1. control of eukaryote transcription

Eukaryotic cells

1.1 what are the different RNA pol involved in the transcription

1. RNA pol I : transcribes ribosomal RNA ex. 18S RNA

2. RNA pol II : transcribes mRNA & different RNA (sRNA)

3. RNA pol III : transcribes tRNA and sRNA,miRNA

1.1 what is the required DNA sequences for efficient initiation of transcription in yeast

Promoter w/ : 

• UAS = Upstream Activation Sequence : binding site for activator proteins

• TATA box at -90

1.1 what is the required DNA sequences for efficient initiation of transcription in mammals 

Promoter w/ : 

• distal : Enhancer/Silencer binding site 

• proximal : multiple binding sites for activator proteins 

• core : TATA box at -30

after TSS/+1 there is the presence of introns (non coding sequences) and exons (coding sequences)

1.2 what is required structure of RNA pol II for minimum transcription/ basal transcription

RNA pol II has 12 subunits with 2 bigger sub units at the C terminal domain (CTD)

The CTD has a sequence repetition where the SER 2 & 5 are subject to phosphorylation —> can impact transcription 

• nb of repetitions depends on the species and complexity 

1.2 regarding the CTD of RNA pol II how does its’ phosphorylation affect its binding to promoter 

Hypo-phosphorylated —> RNA pol II binds to the promoter 

Hyper-phosphorylated —> RNA poll II released from promoter and begins elongation 

1.2 An experiment was carried out to test basal transcription, what was the conclusion ?

that DNA fragments (TATA, TSS etc.) + RNA pol II + nuclear extracts there was minimal transcription 

• wasn’t the case when nuclear extracts were absent 

proteins in the nucleus are involved in the activation of transcription = Transcription Factors 

1.2 How are the transcription factors TF assembled to initiate transcription

• TFIID binds to TATA box ( and other sequences but for a lesser affinity)

• TFII A and B bind to TFIID

• TFIIF helps the fixation of RNA pol II to the TFII B/D/A

• TFII E & F and TFIIH binds to the RNA pol II

  • TFIIH = kinase that phosphorylates SER 2 & 5 on the CTD

==> Pre-Initiation complex PIC

1.3 how does the mediator help in the activation of transcription 

it helps install PIC :

• recrute TFs

• interacts w/ repressors o block RNA pol II , it can block : 

  • fixation and action of activator 

  • action and recrutement of mediator 

  • condense chromatin 

1.4.1 what is the structure of chromatin

organised in nucleosomes : 

• histones wrapped around by DNA

• histone tailed

• linker DNA that connects the 2 nucleosomes  

1.4.1 difference in chromatin condensation and activity 

heterochromatin = condensed and transcriptionally inactive 

euchromatin = loosely packed and transcriptionally active 

1.4.1 how do proteins such as TFII and activators bind to chromatin

Pioneer ACT will bind to the ACT binding sit on the DNA in between the nucleosomes

• ejection or displacement of nucleosomes 

• more ACT binding sites available 

• TATA box available 

TFIID can bind to TATA box and form PIC

1.4.2 what are examples of epigenetic marks

1. DNA methylation

2. Post translational modification of histones

1.4.2 how is the methylation of DNA controlled

a DNMT DNA methyl transferase will methylate a CG s DNMT = writer

a TET ten-eleven translocation will remove the methylation so TET = Eraser

1.4.2 what is methylation usually related to? activation or repression of transcription?

methylation is mostly related to the inhibition of transcription

1.4.2 what are the types of post translational modification histones can undergo ?

on the Nt histone tail : 

• phosphorylation on the Ser

• Methylation of Lys (K) or Arg(R)

• acetylation on K or R

on the Ct histone head : 

• ubiquitination on a K

• sumoylation 

1.4.2 how does histone post translational modification impact the activity of transcription

certain modicifaction done by the writer on the nucleosomes recruit a read that will determine the repression or activation of transcription

1.4.2 how does acetylation and methylation on Lysine of stone tail affect transcriptional activity

• when acetylation of K occurs it looses its positive charge —> liberates tail of the histone —> loosens chromatin

= Euchromatin (genes are accessible and so activation of transcription)

• when K methylated it is done so multiple times and is tri-methylated —> positive charge remains —> cremation remains compact 

= Heterochromatin (genes are inaccessible so inhibition of transcription)

1.4.3 what histone marks are correlated to activation/repression of transcription

activation of gene transcription :

• H3K4-me1,H3K27-ac

• CpG un-methylated 

repression of gene transcription : 

• H3K9-me3

• un-acetylated histones 

• methylated CpG

1.4.4 how is heterochromatin formed and gene expression inhibited 

1. REP binds to REp binding site 

2. a writer = H3K9MT methylates H3K9-me3

3. a reader = H3K9MT reads the repressor methylation signal and continues to ti-methylate H3K9 on each nucleosome 

4. progression of the mark along the sequence 

5. a reader = HP1 recognises the H3K9-me3

6. this recognition recruits DNMT which methylates CpG on DNA —> heterochromatin 

inaccessible genes and therefore of transcription 

1.4.5 how is euchromatin formed 

1. A pioneer transcription factor binds to its specific DNA sequence making the DNA more accessible.

2. The pioneer factor recruits a writer enzyme ( a histone acetyltransferase (HAT) ) which acetylates lysine residues on histone tails (e.g., H3K9ac, H3K27ac).

3. A reader protein with a bromodomain recognises and binds to the acetylated lysines on histones.

4. Chromatin remodeling complexes (such as SWI/SNF) are recruited — these have ATPase activity that uses ATP hydrolysis to slide or evict nucleosomes, exposing promoter and enhancer DNA.

euchromatin formed, genes accessible

1.4.5 how does the formation of euchromatin activate gene expression

1. Closed chromatin (heterochromatin): Histones are deacetylated, DNA is tightly packed, and transcription is inactive.

2. A PTF pioneer transcription factor binds its enhancer site even in condensed chromatin and recruits a HAT (histone acetyltransferase).

3. HAT acetylates histone tails, loosening DNA–histone interaction and marking chromatin for activation.

4. Reader complexes (e.g., Swi/SNF) recognize the acetyl marks and slide or remove nucleosomes, creating nucleosome-free regions.

5. Activator binding: The open DNA allows activator transcription factors to bind promoters/enhancers and recruit more HATs, extending euchromatin formation.

6. Mediator and PIC recruitment: Activators recruit the Mediator complex, which helps assemble the Pre-Initiation Complex (PIC) with RNA polymerase II.

=> Transcription initiation : The open euchromatin and assembled PIC enable RNA Pol II to begin transcription.

1.4.6 How does the repression of gene expression in S.cerevisiae (yeast) by Gal4

absence of galactose (and Glc) : 

• the DBD of Gal4 (Transcription factor) binds to its binding site UAS ( upstream activation sequence)

• Gal 3 remains free and inactive in the cytoplasm -  as its not bound to Gal it doesn’t bind to Gal 80

• Gal 80 binds to AD of Gal4 —> inhibiting its activity and unable to recruit transcription machinery (Mediator and PIC)

• chromatin stays condensed 

—> no transcription of GAL genes

presence of Glc :

• Gal 80 binds to AD of Gal4 —> inhibiting recruitment of transcription machinery

• activation of a repressor that’s binds to its binding sequence

• recruitment of HDAC histone de-acetylase —> removes Ac histone marks and chromatin becomes condense (heterochromatin)

—> no transcription

1.4.6 How does the activation of gene expression in S.cerevisiae (yeast) by Gal4

presence of galactose (absence of Glc) :

• Gal 3 binds w/ Gal 80 —> repressing the activity of Gal 80 and can no longer bind to Gal4

• Gal4 no longer inhibited by Gal80

• Gal4 bound to UAS recruits writer SagA that acylated histone tails

• installation of mediator but no transcription

• reader Swi/SNF recognises Ac histones and allows the sliding of nucleosomes

• promoter is free and TFII can now bind and permit installation of PIC takes place

transcription of GAL genes takes place e.g genes coding for enzymes that metabolise Gal

1.4.7 binding sites of AP1 and NFKB in macrophages to regulate activity of induced genes by an infection by a pathogen

AP1 = DNA binding for FOS-JUN and JUN-JUN dimers

NFKB binding site is located after AP1 BS —> NFKB binds here  

1.4.7 pre-infected macrophage how is the transcription of genes regulated 

pre-infected cell :

• JUN-JUN binds to AP1 

• this allows recruitment of several proteins

  • H4K20MT —> adding me to histone tails 

  • HDAC —> remove Ac from histone tails 

• chromatin remains condensed and genes are unavailable for transcription (nucleus)

• NFKB remains bound to IKBa (inhibitor) in the cytoplasm —> NFKB is inactive

no transcription  

1.4.7 infected macrophage how is the transcription of genes regulated

infected : 

• FOS-JUN dimer binds to AP1 

  • recruitment of HAT Histone Acetylase transferase proteins 

• IKBa is phosphorylated chi liberates it from interaction w/ NFKB

• NFKB is free In the cytoplasm and translocates into nucleus to bind to its BS

  • recruits histone de-methylase

• euchromatin —> chromatin open

transcription of immune response genes takes pace 

1.4.8 transcription of PER and CRY control biological clock

1. Activation Phase:

   • CLOCK and BMAL1 (Act) form a heterodimer and bind to E-box elements in the promoters of Per and Cry genes —> activation of transcription of Per and Cry mRNAs.

2. Translation and Post-translational Modification:

   • Per and Cry proteins are translated in the cytoplasm

   • They undergo phosphorylation

3. Formation of Repressor Complex:

   • Per and Cry form dimers

   • The P-C dimer translocates into the nucleus when there’s an accumulation of P-C dimer in cytoplasm 

4. Negative Feedback:

   • In the nucleus, the P-C dimer binds to CLOCK-BMAL1 on E-box, inhibiting further transcription of Per and Cry

5. Degradation Phase and Cycle Reset:

   • Over time, P-C dimer detaches from Act proteins when too little P or C protein in the cytoplasm

   • new transcription of Per and Cry begin

Cycle Continuation

1.4.8 how is the clock control gene CCG regulate gene expression 

• CCGs have E-boxes elements in their promoters

• When CLOCK-BMAL1 is active (i.e., not inhibited by PER-CRY), it binds to these sites and activates transcription of CCGs

• when C and P accumulates the C-P dimer translocates into nucleus and binds to Act proteins on e-box the transcription of CCGs are inhibited

1.4.8 biological clock : SCN suprachiasmatic nucleus

sigh stimulates SCN neurons

PKA + AMPc —> phosphorylates CREB

P-CREB binds distal binding site (CRE BS)

this along w/ the binding of CLOCK & BMAL1 to e-box activates the transcription of Per and Cry resetting the clock

Separately, the CLOCK:BMAL1 complex regulates circadian transcription independently of light input:

CLOCK and BMAL1 are TF that form a heterodimer and bind to E-box elements in promoters of Per and Cry genes.

PER and CRY proteins accumulate, form complexes, and then inhibit CLOCK:BMAL1 activity — this creates the ~24-hour oscillation.

1.4.9 how is the transcriptional memory established

Activation of gene transcription 

1. A signal activates an activator (TF₁) that binds to its binding site/enhancer in the promoter region of gene A

2. Gene A is transcribed and translated into protein A (TF₂).

3. TF₂ binds to an activator binding site in its own promoter, reinforcing its own transcription

4. This self-sustaining transcriptional loop establishes transcriptional memory, allowing the gene to stay active even after the original signal is gone

Epigenetic marks/histone modifications : 

1. Upon activation due to a signal, the TF recruits a writer (e.g., histone acetyltransferases)

2. These “writer” enzymes add activating histone marks, at the promoter/enhancer

3. These epigenetic marks keep chromatin in an open, transcriptionally active state = euchromatin

4. The marks persist, maintaining continued transcription — even in the absence of the original stimulus.

1.4.9 how is transcriptional memory used in cell division

Epigenetic marks/Histone modification :

• TF/act protein activated due to a signal 

• TF binds to enhancer and recruits writer 

• Histone modification takes place and remains on the newly synthesised cells

• writer can recognise histone tag on these newly synthesised cells

• gene A will have the same signal in new cells  

1.4.10 Control of known transcription initiation by a lncRNA (2 known roles)

known role : 

• lncRNA HOT AIR repression of HOX D gene : recognises specific sequences that will recruit silencing writers methyl transferase and de-methylase —> gene silencing 

• lncRNA Firre : nuclear organiser, 8 different mRNA sequences on lncRNA Firre recognises a specific nuclear matrix proteins. this protein recognises specific sequence on chromosome and brings the chromosomes together to create chromatin contact

1.4.11 3D gene organisation and topology associated domains thanks to activator and mediator

If two genes are too far away on different chromosomes :

• Act will bind to enhancer in chromosome 1 

• mediator recruited which will bring the two chromosomes together and induce transcription go both genes

If two genes are too far away on the same chromosomes : 

• DNA bending protein will bind to the chromosomes and bed the the DNA sequence to bring two genes tother. transcription activated by Act + mediator

1.4.11 how does 3D genome organisation take place

inside chromosomes territories there are TAD domains (topologically associated domain) that are isolated by CTCF insulator proteins so that only the genes present on TAD are transcribed

1.4.11 following a signal what happens the nucleus organisation and TAD reorientation 

in the nucleus we have two compartments : A = compact (heterochromatin) and B = less compact and central (euchromatin)

following a signal TAD domain can change department which will change the expression of genes on TAD

1.4.11 what lncRNAs are involved in 3D genome organisation

• lncRNA Firre 

• lncRNA Xist

1.4.11 how does lncRNA Firre rearrange the genome 

lncRNA Firre which assembles multiple chromosomes together by recruiting nuclear matrix proteins that recognises sequences on chromosomes along with specific sequences repeated RNA domains on lncRNA Firre 

1.4.11 how does lncRNA Xist switch between active/inactive transcription

lncRNA Xist acts on one of the chromosomes X in females where only one chromosome X remains active during the embryonic development  

Xist condenses the chromosome to inactivate it : 

• Xist is only expressed but the chromosome that will be inactivated 

• Xist will recognise a protein SafA present on chromosome X which helps it attach to chromosome

• once bound to SafA —> recruitment of writers that silence gene expression e.g H3K27MT and HDAC

• the inactivated chromosome X is brought o compartment B (Heterochromatin)

• Xist interacts w/ lamin receptor on nuclear membrane which allows the inactive chromosome to remain in the heterochromatin/B heterochromatin

1.5 how does alternative promoters control transcription initiation in simple genes

• 5’ CAP & 3’ Poly A : protects against exonuclease

• 5’ CAP facilitates loading of mRNA on ribosome (translation)

• 3’ polA export of mature mRNAas

• regulate mRNA translation and stability

1.5 how does alternative promoters control transcription initiation in complex genes

Alternative promoters allow complex genes to initiate transcription from different start sites under different regulatory conditions.

alternative promoter can change protein addressing e.g nuclear/mitochondrial localisation signal

2. the role of RNA Pol II CTD after transcription initiation 

hybridisation of PIC to promoter = initiation of transcription

phosphorylation of S5 by TFIIH on RNA pol II CTD recruits Capping enzyme —> capping of mRNA during translation

once capping is done the phospho-SER5 is dephosphorylated

phosphorylation of ser2 : phospho-S2 recruits splicing complex (SPL + CPA) —> co-transcriptional splicing takes place

SPL dissociates at the end of transcription and CPA remains and stops transcription at polyA site

3.1 how does the cleavage cleavage at polyA on mRNA take place

DNA sequence : 

• USE = upstream stimulatory element (T/A)

• polyadenylation motif (T/A)

• polyadenlyation/cleavage site 

• DSE =down stream stimulatory element 

mRNA transcribed : factors recruited due to specific sequence on mRNA from DNA 

• CPSF (cleavage and polyadenylation specific factor) binds to polyadenylation motif 

• CF I/II (cleavage factors) and PAP (polyA polymerase) binds to polyadenylation site  

• CstF (cleavage stimulation factor) binds to DSE

the further the DSE sequence resembles the consensus sequences the healer it is and therefore polyA is less

3.2 polyadenylation efficiency can be controlled by protein such as U1A protein, how?  

When U1A protein levels are high (few snRNA U1 complex):

• Excess U1A now binds to both U1A binding sites in its own mRNA’s 3′ UTR.

• When two U1A molecules bind, they interact with poly(A) polymerase (PAP) and inhibit its activity.

• Result:
  ❌ No (or very short) poly(A) tail added →
  ❌ mRNA is unstable and rapidly degraded by exonucleases.

• This reduces production of more U1A protein.

When U1A protein levels are low (lots of snRNA U1 complex):

• There is little U1A available in the cell as lots bound to snRNA  

• The U1A mRNA has two weak U1A binding sites in its 3′ UTR, near the polyadenylation signal (AAUAAA).

• Because U1A levels are low, these sites remain mostly unbound.

• This allows poly(A) polymerase (PAP) to bind normally →
  ✅ Efficient polyadenylation → stable U1A mRNA → translation of more U1A protein.

3.3. strength of alternative polyadenylation sites 

proximal sites PA1 = weak

distal sites PA2 = strong

3.3. 3’ UTR (untranslated region) and alternative PA

if PA1 used : little to no effect on stability, localisation or ability to be translated 

if PA2 used : can effect stability, localisation and ability to be translated 

=> final protein remains unchanged but its quantity and localisation can vary depending on PA used 

3.3. CD coding region and alternative PA

if PA are places in different coding regions

PA1 used —> shorter coding sequence translated 

PA2 used —> longer coding sequence translated

=> depending on PA used proteins may differ

3.3. how does the choice of alternative PA effect transcription

APA (alternative polyadenylation) modulates :

1. level of cstF (cleavage stimulation factor)

2. elongation rate of RNA pol II

3. splicing

3.3.1 how does the choice of APA in UTR effect CstF levels

CPSF (cleavage and polyA specificity) and CstF (cleavage stimulation factor)

USE Upstream stimulator element

DSE downstream stimulator element

Quiescent cells : non proliferating cells so need less CPSF and CstF

• CPSF binds to USE and little CstF binds to strong distal DSE PA2

• mRNA is longer and has motifs that control stability and ability to be translated 

  • longer transcript —> instable and few proteins translated

proliferating cells : needs more CPSF and cstF

• CPSF binds to USE and CstF binds to both DSE of PA1 and PA2

• increased polyA at PA1 —> short transcript w/out motifs that control stability and ability to be translated

  • proteins translated are stable and many are produced (necessary for proliferation )

3.3.1 how does the choice of APA in CR coding region effect CstF levels

CstF cleavage stimulator factor 

DSE downstream stimulator element 

few CstF : 

• CstF binds to DSE to strong distal PA2

• coding sequence between PA1 and PA2 excluded 

—> Ig in the membrane of Bcell = BCR

lots of CstF : 

• CstF binds to DSE of PA1 and PA2 —> PA1 favoured 

• coding sequence between PA1 and PA2 spliced/included 

—> secreted Ig from pasta cell 

this can effect whether Ig will be transmembrane or cytoplasmic 

3.3.2 how does the choice of PA effect elongation rate of RNA pol II

protein attached to RNA pol II can modify its conformation which changes its speed of transcription 

A slower Pol II gives RNA processing complexes more time to act, promoting use of proximal poly(A) sites/PA1
faster Pol II often skips these and uses distal sites/PA2, producing longer mRNA

3.3.2 how does the speed of RNA pol II effect the use of APA and so change the expression of cyclin D1

normal cell :

• ARN pol II moves rapidly

• so proximal PA1 is skipped and distal strong PA2 is used 

• long mRNA w/ 5 exons corresponding to cyclin D1

Cyclin D1 >> cyclin D1b

tumour cell : 

• RNA pol II moves slow 

• proximal PA 1 used as factors have more time to bind and induce polyA

• short mRNA w/ 4 exons expressed corresponding to cyclin D1b

Cyclin D1 << cyclin D1b

3.3.3 how does splicing take place

premRNA :

• nuclear Cap binding complex binds to 5’ CAP —> define beginning of exon and prevents degradation

• snRNPU1 at the 5’Splicing Site SS of the intron

• U2AF binds to 3’SS = auxiliary factor

• SF1 and snRNP U2 is recruited at branch point thanks to U2AF and binds to branch point

• snRNP U2 recruites other snRNP U4/5/6 and SF1 is removed

= spliceosome

3.3.3 how does splicing change the choice of polyA (APA)

the interaction of U2Af at 3’SS w/ CFII (cleavage factor)

+ the interaction of snRNP U2 at branch point w/ CPSF (cleavage and polyA specific factor) triggers polyA complex installation 

this causes the exon to be caught in this crosslink

4. is the control of splicing another way of controlling transcription ?

YES

splicing sites can have different strengths depending on how close they are to the consensus sequence : pyr (C/U)

weak sequence : pur (A/G)

4. what are the different types of alternative splicing 

1. optional exon skipping 

2. mutually exclusive exons 

3. choice between two SS

4.1 how does alternative splicing change the expression of genes for totipotent and differentiating cells 

mutually exclusive splicing of TF FOXP1 where gene 18A/B codes for DBD DNA Binding Site

differentiated cells : 

• gene 18A is spliced/included 

• FOXP1 w/ 18A DBD will bind to promoter for genes for differentiation 

embryonic totipotent stem cells : 

• gene 18B is spliced/included 

• FOXP1 w/ 18B DBD binds to promoter for totipotency genes

4.2 how is alternative splicing controlled by activator/repressor

activation model : 3’ and 5’ SS are weak

• act proteins bind to E/ISE exonic/intronic splicing enhancer

• insuring the binding of splicing factors (U2Af and snRNP U1) to 3’SS and 5’SS

repression model : 3’ and 5’ SS are too strong

• Rep binds to E/ISS exonic/intronix splicing silencer

• inhibits the installation of splicing factors (U2AF and snRNP) —. no spliceosome 

4.2 what are examples of activators and repressors

Activator : 

• SR protein w/ SR domain that’s contains Arg-phosphoSer

• RBD ad RNA binding domain 

Repressors : 

• hnRNP —> ESS

• PTB —> ISS

4.2 using the example of pyruvate kinase M how does the abundance of repressors effect transcription 

Mutually exclusive splicing

Adult : PKM1 expression 

• exon 9 spliced and exon 10 excluded 

embryo and Cancer : PKM2

• exon 10 spliced and exon 9 excluded 

• due tosplicijg silencers PTB and hnRNP A1 that bind to ESS in exon 9 (silencing of exon 9 splicing) and ISS 

• in cancer MYC = TF over expresses PTB and hnRNP A1 (splicing silencers)

4.2 using an example of MCL1 (involved in apoptosis) how does the abundance of activators the transcription 

3’ and 5’ SS of exon 2 are weak 

• MCL1-L (Long): anti-apoptotic.

• MCL1-S (Short): pro-apoptotic (lacks transmembrane domain).

• High SR protein ASF/SF2 favors MCL1-L → cell survival.

• Stress or pro-apoptotic signaling ↓ ASF/SF2 → exon skipping → MCL1-S → apoptosis.

4.2 auto-regularion of SR proteins - change of splicing factor activity

• SR proteins promote inclusion of a “poison exon” with a premature STOP codon.

• Produces a transcript degraded by Nonsense-Mediated Decay (NMD).
  → Negative feedback keeps SR levels stable.

4.2 how is the activity of SR changed by SRPK

SRPK kinases (activated by EGF signaling) phosphorylate SR proteins, affecting localization to translocate inside nucleus and activate splicing activity

• Heat shock → activates HSP70 → changes SR protein phosphorylation → global splicing reprogramming.

• EGF/AKT pathway → activates SRPK kinases → SR proteins phosphorylated → new exon inclusion pattern.

4.2 change of localisation of splicing factor : hnSNP A1

Under osmotic stress, hnRNP A1 shuttles to cytoplasm → less nuclear repression → splicing patterns change.
→ Rapid environmental adaptation.

4.2 Immune example: CD45 gene (RA/RO isoforms)

• T-cell receptor (TCR) signaling affects hnRNP LL and PSF activity → regulates inclusion of exon A.

• Naive T cells (CD45RA) vs activated T cells (CD45RO) produce different CD45 isoforms.

SO 

• CD45RA (naive T cells): exon A included.

• CD45RO (activated T cells): exon A skipped.

• Controlled by hnRNP LL and PSF, regulated by TCR-GSK3 signaling.
  → Allows T cells to adapt splicing during activation

4.2 how does speed of RNA pol II

• Slow elongation: spliceosome has more time → inclusion of weak exons.

• Fast elongation: spliceosome skips weak sites → exon exclusion.
  → Splicing is co-transcriptional; transcription kinetics directly influence it.

4.2 Epigenetic marks: FGFR2 example

• H3K36me3 recruits MRG15, which attracts PTB to silence exon IIIb.

• Tissue-specific histone modification determines exon inclusion (e.g. IIIb vs IIIc in FGFR2).

• Two mutually exclusive exons (IIIb, IIIc).

• H3K36me3 histone mark recruits MRG15, which binds PTB, repressing IIIb in mesenchymal cells.
  → Epithelial vs mesenchymal tissues express different FGFR2 isoforms due to chromatin context.

4.2 RNA pol II partners

Different co-factors (X or Y) associated with Pol II determine whether a weak exon is spliced in or skipped.

• Pol II’s C-terminal domain (CTD) recruits RNA-processing factors (capping enzymes, splicing factors).

• Depending on its phosphorylation state (Ser2/Ser5), it interacts with different splicing regulators → cell-specific outcomes

4.3 splicing defects and genetic disease general mechanism 

mutation that can : 

• inactivate 3’SS or 5’SS

• create a 5’SS or 3’SS

• creating ESS/ISS/ISE/ESE

4.3 myotonic dystrophy Type 1 - splicing defect

• Expansion of CTG repeats in DMPK 3′UTR.

• RNA with long CUG repeats sequesters MBNL1 protein (a splicing regulator).

• Loss of MBNL1 → mis-splicing of many pre-mRNAs (e.g., ClC-1, INSR).
  → Muscle weakness, myotonia, insulin resistance.

4.2 Duchenne muscular dystrophy DMD - splicing defect 

• Mutations create cryptic splice sites in introns → inclusion of pseudoexons → nonfunctional dystrophin.

• Therapeutic approach: antisense oligonucleotides to block aberrant sites and restore the reading frame

4.2 cancers - splicing defect 

MYC overexpression alters splicing factor levels → favors pro-tumor isoforms (e.g., PKM2).
→ Aberrant splicing = hallmark of cancer metabolism and signaling

5. how does a mRNA exported to cytoplasm look like ?

At the 5′ end — the “Cap Structure”

• The 5′ cap (7-methylguanosine) is bound by the nuclear Cap-Binding Complex (nCBC) made of:

  • CBP20

  • CBP80

Within the coding region — Exon Junction Complexes (EJCs)

• After splicing, an EJC (Exon Junction Complex) is deposited ~20–24 nucleotides upstream of each exon–exon junction.

• Core components:

  • eIF4AIII

  • MAGOH

  • Y14 (RBM8A)

  • MLN51 (CASC3)

• Associated with UPF3 (and later UPF2, UPF1) for Nonsense-Mediated Decay (NMD) control.

Throughout the mRNA — hnRNPs and SR proteins

• hnRNPs (heterogeneous nuclear ribonucleoproteins) coat the mRNA to protect it and help in packaging.

  • e.g., hnRNP A1, hnRNP C

• SR proteins (splicing activators) remain bound and help recruit export adaptors.

= shuttling proteins

At the 3′ end — Poly(A) tail and Poly(A)-binding proteins

• The poly(A) tail (≈200 adenines) has two major binding proteins:

  • PABPN1 (nuclear) — binds the tail in the nucleus during processing.

  • PABPC1 (cytoplasmic) — replaces PABPN1 after export and interacts with eIF4G to stimulate translation.