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
Q
Circular RNAs
A
- 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
Q
Transcription + processing
A
- 3’ end of pre-mRNA needs to be processed
- Occurs by cleavage + polyadenylation
- 2 outcomes = matures 3’ end, instigates transcription termination
27
Q
End of protein coding gene
A
- 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
Q
What signals direct 3’ end formation
A
- 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
Q
Recognition of poly A site
A
- 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
Q
5’ 1/2 of cleaved RNA = polyadenylated
A
- 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
Q
Most but not all mRNAs are polyadenylated
A
- 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
Q
Regulation of replication dependent histone gene expression
A
- 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
Q
3’ end formation + termination
A
- 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
Q
Effects of processing on transcription
A
- 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
Q
Increasing complexity
A
- 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
Q
Expanding the size of the proteome
A
- ↑ 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
Q
Expansion of protein by alternative splicing
A
- 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
Q
Common alternative splicing mechanism
A
- Exon skipping (certain exons skipped or included)
- Intron retention (mRNA differ depending on whether or not they have introns in mature mRNA, creates mRNA w/ diff coding capacity)
- Alternative 5’ donor sites of 3’ acceptor site (additional sites activated in certain circumstances → cystic exons, creates variety of transcripts)
- Mutually exclusive exons (inclusion/ exclusion of exons → mRNA that differ in coding capacity)
- Alternative promoters (makes greater variety of possible transcripts)
39
Q
Alternative splicing leads to diversity
A
- 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
Q
Trans factor
A
- 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
Q
Recognition of splice sites in introns alone x explain alternative splicing
A
- 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
Q
Regulating splice site recognition w/ SR proteins
A
- 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
Q
Initiation of exon regulation via silencer sequences
A
- 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
Q
Splicing of individual exons
A
- 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
Q
Example - sex determination in Drosphilia
A
- 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
Q
DNA methylation
A
- 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
Q
Alternative polyadenylation
A
- ↑ 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
Q
Alternative cleavage + polyadenylation
A
- 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
Q
RNA editing
A
- 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 - 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
Q
Cap snatching by influenza
A
- 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
Q
Capping, early checkpoint release + disease
A
- 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
Q
Mutations that affect pre-mRNA processing
A
- 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
Q
Mis-splicing in cancer
A
- No. of mutations that inactivate splice sites either directly or indirectly by affecting splice regulatory sequencing
- Mutations to spliceosome e.g. U2AF
54
Q
3’ end formation + disease
A
- 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
Q
Influenza A virus
A
- 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
Q
Nuclear-cytoplasmic transport
A
- 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
Q
Karyopherin family
A
- 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
Q
Import/export of non-coding RNAs
A
- Exportins specific for ncRNA, also use karyopherin
- E.g. tRNA assoc. w/ exportin-t
- SnRNA exported by Phax/CRM1
59
Q
Export of mRNA/mRNPs
A
- 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
Q
Link between transcription, exosfome + mRNA transport
A
- 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
Q
RNA localisation
A
- 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
Q
Transport of RNA
A
- 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
Q
Eukaryotic vs prokaryotic translation initiation
A
- 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
Q
Translation initiation in prokaryotes
A
- 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
Q
Translation initiation in eukaryotes
A
- 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
Q
Translational control
1. Global
A
- 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
Q
Translational control
2. Individual
A
- 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
Q
Translational control
2. iii Control of factors by binding UTR
A
- 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
Q
Translational control
2. iv Role of CPE in translation inhibition
A
- 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
Q
Translational control
2. v Translational regulation by MiRNA
A
- 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
Q
Translational control
2. vi Selenocysteine
A
- 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
Q
RNA degradation
A
- 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
Q
Regulating stability of mRNA
A
- 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
Q
Nonsense mediated decay
A
- 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
Q
mRNA as regulatory target
Iron metabolism
A
- 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
Q
Processing of primary ribosomal RNA transcript
A
- 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
Q
Processing of rRNA precursors in nucleolus
A
- 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
Q
snoRNP cleavage
A
- 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
Q
tRNA modification
A
- 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
- BoxH/ACA snoRNAs (contain conserved H box consisting of consensus ANANNA btw 2 stem loops + ACA 3 nuc away from 3’ end)
80
Q
5S rRNA
A
- 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
Q
tRNA processing in eukaryotes
A
- 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
Q
snRNA/ RNP processing
A
- 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
Q
Very small ncRNA
A
- miRNA = 22nt, assoc w/ 3’UTR
- siRNA = 20-25nt, similar to miRNA
- piRNA = 25-30nt long, assoc w/ PIWI → piwi induced silencing
84
Q
miRNA processing
A
- 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
Q
RNAi as a tool
A
- Exogenous delivery of dsRNA, comp to sequences of mRNA, mRNA knockdown
86
Q
Regulating gene expression by ncRNA
A
- 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 - 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
