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Paper 3 > RNA biology ALL > Flashcards

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
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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
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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)
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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
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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)
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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
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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
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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
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9
Q

Capping

A
  • Starts after 20-30 nucleotides
  • Involves 3 reactions, finalised by assoc. of modified cap nucleotide
    1. (RNA triphosphatase carries out 5’ triphosphate → diphosphate)
    1. (guanyltransferase transfers GTP to GMP w/ diphosphate, GMP attached to 1st nucleotide)
    1. (methytransferase uses 5-adenosylmethionie to methylate G at pos 7)
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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
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11
Q

Capping function

A
  • Protects mRNA from degradation by 5’-3’ exonuc
  • Stimulates splicing
  • Nuclear-cytoplasmic export
  • Translation initiation
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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

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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
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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
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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
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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
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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
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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)
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
  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
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
  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
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
  1. 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
  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

Paper 3 (29 decks)

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