Day 3: Notch, Beyond coding genes Flashcards
HC 08, 09, 10
Discovery Notch
Drosophila wing variations in shape: smooth wing or notches in wig
> Crossing experiments to find spot in genome
> Notch gene
> Notch gene important in development
Loss Notch
Important role embryo development
> patterning nervous system lost in embryo
Protein Notch
-Transmembrane protein
-In plasma membrane, partly intracellular and extracellular
- EGF like modules: repeats
Humand have .. Notch genes
4
Conservation Notch gene
highly conserved
Developmental roles Notch
- Differentiation
- Proliferation
- Cell death
Cell fate is controlled by …
Long range and local signalling
How does Notch act?
Local interactions
> contact-dependent signalling
How many human Notch receptors and ligands
4 receptors and 5 ligands
> all transmembrane
> extracellular domains can interact
Ligands Notch
Delta
> Dll1 & Dll4
> Jagged1 & Jagged2
> Dll3: decoy (inhibitory for signalling, binds but no signal transduction)
Activation Notch pathway
1 - Ligand binds to Notch receptor (bind)
2 - The NRR (negative regulatory region) domain becomes accessible (pull, ligand pulls receptor a bit out so that NRR is exposed)
3 - The ADAM protease cuts the extracellular part (cut)
4 - Gamma secretase cuts the intracellular part (cut)
5 - The NICD (Notch Intra-Cellular Domain) travels to nucleus and activates transcription
Where is the ADAM protease located
Transmembrane protein on cell with Notch receptor and cleaves NRR with extracellular protease domain
Cis inhibition (Notch)
Notch receiver cell or sending cell determined by ratio receptor and ligand
- Notch > ligand: receiver
- Notch < ligand: sender
> No activation sending cell when binding ligand to receptor: the pulling part is missing, ligand cannot pull for ADAM to cut, not the right conformation (own delta coupled to all notch, cannot pull)
Lateral inhibition
- Notch activation inhibits ligand expression through pathway
- Driven down by reception of signal > to receiving state
- Locks cells into fate into Notch context
- Through filopodia, sending cells can inhibit cells further than the neighbouring cells
Lateral inhibition: are the cells only on or off for Notch?
- A lot of context surfaces possible: not on or off, but stronger and weaker senders and receivers based on spatial conformation
Organs where Notch is important for development
Heart, skeletal muscle, blood vessels, inner ear, nervous system
Role Notch signalling in gut
- Stem cell maintenance in crypts
- Cell fates
Organisation stem cells in intestinal crypt and notch
Stem cells and Paneth cells in alternative organisation
> stem cell is receiver
> Paneth cell is sender
> cells in locked states because lateral inhibition
Notch lateral inhibition in differentiation
-Secretory cells: receivers
-Absorptive cells: senders
> few receiving cells surrounded by all senders
Role Notch in cancer
Mostly oncogene
> in rare cancers: tumor suppressor
Role Notch in hallmarks cancer
> Metastatic behaviour: in EMT (oncogenic)
Notch is P53 suppressor (oncogenic)
Tumor suppressor role: Notch needed for macrophage polarization, immune system is changed when loss Notch
Change of Notch in cancer
Putative driver mutations in Notch1 > hotspot near C-terminus (possible gain of function): small domain/region
> many tumors have this same oncogenic mutation
(> tumor suppressor mutations more spread and not concentrated on one spot)
Where in Notch gene loss-of-function mutations
Middle and N-terminal region (spread, not concentrated same spot)
Other activation method of Notch except gain-of-function mutation
Notch copy number
> more copies: worse prognosis in metastatic CRC numbers
> drive Notch activation
Is Notch mutation solely enough to cause cancer?
No, it is part of driving tumorigenesis, is dangerous in combination with APC loss
Notch and implantation of cancers (xenograft)
Notch positive cells have much better ability to establish tumor after implantation
Notch and stemness of cell
Induced by Notch (also in normal cells)
KPN mice model
KRAS, p53 and Notch mutations > increased metastatic behaviour
> more EMT
> more mesenchymal gene expression profiles
Notch and immune system
Notch activation: make and secrete cytokines
> recruit neutrophils
> downregulate immune cells in tumor (T-cells)
> T-cells do not attack cancer, better survival of cancer
> more neutrophil infiltration
Blocking Notch leads to less neutrophil recruitment, why still useful for cancer
More T-cells infiltrate tumor > the CTLs are the ones that attack the tumor
Crosstalk Notch with other morphogen pathway
Wnt
Notch as target in cancer: which targets
- Ligands > mAbs
- Receptors > mAbs
- Cleavage > Gamma secretase inhibitors
- Transcriptional activation by NICD
Gamma secretase as target
Inhibit second cleavage to release NICD
> not a lot of success achieved: still progressive tumors
> increased dose shows increased Notch inhibition
> still not effective
> result in very few patients
Combinational therapy crenigacestat (gamma secretase inhibitor) with other drugs
Works better
Side effects Notch inhibition: where first problems?
GI tract first affected
> early effect because rapid dividing
> role Notch in stem cell environment and differentiation (gut morphology and homeostasis)
Which part of Notch is directly involved in transcriptional response upon Notch activation
NICD
How does a cell become a Notch sending cell (disregarding outside influence: lateral inhibition)
Cis-inhibition
Given that Notch has a role in maintaining stem cell state, do you expect it to be a oncogene or tumor suppressor gene?
Oncogene
HC09: Evolution and noncoding part DNA
This part increases with the complexity of organisms
Noncoding part human genome is 98%, how large part can be transcribed though?
over 70%
Discovery nuclein
Isolated nuclein > presence uracil > discovery DNA and RNA
Most abundant produced RNA
rRNA
rRNA function
> Form ribosomes with ribosomal proteins
Where production rRNA and systhesis ribosomes with proteins?
In the nucleolus
> organelle in nucleus by phase to phase separation
Formation nucleolus
By five acrocentric choromosomes
> short p-arm of chr. 13, 14, 15, 21, 22
How is the short p-arm from the acrocentric chromosomes which contribute to nucleolus called?
NOR: nucleolus organizing region
NOR consists of
Telomere, rDNA, centromere
> rDNA genes located at NOR (tandem repeats)
> trancription of the coding region only
Intergenic sites of rDNA is … transcribed
Not, only coding parts
Transcription rDNA genes
Transcription by RNA polymerase 1
> 18S rRNA
> 5.8S rRNA
> 28S rRNA
Transcription by RNA polymerase 3
> 5S rRNA (chromosome 1, not in nucleolus)
Assembly ribosomes
Transcription rRNA genes into large 45S pre-rRNA and 5S rRNA separately
Processing/modifications of pre-rRNA
Assembly to ribosomal subunits: 18S to 40S subunit with proteins and 5.8S, 28S and 5S with proteins to 60S ribosomal subunit
Export
mt-rRNAs
2 mt-rRNAs
> 16S mt-rRNA and 12S mt-rRNA
> encoded by mitochondrial DNA (mtDNA)
> Transcription by POLRMT
> (for mitochondrial translation)
Deregulation of rDNA in CRC
Intracellular increase Ca2+ in CRC cell from ER (together with Wnt pathway): emptied storages
> upregulation p-CaMKII (CaMK pathway) and p-UBF (via CaMK pathway)
> UBF involved in transcription of rDNA (part transcription complex to make 45S pre rRNA) > more rRNA made
Block upregulation rRNA transcription by:
Producing BAPTA-AM: chelator of Ca2+ (binder)
KO pre-45S rRNA
Induce cell cycle rest
Upregulation 45S pre-rRNA in cancer
Via pUBF
> for transcription of it in cancer cells
Cancer cells inhibit cell cycle arrest through
Inhibiting p53 through a pathway:
> APC inactivation
> Increased Ca2+
> Activation UBF
> Increasing rRNA levels
> maintain high level translation and prevent p53 dependent cell arrest
tRNA function
Coupling with amino acid and recognize mRNA codons
> close interaction tRNA with ribosome
> to assist elongation of polypeptide chain by supplying amino acids and recognizing genetic code
Ribosome EPA
Exit site, peptidyl site, aminoacyl site
tRNA has … codons
64 codons
> 61 AA codons
> 3 stop codons
tRNA is transcribed by
RNA polymerase 3
> will stop after a stretch of 5 As (adenosines), forms U tail at 3 prime of tRNA
Modifications of pre-tRNA
- Cleavage 5’ leader (RNase P)
- Cleavage 3’ poly U (RNase Z)
- Splicing (TSEN/HSPC117)
- Addition CCA triplet at 3’ prime
- Aminoacylation at 3’ prime
tRNA-derived small RNAs: tsRNA fates
tRNA halves (tiRNAs)
Small tRNA fragments (tRF)
Formation tsRNAs is mediated by ….
Endoribonucleases
> DICER
> ANG (angiogenin)
> Rny1p
tsRNA-dependent regulation of gene expression
- Post-transcriptional silencing
- Nascent RNA silencing
- Transcriptional gene silencing
tsRNA functions
- Regulate stability of transcription
- Histone tail modification
- Translation regulation: prevent ribosome from translating, get RNA trapped in stress granules (YBX1 dependent), stall translation (decoy for elongation factors)
- Cell cycle and proliferation regulation: decoy for cytochrome c
5’tiRNA-His-GTG promotes CRC tumor growth, how?
- Upregulated when hypoxia
- Increase vasculature
- Regulate LATS2 expression
> Hypoxia: HIF-1a activates ANG transcription, ANG processes tRNA to form tiRNA
> LATS2 is known player in hypoxia pathway and phosphorlates YAP for degradation or inactivation: the tiRNA inactivates transcription LATS: YAP actively to nucleus for target genes
> anti-apoptosis: tumor survival and pro-proliferation
How does 5’tiRNA-His-GTG interfere with LATS2
Interfere with endogenous DNA fragment for transcription block
Tumor grows faster than vasculature can handle leads to …
hypoxia
> pathway initiated for 5’tiRNA-His-GTG via HIF-1a and ANG
Which RNA type is part of the central dogma
mRNA
RNAs are the …. of DNA
final effectors
> because also role noncoding RNAs
snRNA (small nuclear RNA) function
Involvement in splicing
RNA splicing by the spliceosome, name the route
5’ exon-intron junction: U1 binds
3’ exon-intron junction: U2 binds
Formation spliceosome: U4, 5, 6 recruited (snRNPs, small nuclear ribonucleoproteins, with protein)
Excised intron in lariat shape
Name the snRNAs from the major spliceosome, minor spliceosome and histone splicing
Major spliceosome
> U1
> U2
> U4
> U5
> U6
Minor spliceosome
> U11
> U12
> U5
> U4atac
> U6atac
Histone splicing
> U7
Difference major and minor spliceosome in humans
Recognize different introns
Transcription snRNAs by…
RNA polymerase 2
only U6 by RNA polymerase 3 (stops after 5 T segment)
What happens with snRNA after transcription
Quickly exported to cytoplasm: interaction with different proteins
> 3 prime trimming, maturation
> nuclear import snRNA: accumulate in splicing speckles
Deregulation U2 snRNPs in CRC
SF3B3 upregulated in cancer (part of U2, splicing factor)
> mTOR splicing: from exon skipping to inclusion new exon (that is the mutant, no alternative splicing of mTOR pre-mRNA)
> mTOR no longer promoting apoptosis but mitigating it because exon 8 inclusion
> induce EMT features because inclusion mTOR exon 8 by high SF3B3
> aggressive cancer
snoRNA discovery (small nucleolar RNA)
Released from 28S rRNA by heat
> rRNA interacts with snoRNA in nucleolus
Transcription snoRNA by …
RNA polymerase 2
snoRNAs DNA are often located in …
introns
Types of snoRNAs
C/D (SNORDs) and H/ACA
Difference C/D and H/ACA snoRNAs in maturation process
Associate with different proteins
> different snoRNPs (ribonucleoproteins)
Production process snoRNPs
Transcription > splicing > debranching > exonucleolytic processing
Different complementariy C/D box and H/ACA box for snoRNA
C/D box is triggered to methylation after complementary annealing rRNA (2’O-methylation)
H/ACA box: triggered by target rRNA binding > uridine to pseudouridine (pseudouridinylation)
Functions snoRNA for rRNA
Catalyze modifications of rRNAs (2’O-methylation and pseudouridylation)
> rRNA modifications impact RNA stability and translation efficiency of ribosomes
snoRNAs and cleavage
Some snoRNAs (both C/D and H/ACA box) required for cleavage pre-rRNAs
> interactions at cleavage sites
snoRNAs have diverse roles. What is the primary function
Modification of rRNAs and snRNAs (including cleavage of rRNAs)
> Targeting is guided by complementarity
Silencing and KO of snoRNAs
Silencing: cell proliferation goes up
KO: regulate oncogenes
Upregulated snoRNA in CRC
SNORD11B
(> present: more methylation, more repression MeUA in primer before RT in experiment when low dNTP: RT more efficient)
> SNORD11B dependent (2’-O) methylation of miRNA let-7 promotes its degradation, prevents downregulation of key oncogenic proteins in CRC (RAS, MYC, YAP)
» C/D box
H19 is first discovered ncRNA in human. Only expressed in … allele and IGF2 only in … allele
Maternal, paternal
> noncoding RNAs
> different enhancers
Xist only expressed when
XX > for X chromosome inactivation
> shield the X chromosome
> an RNA, no open reading frame
HC10: Discovery of the first miRNA: Lin-14 and Lin-4
Expression during C. elegans larval stages
> LacZ gene fused with 3’UTR of interest: blue staining
> Lin-14 only expressed in early larval stages
> Lin-4 (miRNA) mutant, also in later stages, no degradation RNA of Lin-14 via miRNA Lin-4
Regulation of miRNA
By transcription of miRNAs
Expression Lin-4 on western blot
> 61 and 22 nts fragments on western blot
Hairpin pre-miRNA (66 nts) and muture active miRNA (22 nts)
Large gap between identification first and second miRNA (lin4 and let-7), why?
Technical limitations and lack of awareness of miRNAs, more focus protein coding
Let-7 expression in larval stages C elegans
Similar role as Lin4
> Let-7 expression goes up from L4>adult stages of C. elegans
> Lin4 from L1>L2
The let-7 sequence is very … in many species
conserved
Length miRNAs
21-24 nts
> 2600 encoded in human genome
miRNAs are relatively … ncRNAs
small
Biogenesis miRNAs and function
-RNA polymerase 2: transcription
-Highly structured hairpins in pri-miRNA (primary) are recognized by proteins which remove separate hairpins (pre-miRNA)
-Export to cytosol
-Cytosolic proteins: cleavage to mature miRNA and strand selection (one strand selected) > (RNA Induced Silencing Complex (RISC) formed with proteins)
-Translation inhibition or mRNA degradation by binding target mRNA (complementary) (removal cap)
> degradation preferred
miRNA target sites
What is the most important feature to predict miRNA target sites?»_space;
-5 prime
-Seed sequence nt-2-8 is conserved: 5’
-3’ prime supplementary pairing lacks predictive values
-miRNA and mRNA are partially complementary
miRNA in cancer
Downregulated
> miRNA promotes differentiation
> miRNA inhibited, no differentiation, stemness kept
Repression of miRNA expression levels in cancer
-Mutations (biogenesis route)
- DNA methylation (CpG island on gene promotor/enhancer)
- Uridylation (TUT4/TUT7) > target miRNA for degradation (add stretch of urindine U on miRNA)
- Upregulation snRNA (SNORD11B)
miRNA as tumor suppressors: let-7
Reduction of let-7 levels prevents negative regulation of RAS
> promotes cell proliferation and tumor growth (MAPK activation, Mek Erk route)
Oncogenic miRNAs
Upregulated in cancer (this is the minority, majority is tumor suppressor)
> miR17-92 cluster in lymphoma
> similar seed sequence in cluster (same transcripts targeted?)
> Overexpressing Myc: get lymphoma in mice model: more severe and faster tumor growth and transformation of cells when lacking let7
Tsix
ncRNA
> important for regulation Xist transcription
> Reverse transcription on Xist site (transcriptional interference): inhibit expression Xist > Tsix masks Xist RNA domain
> Tsix enhances Xist RNA degradation
Air ncRNA
Together with Igf2r receptor expressed (maternal allele)
> Air only on paternal allele
> Forces Igf2r receptor to be expressed on maternal allele
> forcing paternal and maternal alleles to interact with each other
Big boom miRNA discovery around 2000s, why?
NGS available
HOTAIR
Interacts with chromatin modifiers: modulate histone tails and condensation of DNA
> lncRNA
PCGEM1
Interacts with TFs which interacts with enhancers and promotors
> help with transcription endogenously
> linker between enhacer and promotor via TFs
> lncRNA
MALAT1
lncRNA
> interaction with miRNAs and splicing factors (snRNA)
Mechanisms of lncRNA actions
-Guide: for chromatin modifying enzyme
-Scaffold: for transcriptional proteins
-Decoy: bind trancriptional protein so that it does not bind DNA
-lncRNA sponging miRNA: bind and occupy it (clean it)
-lncRNA as miRNA precursor
-Chromatin looping (like PCGEM1)
lncRNA biogenesis
-Transcription by RNAP II
-Capped and polyadenylated
-Little ORF (open reading frame)
-Some ORFs are a bit translated to little peptides
> Not entirely noncoding always
-RNase P cleaves 3’ prime UTR to get tRNA-like structure and release it
-Or release miRNA like structures (stem loop/hairpin)
lncRNA CRNDE function in CRC
Epigenetically repress cdk1a (cell cycle inhibitor is repressed for G1 progression)
> associate with negative transcription proteins (PRC2 complex) to epigenetically repress cdk1a gene
> lncRNA
KO CRNDE
The complex (PRC2 complex) which can bind p21 is less present
> reduction repressive mark
> switch off transcription cdk1a is stopped: increase cdk1a > inhibit cell proliferation
CCAT2 in CRC
CCAT2 activates Myc expression (upregulated in CRC)
> lncRNA
> CCAT2 interacts with TCF7L2 > for Wnt target gene transcription, needed for full expression of Myc
> other factors are attracted which support CCAT2
Enhancers are much bound by factors like CBP (Creb binding protein, cofactor for RNA pol 2) and POL2, which kind of RNAs support these
eRNA: enhancer RNA
Enhancer RNA biogenesis and types
Short-bidirectional eRNAs (more rare)
> short half life
> Bidirectional transcription
> unspliced
> nonpolyadenylated
Long-unidirectional eRNAs (more widespread, capped)
> Long half life
> Unidirectional transcription
> spliced
> polyadenylated
Where production eRNAs?
Enhancer regions bound by RNA polymerase 2
eRNA biogenesis
- Enhancer domain active (eg H3K27ac)
- Enhancer bound by TFs/GTFs/Pol2
- Transcription initiation leading to RNA capping by CBC (cap binding complex)
- Elongation is regulated by p-TEFb
- Termination is mediated by Integrator (endonuclease) and WDR82/PP1 (pol2 dephosphorylation)
- RNA exosomes degrade eRNAs from 3’ to 5’
Mechanisms of eRNAs o influence transcription
- establishment and/or stabilization of enhancer-promotor looping
- intervening with trancription machinery
- trapping transcription factor and co-activator
- regulation of histone modifications (eg recruit CBP (acetylation) or PRC2 (me3) to promote or inhibit modifications)
ETS2 eRNA (ETS2e) in CRC
Upregulated in CRC
> super-enhancers activated (many enhancers close to each other)
> DNA gets opened up (eRNAs produced)
> in graph, much H3K27ac upstream (or downstream) promotor
SNP involved in Crohns disease> increased risk CRC > why?
T-variation ETS2e (ETS2 eRNA): more ETS2 produced
> eRNA in super-enhancers upregulated
> more ETS2 transcription because high ETS2e
> > ETS2 is oncogene
ETS2e variation C/T SNP
SNP (C/T) in ETS2e creates binding motif for MECOM which drives the expression of eRNAs and higher expression of ETS2
> more ETS2 > IBD, CRC
circRNA function
circular RNA
> can sponge (clean, get rid of) miRNA (loss of function)
Biogenesis circRNA
- During transcription ncRNA or mRNA
- During splicing
- Embedded elements interact > close together to transcript
- back splicing favoured (instead of linear splicing in mRNA): circle formed
- Only with exons, can be one exon, can include introns
Mechanisms of action circRNAs
- miRNA sponges or decoys
- protein sponges or decoys
- enhancer of protein function
- protein scaffolding
- protein recruitment (for example TF to loci or to subcellular component)
- templates for translation (production unique circRNA peptides)
circRNA and CRC
Can increase cell proliferation
> circRNA from SKA3 mRNA (back-splicing of one of the exons) > has-circ-0000467
» overexpression: Myc goes up (proliferation)
» E-cadherin goes down (EMT induced)
» cdkn down (less cell cycle control, proliferation)
How does circRNA help with translation of Myc
- Interact with 3’UTR of Myc
- Bind elongation factor
- Interact with translation initiation factor for Myc
- More Myc > more metastasis, proliferation and migration
Relative RNA expression circ467 and SKA3 when RNase R+ or RNase R- (RNase R treatment)
(skip this maybe)
RNA expression circ467 for both present
RNA expression SKA3 absent for RNase R+
C/D box snRNAs main function
methylation rRNA
Which nucleotides is for binding of miRNA
Seed sequence: nt2-8, (first ten)
lncRNA can interact with?
-Protein
-mRNA
-miRNA
eRNAs mainly function … (cis/trans)
in cis
Name functions rRNA, tRNA, snRNA and snoRNA in cancer
- rRNA: transcription increases (Ca2+ dependent) to support proliferation and prevent p53 stress response
- tRNA: fragments with different functions (eg miRNA like) can be generated by endonucleases (eg Dicer and Ang)
- snRNA: splicing can be influenced (inclusion vs skipping) by deregulation of splicing factors
- snoRNA: influence stability of other RNA (eg let-7a miRNA)
miRNA function name (RNA?)
Post-transcriptional regulation: RNAi
lncRNAs are gene expression regulators: how
- Bind chromatin modifiers (eg PRC2)
- Interact with TFs (eg AR)
- Sponge/decoy miRNAs or proteins (eg malat1)
eRNAs (1D, 2D (directional, unidirectional long living or bidirectional short living) functions as transcription regulators
- Bind chromatin modifiers (eg PRC2, CBP/p300)
- Stabilize chromatin structure (eg cohesin, mediator)
- interact with TFs (eg YY1)
- interact with transcription cofactors (eg BRD4)
circRNA as gene expression regulators
- bind chromatin modifiers (eg TET1)
- sponge/ decoy miRNAs or proteins
- serve as scaffold
- can be translated to produce peptides
miRNAs, lncRNAs, eRNAs and circRNAs in CRC
- miRNAs: mostly downregulated in CRC (mutations/biogenesis), can also exert oncogenic functions: miR17-92
- lncRNAs: often deregulated in cancer (eg CRNDE/PRC2 and CCAT2/TCF7L2)
- eRNAs: often epigenetically deregulated in cancer (activated or repressed), occasionally, genetic changes or predispositions (SNPs) can modulate eRNA expression (eg ETS2e)
- circRNA: back-splicing as well as function of circRNAs can be deregulated in cancer (eg has-circ-0000467/MYC)
insert kahoot questions
