for transport
- Other functions = prevent pre-mature assoc. of mRNA onto ribosome where mRNA shouldn't be
63
Eukaryotic vs prokaryotic translation initiation
- Organisation of genes on mRNA = different
- prokaryotes = mRNA poly-cistronic, each gene has SD, co-transcriptional translation
- Eukaryotes = mRNA monocistronic, 1 gene but through alternative processing make ↑ mRNA, 5' cap + 3' tail
64
Translation initiation in prokaryotes
- SD/rbs = us of Aug start
- 16S RNA associates via compl. RNA-RNA interactions
- This also positions P site of ribosome in region of AUG
- Additional factor join
- Complex recognised by 50S, finalise assoc. of ribosome onto ORF of mRNA
- Release 1F1/3 to open up A site
- ↑ ORF can be translated from 1 mRNA
65
Translation initiation in eukaryotes
- 5' cap + 3' tail for regulation efficiency Cap-5'UTR-ORF-3'UTR-tail)
- CAP binds w/ CBP20/80, poly tail = occupied by PABP
- CBP + PABP interact w/ each other → circular mRNA
- In yeast, mRNA exported circular
- In cytoplasm, CBP + PABP exchanged for cytoplasmic versions
- Initiation = dependent on factors, eIF4F = 3 components, make up CAP-binding complex (cyt)
- eIF4e contacts CAP, eIF4G enables bridging btw PABP + eIF4E
- Mechanism = disc of 50S ribosome → 60S + 40S subunit → 40S trapped, 40S-3-IA assoc w/ ternary complex → 43S complex, mRNA occupied by 4F CAP binding complex, this complex assoc w. 43S → 48S ribosomal subunit, 48S scans RNA for AUG, at AUG forms complex where 60S joints → 80S initiation complex
- In eukaryotes, assoc of ribosome + cap means Aug can be 1000s of bp away from CAP site
66
Translational control
| 1. Global
- E.g. by phosph of translational apparatus like initiator factors
- Formation of 3o complex, EIF2-GDP needs GTP, phosph of a-subunit of eIF2 on Ser51 → inhibition as eIF2B-catalysed exchange of GDP for GTP x
- Kinase = way regulation = integrated
- E.g. oxidative stress activates HRI, respond to environmental changes
- When goes wrong: x phosph Ser51 of eIF2 → T2D, sub of Ser51→Ala in mic → glucose intolerance
E.g. association of CAP
- Unphosph eI4F4e binds some CAPs, others only bind phosph CAP
- eI4F4E = phosph on Ser209
- 4E-Bp associates w/ eIF4E which blocks interaction btw eIF4e + eIF4G, preventing formation of eIF4F + recruitment of 43S
- phosph of 4eBP by mTOR Δ structure of 4E-BP → releases eIF4fe, associates w/ eIF4G
- Insulin signalling → MAPK → MTOR phosph → 4E-BP phosph, CAP x form
67
Translational control
| 2. Individual
- Circular structure of mRNA = key as brings sequence in 3'UTR close to cap
- Regulation of mRNA w/ TOP sequence (found in proteins assoc. w/ translational apparatus, TOP mRNAA have C followed by 4-14 pyrimidine, unusual 5', in growing cells, translated w/ ↑ efficiency)
- Regulation at transcript level = assoc w/ regulation of intracellular [ion]
- Ferritin + ferritin encoded protein sequester ion
- IRP = if iron around associates w/ it, if x IRP=free
- Iron dangerous, produces free radical
- Ferritin encoded by mRNA w/ classic stem loop, has bs for IRP (only binds when x bound IRP)
- Hinders assoc w/ 5' UTR of ferritin, blocks translation initiation
68
Translational control
| 2. iii Control of factors by binding UTR
- Sequence elements that can be recognised by SXL
- Dosage compensation in Drosphilia ↑ transcriptional output from genes from signal X chromosome in males= 2X in female
- Dosage compensation in females = prevented by repression of MSL2 by SXL
- SXL binds 3'UTR + recruits protein that prevents assoc of 43S w/ 5' cap, also binds to 5'UTR
69
Translational control
| 2. iv Role of CPE in translation inhibition
- Some maternal mRNAs not only involve controlled adenylation of mRNA, also CPEB-mediated inhibition of translation initiation
- Fertilisation = development driven by maternal mRNA, period of transcriptional inactivity
- In oocyte cytoplasm, mRNA transcripts have 3'UTR elements like CPE that sequesters CPEB, inhibits assembly of complex that makes functional CAO
- When signals activate kinases, phosph CPEB → inactivation of pARN, activation of Pol that extends poly A tail
70
Translational control
| 2. v Translational regulation by MiRNA
- miRNA associate w/ complex in cytoplasm that enables them to associate w. compl sequences mostly in 3' UTR
- Can inhibit Cap recognition or repression 60S joining
- Can repress translation at post-initiation stage by slowing elongation
71
Translational control
| 2. vi Selenocysteine
- 25% proteins incorporate selene-cysteine at UGA stop
- Controlled by SECIS structure in 3'UTR that binds SEBP2 + tRNAsec allows incorp into peptide at stop
- 3' UTR sequences can make specific proteins
72
RNA degradation
- CAP + polyA tail protect proteins, need to remove
- PARN - 3'-5' exoribonuclease degrades polyA tail, free 3' targeted by EXOSOM
- Decapping E removes guanosine cap, exposes free 5' end, degraded by 5'-3' exoribonuc like XRN1
- Deadenylation independent pathways, mRNA cut in 1/2 w/ endonucl, exposes unprotected 5' on 31/2 targeted
73
Regulating stability of mRNA
- Key for overall expression levels
- mRNA stab + de-stab cis elements that control expression, if ↑ stable, repeatedly translated
- e.g. destabilise sequences like ARES = in 3'UTR, interact w/ proteins that recruit components of decay machinery, stability = Δ by factors that compete for ARE biding
74
Nonsense mediated decay
- Assoc w. degradation of aberrant mRNA e.g. premature stop
- EJC denotes exon-exon junction
- After 1st round of translation, mRNA engages w. cytoplasmic CBP + PABP → repeated rounds of translation
- Immature stop codon likely flanked by EJC
- In primary round, stop is recognised as stop codon, release factors assoc w. ribosome
- Us of EJC, cells signal faulty mRNA transcript, recruit endonuc + upf1
75
mRNA as regulatory target
| Iron metabolism
- Translation initiation inhib by IRP
- IRP also recog 3'UTR of Tfr
- Intracellular Fe brought into cell by serum transferring
- Transferrin receptor assoc w/ iron → release Fe in cell
- Tfr mRNA has stem loop in 3'UTR, prevents mRNA degraded by exonuc
- ↓ Iron, need ↑ Tfr to get more iron in, IRP free, assoc w/ 3' UTR of Tfr, prevents endoncucl
76
Processing of primary ribosomal RNA transcript
- rRNA = 80% of cell RNA mass
- 100s of rDNA genes = tandem arrays on chromosomes 18,14,15
- Regions form nucleolus organiser region
- After transcription by RNA pol 1, no. of nucleotides in Io RNA are mod
77
Processing of rRNA precursors in nucleolus
- rRNA has PolI-specific promoter, recog by TF SL1
- Pol I transcribes genes - pre-ribosome RNA
- Transcription stopped at terminators
- ITS = btw 18S + 5.8S, ITS-2 = btw 5.8S-28S, 3'ETS = after 28S
- Primary transcript = modified → 18, 5.8 + 28S RNA
- 5S RNA transcribe by RNA Pol III in nucleoplasm, imported to nucleolus
- In nucleolus, assemble w/ ribosomal proteins
78
snoRNP cleavage
- pre-rRNA sequences like in ITS-1 = recognised by endoribonuc MRP, releases 20 + 32S ribosomal precursor
- U8 snoRNA recognises ITS-1 + 3'ETS
- tRNA modified by methyl + pseudouridyl, guided by SnoRNA
- Las1 endonuc uses seq in ITS2 to make precursors for 5.8 + 28S rNRA
- 5'-3' of XRN2 also helps
79
tRNA modification
- SnoRNAs, 2 types
1. BoxC/D snoRNAs (have BoxC, sequences after = conserved, sequences btw C+D boxes in snoRNA hybridise w/ equivalent sequences on rRNA, identifies nucleotides that can be subject to modification in tRNA
2. BoxH/ACA snoRNAs (contain conserved H box consisting of consensus ANANNA btw 2 stem loops + ACA 3 nuc away from 3' end)
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5S rRNA
- Transcribed by RNA Pol III in nucleolus
- Intragenic promoters, recognised by TFIIIA,B+C that recruit RNA Pol III
- Assoc w/ LA protein, protects from degradation
- Ribosomal proteins involved in transfer of 5' to nucleoplasm
- 5S RNA expression regulated by altered reg. feedback
- Zinc finger of TFIIIA can associate w/ 5S RNA when in surplus
81
tRNA processing in eukaryotes
- Precursor tRNA have extended 5' + 3' ends, can have introns
- Trimmed by endonucleases
- Nucleotidyl transferase adds CCA to 3' end
- Introns spliced by complex of sen proteins, leaves tRNA at 3' w/ cyclic phosph + 5'OH
- Phosphodiester modifies 3', kinase modifies 5'OH
- Now express anticodon
82
snRNA/ RNP processing
- snRNPs inc U1, 2, 4 + others U7 that process replication-dependent histone genes
- Most transcribed by RNAPII in nuc then exported using Fox to cyt where assoc w/ SMN
- Mutations in MSN = spinal muscular atrophy
- SMN charge snRNAs w/ SM binding proteins, cap of snRNA added m7G
83
Very small ncRNA
- miRNA = 22nt, assoc w/ 3'UTR
- siRNA = 20-25nt, similar to miRNA
- piRNA = 25-30nt long, assoc w/ PIWI → piwi induced silencing
84
miRNA processing
- Transcribed from Pol II
- pre-miRNA folded into specific structures, recognised by DICER, chop stem loop
- Smaller stem loops exported to cytoplasm where assoc. w/ dicing complex inc Argo1 + Dicer1, process 80 nt pre-miRNA → 28nt miRNA
- One ds ejected, remaining ss-RISC complex assoc w. target mRNA to inhibit mRNA
- siRNA assis w. Argo2/Dicer2 →21nt in siRISC
85
RNAi as a tool
- Exogenous delivery of dsRNA, comp to sequences of mRNA, mRNA knockdown
86
Regulating gene expression by ncRNA
1. snRNA in complex that phosph CTD of RNA pol II
- U1 snRNA involved in both spliceosome + TFIIH
- Interaction of U1 snRNA-TFIIH stimulates transcription initiation + re-initiation
- pTEFb = -vely regulated by 7SK snRNA, inhibit Cdk9
2. B2 RNA inhibits transcription of RNA pol II
- Species specific
- Heat shock in mouse cells induces Pol III transcribed B2 RNA synthesis
- B2 RNA directly assoc w/ RNA pol II + inhibits transcription of NON heat shock genes
- Expression of heat shock = unaffected
- B2 assoc w/ RNA pol II, incorp into PIC of non-heatshock gene, prevents CDk7 phosph of CTD → prevents promoter escape
