3 Post transcriptional gene control PDF

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MCB 2050 gene control 1 Review o o o o o is capped, polyadenylated, spliced, associated with ribonucleoproteins before it is exported to cytoplasm for translation Transcription: nucleus Translation: cytoplasm Splicing: ribonucleoprotein spliceosome complex catalyzes intro removal (2 exons join) Inte...

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MCB 2050 Post-transcription gene control

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Review o o o o

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Pre-mRNA is 5’ capped, 3’ polyadenylated, spliced, associated with ribonucleoproteins before it is exported to cytoplasm for translation Transcription: nucleus Translation: cytoplasm Splicing: ribonucleoprotein spliceosome complex catalyzes intro removal (2 exons join) Interactions between 1. SR proteins (RNA splicing proteins) 2. snRNP (form spliceosome with pre-mRNA) Cross-exon recognition complex 3. Splicing factors specifying correct splicing sites How is gene expression regulated beyond DNA/histones?

RNA processing, post-translational gene control

1) 5’ cap added, pre-mRNA splicing (remove introns, alternative exons remain) 2) Polyadenylation at 3’ end (protect from degradation, termination end), use alternative polyA sites 3) Improperly processed mRNA goes to exosome while properly processed mRNA goes to cytoplasm for translation 4) In cytoplasm, translation initiation factors bind to 5’ cap, while polyA binding protein on 3’ end, translation starts 5) Cytoplasmic P-bodies degrade mRNA to repress translation (polyA, 5’ cap removed), exosomes degrade mRNA, mRNA degradation controls translation rate 6) If mRNA lacks polyA tail, polyA polymerase adds tail 7) miRNA inhibits translation of mRNA 8) a. tRNA (translate mRNA to AA): transcribed by Pol III, processed in nucleus b. rRNA (build ribosomes): transcribed by Pol I, processed in nucleolus 9) Regions from pre-tRNA are degraded by exosomes

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Key points of translation regulation: 1. Splicing, alternate splicing = variety of mRNA made 2. miRNA, siRNA regulate mRNA stability and translation = usually inhibit translation 3. Polyadenylation in cytoplasm 4. Cytoplasmic P bodies perform mRNA surveillance and degradation (5’ cap, 3’ polyA tail removed)

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mRNA processing overview  Various proteins produced by different exon combinations (alternate splicing)  3 modifications: capping + 3’ cleavage/polyadenylation, RNA splicing

1. 5’ cap attached (7-methylguanosine)  5’ cap attached immediately after RNA Pol II initiates transcription at first nucleotide of first exon 2. Cleavage at polyA tail  Enzyme chews off mRNA transcript at polyA site to add tail 3. Polyadenylation at 3’ end  Adenosine residues (250) 4. RNA splicing (introns removed, exons stay)

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snRNP associate with exon/intron boundaries, excise intron RNA  Exons spliced together, 2 introns removed  5’ 7-MG cap  3’ polyA tail  Introns removed  Alternative splicing of exons occurs (could have E1-E2-E3, or just E1E3)  Sequences in untranslated blue regions (stay in mRNA, but not in AA) control mRNA stability, translation efficiency

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mRNA processing is co-transcriptional o o o

Pre-mRNA processing occurs while mRNA is made by Pol II Processing + transcription at same time CTD subunit of RNA Pol II controls transcription  Unphosphorylated CTD = transcription initiation  Phosphorylated CTD = elongation

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Phosphorylated CTD recruits with factors involved in RNA processing 1. Capping enzymes 2. Processing factors (splicing proteins, SR) 3. Cleavage/polyadenylation factors 4. Nuclear export factors

mRNA alternative splicing

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How are different mRNAs made from same gene?  Alternative splicing: RNA-binding proteins bind near specific splice sites to regulate alternative splicing  Alternative promotors on same gene  Alternative cleavage at polyA site

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Splicing regulation by Sxl/Tra/Dsx  Female splicing = red Male splicing = blue  Red line = stop codon, non-functional protein made  Boxes = exons Lines = introns  Splicing controls sex determination (which exons stay determines gender in Drosophila)  Lack splicing proteins, lack functional genes since exons with stop codons not excised

Sex-lethal (Sxl): splicing silencer, inhibits translation

 RNA-binding protein

 Suppresses splicing (translation slowed)     

Only in females Sxl protein controls its own translation Non-functional Sxl protein in males Binds to sequence near 3’ end of intron between exon 2-exon 3 Sxl gene controls own splicing to produce exons 2-4 mRNA

 Females: deletes exon 3 (exon 3 has stop codon)… functional Sxl protein  Males (no SXL protein): retain exon 3, truncated protein since have stop codon in exon 3, non-functional Sxl protein

MCB 2050 Post-transcription gene control

Tra: activates splicing

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 RNA-binding protein

 Activates splicing (translation quicker)  Specific Dax splicing  Sxl controls Sxl and Tra splicing  Non-functional Tra in males  Females: deleted exon 2, functional Tra  Males: non-functional Tra since retain exon 2 (stop codon in exon 2 of Tra)

Dsx

 Transcription activator or repressor (isoforms in M/F)  Both females/males have functional Dsx protein (different exons)  Tra protein regulates alternate splicing of Dsx in male/female isoforms  Females: Rbp1 + Tra2 + Tra proteins bind, exon 3-4 retained, polyadenylated at 3’ end of exon 4 (exon 5 not made) - Translation of Dsx is activated by Tra - Activates genes with Dsx-binding sites - Exon-4 encoded sequence acts as transcription activator - Exon 4 has polyA tail (end transcription)  Males: exon 4 deleted, exon 3 – exon 5 - Represses genes with Dsx-binding sites - No functional Tra protein, no Rbp1-Tra2 binding, exon 3-5 retained - Exon-5 encoded sequence acts as transcription repressor

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Dsx: TF binding to DNA in same DNA-binding domain in M/F isoforms (same Dsx gene but encodes both activator and repressor) Alternative splicing regulates critical  Females: Dsx has transcription activation domain neuronal processes and even sex determination.  Males: Dsx has transcription repression domain

Alternative splicing in hearing o

Different isoforms (products of alternative splicing, various exon combinations) allow perception of different frequencies  576 possible isoforms of membrane Ca2+ channel from 1 mRNA  Channel isoforms encoded by alternatively spliced mRNA…all from 1 mRNA transcript  S0-S6 transmembrane domains  Detecting sounds at different wavelengths, but all regulated by 1 gene which was spliced 576 different ways

Alternative splicing linked to neurological disorders o o

More microsatellite repeats in transcribed gene regions alters [alternatively spliced mRNA] Neurological disorders related to abnormalities in  Nuclear RNA-binding protein function (splicing proteins)  More microsatellite repeats generating binding sites for splicing factors

Drosophila DSCAM gene has 38 016 isoforms o o o o o

95 exons = 38 000 isoforms MOST EXTREME example of regulated alternative RNA splicing Specify millions of different synaptic connections between neurons in Drosophila brain 12 x 48 x 33 x 2 transmembrane exons = 38 016 isoforms from 1 mRNA Ig domains, fibronectin domains, transmembrane domains

Ig domains

Fibronectin

Transmembrane

MCB 2050 Post-transcription gene control Post-transcriptional control in cytoplasm (translation site) o

mRNA stability controlled by 1. polyA tail length 2. Binding of various proteins to 3’ untranslated sequences

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mRNA translation controlled by 1. micro-RNA – bind to 3’ untranslated region, repress protein production (destabilize mRNA) 2. RNA interference via siRNA 3. Degradation – don’t want mRNA to persist in cytoplasm if don’t want more protein 4. Cytoplasmic polyA mechanisms

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mRNA transported to specific locations by RNA-binding proteins  These proteins bind to 3’ untranslated sequences

mRNA stability controlled by polyA tail length o

Poly-A binding protein I (PABPI) binds to polyA tail  PABPI interact with initiation factors binding to 5’ cap  Initiation factors: eIF4E, eIF4G  polyA tail binds to 5’ cap via PABPI  Circular mRNA structure makes translation more efficient, helps ribosome subunits rebind to TSS  Shorter polyA tail = less stable mRNA, mRNA becomes linear= less PABPI protein binding = mRNA degraded

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Short-lived mRNA have copies of destabilizing AUUUA sequence in 3’ untranslated sequence interacting with de-adenylating enzyme (shorter polyA tail)  Translation is repressed since no polyA tail (mRNA becomes linear)

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Long-lived mRNA (e.g. globin) has stabilizing CCUCC in 3’ untranslated sequence (longer polyA tail, mRNA circular)

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Length of polyA tail determines translation  Short polyA tail = translation repressed  Factors elongating polyA tail = circularization of mRNA = translation activated

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mRNA half-life varies  Many stable for hrs  Bacterial mRNA unstable (few minutes)

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5’ end degraded before 3’ end synthesized

Stability and half-life of mRNA determined by length of polyA tail  Normal length = 200 A  Destabilizing sequence = AUUUA = shorter half-life = de-adenylation  Deadenylation-dependent pathway: nucleases shorten polyA tail so that less PABPI bind - mRNA 5’ cap lost, nuclease digestion - this pathway explains how proteins produced in bursts over short time - Deleting AUUUA increases half-life (malignant cancer cells has less AUUUA, longer half-life)  Stabilizing sequence = CCUCC = longer half-life = adenylation prolonged

3 mechanisms of normal mRNA degradation 1. Deadenylation-dependent pathway (most common)  Nucleases shorten polyA tail… less PABPI bind… mRNA linear  polyA tail is shortened!  mRNA degraded 2. Deadenylation – independent  polyA tail is not shortened

3. Endonuclease-dependent  Endonucleases cleave middle of mRNA (not related to polyA tail)

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Deadenylation-dependent pathway (MIDTERM)

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Most common pathway: remove polyA tail (serves to protect mRNA), mRNA unprotected, mRNA linear, degraded 1. Deadenylase enzyme complex shortens polyA tail from 200 to 20 A 2. Destabilized/less PABPI binding (polyA binding protein). PABPI normally interact with 5’ cap initiation factors. 3. Weakened interactions between 5’ cap and translation initiation factors 4. Decay occurs from both ends at same time as mRNA opens up 5. 5’ to 3’ decay:  DCP1/DCP2 decapping complex  mRNA degraded by XRN-1 (a 5’ – 3’ exonuclease) 6. 3’ to 5’ decay:  3’- 5’ exonuclease in cytoplasmic exosomes

MCB 2050 Post-transcription gene control Deadenylation independent mRNA decay

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Degradation doesn’t depend on removal of polyA tail mRNA decapped, then de-adenylated only 5’ to 3’ decay 1. mRNA 5’ cap removed by DCP2-DCP1 complex 2. Edc3 and Rps28B factors bind to polyA tail 3. XRN-1 5’-3’ exonuclease degrades DNA

Endonuclease-dependent mRNA decay

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mRNA is cleaved internally by endonuclease to reveal 5’-3’ and 3’-5’ ends mRNA decayed in 5’ to 3’ and 3’ to 5’ direction Fragments of mRNA degraded by cytoplasmic exosome + XRN-1 exonuclease

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Purpose: if protein harmful to cell or unnecessary, mRNA is destroyed using endonuclease

*5’ cap: assists in ribosomal binding during translation, prevents mRNA degradation

mRNA degradation occurs in P-bodies (cytoplasm) o

P-bodies have cytoplasmic exosomes  Decapping enzymes (remove 5’ cap)  Deadenylase (removes polyA tail on 3’ end)

miRNA and siRNA regulate mRNA stability & translation o

miRNA/siRNA: small RNA molecules  21-28 bp long  Made from longer ds RNA molecules which are processed to produce shorter, functional miRNA/siRNA

miRNA = translation inhibition, mRNA degradation -

3000+ different Bind different mRNA transcript RNA silencing, post-transcription regulation mRNA bind many miRNA 33% of mRNA have functional miRNA binding sites Abnormal miRNA = pathogenesis

 Bind IMPERFECTLY to target DNA to repress translation, with mismatches  Bind at 3’ untranslated end  Many miRNA needed to repress translation  2-7 seed sequence on miRNA critical for binding to specific mRNA 3’ UTR  Loops form

siRNA = RNA degradation/cleavage -

degrade viral RNA degrade transposon RNA interfering RNA, prevent protein production

 Bind PERFECTLY to target DNA, no mismatches  Degrade mRNA quickly through RNA interference  Made from cleaved ds RNA  Control many nucleus processes  Linear process w/o loops

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Biosynthesis of miRNA and siRNA – RNA interference o

miRNA, siRNA made as large pre-miRNA  Made from longer ds RNA by RNA Pol II  Primary transcript: 5’ cap, polyA tail  Folds back to form long, ds RNA hairpin structure = pre-miRNA  How is miRNA produced? 

Drosha5 – Dicer – RISC – Ago2 – guide 1. 2. 3. 4. 5.

RNase Drosha cuts hairpin ds RNA. Pre-miRNA exported from nucleus by exportin-5 RNase Dicer cuts dsRNA to 21-24 bp fragments Shortened ds RNA binds to RNA-induced silencing complex, RISC Argonaut helicase protein Ago2 (in RISC) unwinds one RNA strand, degrades RNA Remaining guide RNA strand inhibits target mRNA in 2 ways - Hybridizes to homologous mRNA sequence - Impairs mRNA translation

Nucleus: drosha - exportin 1. Drosha: cuts and removes hairpin in ds RNA 2. Exportin: transports miRNA to cytoplasm

Cytoplasm: dicer – RISC - argonaut – Ago2 3. Dicer (endonuclease): cuts ds RNA to small pieces 4. RISC: small ds RNA binds to silencing complex to form inactive complex 5. Argonaut helicase of RISC: unzips ds RNA to form ssDNA, form active RISC complex, removes one of strands of miRNA, degrades it. RISC guided by ss siRNA cleaves target mRNA through Argonaut slicer activity. Cleaved RNA and RISC target other mRNA. Other strand is guide miRNA, binds to complementary mRNA. If miRNA binds imperfectly to RNA = translation repressed If siRNA binds perfectly to RNA = RNA degraded

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miRNA function in gene repression in limb development WT – dicer normal o Dicer present, ds RNA fragmented, miRNA made (RISC, Ago2…)

Dicer mutant – abnormal limb development o No dicer, no miRNA made o No mRNA degradation o Proteins are synthesized o Embryonic lethal mutation

Polyadenylation in cytoplasm = stimulated translation o

mRNA made, transported to translation site (cytoplasm) but sometimes not translated if polyA tail too short  Short polyA tail means mRNA must receive for positive signal to be translated  Receive signal, mRNA polyadenylated in cytoplasm  RNA site: cytoplasmic polyadenylation element (CPE) - CPE binds to CPEB protein - Recruits cytoplasmic polyA polymerase (PAP) - This enzyme lengthens polyA tail, adds A residues - Increase translation & protein synthesis - Neuron development! No translation: short polyA tail, CPEB recruits Maskin which binds to eIF4E on 5’ cap, Maskin protects 5’ cap, translation blocked Translation: CPEB recruits PAP, polyA tail lengthened, no Maskin, translation begins

Translationally active: 1. CPEB is phosphorylated by kinase. 2. Maskin released. 3. Recruit CPSF (cleavage & polyadenylation specificity factor)

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4. CPSF recruits PAP to lengthen polyA tail. 5. Longer polyA tail recruits more PABP1, more CPSF, more PAP. 6. Recruit translation initiation factors Conclusion: always thought that polyadenylation only occurs in nucleus right after transcription, but it also happens in cytoplasm. May want to store mRNA in cytoplasm so proteins can be made later.

Global translation regulation through TOR o o

TOR (target of rapamycin) activates translation TOR: large and conserved cytoplasmic kinase with many substrates  Senses [nutrient, AA, sugars] to determine protein synthesis, cell growth  TOR is master regulatory of cell growth  Important in lymphocytes  TOR hyperactive in cancers (if TOR too active… cancer)  Rapamycin = immunosuppressor  Rapamycin extends lifespan of model organisms

o TOR phosphorylates/activates key translation regulator = S6K  mTORC1 responds to signals from cell-surface signaling proteins to control cell growth

Mammalian TORC1 pathway – active TOR 1. Rheb-GTP activates TOR 2. Phosphorylates/inactivates 4E-BP to release eIF4E, which starts translation 3. Phosphorylates/activates S6 kinase, which phosphorylates ribosomal proteins to start translation 4. Active transcription factors for RNA Pol I/II/III 5. More ribosome, tRNA, translation factor activity Inactive TOR 1. Low energy: AMP kinase phosphorylates/actives Rheb-GAP 2. Stress response: activate Rheb-GAP 3. Rheb-GAP inactivates TOR

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Rheb-GTP = TOR activated

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Rheb-GAP = TOR deactivation

o TOR senses stress, [oxygen], [temp.]  If temp. high, oxygen low: TOR suppressed, translation reduced, only survival proteins made… survival mode  TOR phosphorylates 2 factors

o Steps of TOR activation: Rheb-GAP, Rheb-GTP 1. Growth factor binds to receptor 2. Signal transduction pathways phosphorylate/inactivate Rheb-GAP, so TOR is not deactivated 3. Rheb-GTP activates TOR protein kinase activity (S6K phosphorylated) o TOR: master orchestrator of cell growth irt nutrients    

Controls many processes TOR regulates mRNA translation, rRNA/tRNA transcription Affects cell cycle growth Regulates autophagy: if insufficient nutrients, degrade own proteins to gain AA

Surveillance mechanisms prevent translation of wrongly processed mRNA

o E.G. Wrongly spliced, not all introns removed o All are terminated/edited by mRNA surveillance mechanisms 1. Nonsense mediated decay 2. Non-stop decay 3. No-go decay

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1) Nonsense mediated decay (early stop codon)

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Unspliced intron shifts open reading frame Results in nonsense codons (early stop codon = truncated protein)

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Normally: stop codon after last splicing event (last exon-exon junction)  Exon junction complex (EJC) bound to last exon-exon junction during transport to cytoplasm  Last intron is removed  EJC removed by translating ribosome  Properly processed mRNA translated

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What if intron left? Incorrect stop codon.  Pre-mature stop codon prevents EJC removal  EJC not yet at last intron  Persisting EJC signals mRNA degradation  EJC stays, acts as surveillance mechanism

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2) No-stop decay (no stop codon)

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Normally, stop codon is upstream of polyA tail If no stop codon, ribosome not stopped, goes over polyA tail PolyA tail removed, mRNA exposed to enzymes, instantly degraded Ribosome stalls, causes degradation

3) No-go decay

Damaged mRNA or 2ndary structure o o o o

Intron with secondary structure (e.g. hairpin, loop), which is hard to translate Ribosome stalls on damaged mRNA or on secondary structure Stalled ribosome recruits RNA endonuclease Endonuclease cleaves RNA… degradation...