How to make RNA? TRANSCRIPTION

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TRANSCRIPTION How to make RNA?

Institute of Genetics and Biotechnology University of Warsaw

Cbp20 Cbp80

CTD

Pol II

m

7

G CP

(2)

1) chromatin

5) translation (mRNA) 6) protein stability

3) RNA processing 2) transcription

4) RNA export

7) RNA degradation

1

2 3

3

4 5

6

REGULATION OF GENE EXPRESSION

7

(3)

All steps of RNA processing affect

gene expression

Manning et al, NatRevMolCellBiol 2016

(4)

Transcriptome diversity and gene expression

Pelechano, Yeast 2017

(5)

Processing

Export

Transcription

Translation

eukaryotic cell

RNA

coding- mRNAs

non-coding- ncRNAs - housekeeping

- regulatory - short

- long

polyadenylated

non-polyadenylated

• stable

unstable

capped

uncapped

(6)

1. Chromatin structure and modifications, histones, nucleosomes

2. Eukaryotic polymerases

3. Promoters, activators, enhancers 4. Factors, reguators, complexes

5. Initiation, elongation, termination 6. Co-transcriptional processes

TRANSCRIPTION

(7)

CHROMATIN

transcription

silencing

(8)

Caterino and Haye, 2007, Nature

H2A

H2B

H3 H4

~147 bp DNA wrapped around histone octamer

CHROMATIN: STRUCTURE

8 histones:

2x {H2A, H2B, H3, H4}

linker DNA (50 bp)

linker histone H1

(9)

CHROMATIN: STRUCTURE

Heterochromatin

• constitutive

- always present in a cell, - devoid of genes

(centromeric, telomeric regions)

• facultative - temporary

- often tissue or cell specific - during some cell cycle phases

(DNA during mitosis is

heterochromatic)

C EN TR OM ER E

euchromatin

- loosely packed

- contains transcriptionally active genes

euchromatin DAPI cetromeric

heterochromatin

Deal, et al, 2007, Plant Cell

H2A.Z antibody

(10)

DNA chromatin

histones

DNA methylation histone postranslational modifications

ATP-dependent chromatin

remodelling histone

variants non-codnig RNAs

Dulac, 2010, Nature

CHROMATIN: LEVELS OF REGULATION

(11)

TTCGCCGACTAA

5-meC

Covalent DNA modification in mammals and plants

O N NH2 N

O N

N NH2

CH3

cytosine 5-methylcytosine

Function of DNA methylation: imprinting, X chromosome inactivation,

embryonic development, silencing of repetitive sequences and transposons

DNA METHYLATION

MET1 (METHYLTRANSFERASE1) – 5'-CG-3’ i 5’-CNG-3’

- silencing of transposons and DNA repeats - genomic imprinting

CMT3 (CHROMOMETHYLASE3) – 5'-CHG-3’ (H= A, C or T ) - plant specific

- can be recruited by histone methyltransferase SUVH4 (KYP) - correlated with histone modification

DRM1/DRM2 (DOMAINS REARRANGED 1/2) – 5'-CHH-3’

- de novo methylation – DRM2

- methylation of DNA repeats silenced by siRNA

METHYLATION IS REVERSIBLE (demethylation)

(12)

DNA (DE)METHYLATION - ENZYMES

Law and Jacobsen, 2010, NatRevGenet

(13)

DNA METHYLATION

Piccolo and Fisher, 2014, TiCB; Bergman and Cedar, 2013, Nat Str Mol Biol.

Targeted de novo site-specific DNA metylation involves histone methylases

(14)

Symmetric CG methylation is maintained by replication (typical for mammals)

MET1

5’ A T G C G T A C T

A T G C G T A C T T A C G C A T G A

conservative methylation

A T G C G T A C T T A C G C A T G A

A T G C G T A C T T A C G C A T G A 3’ T A C G C A T G A

A T G C G T A C T T A C G C A T G A

Initiation and maintenace of assymetric methylation (CHH) depends on

histone modifications and occurs via RNA-directed DNA methylation (RdDM)

5’ A T G C A A A C T

A T G C A A A C T T A C G T T T G A 3’ T A C G T T T G A

A T G C A A A C T T A C G T T T G A

A T G C A A A C T T A C G T T T G A

siRNA DRM2

Methylation of some cytosines is maintained by siRNA and RdDM

DNA METHYLATION

(15)

DNA METHYLATION

Jones, 2012, Nat Rev Genet

CpG methylation: - TSS are unmethylated, when methylated -> silencing (XCI) - methylation of repetitive elements-> silencing (transposons, LINEs, Alu)

- methylation blocks transcription start not elongation

- methylation in gene bodies is not associate with repression

- other methylation sites: enhancers, insulators, splicing

(16)

DNA METHYLATION: SILENCING (plants)

Law and Jacobsen, 2010, NatRevGenet

ssRNA RNA Pol IV transcripts ocnverted to dsRNAs by RDR2 are processed

to siRNAs by DCL3 and associate with AGO4. Nascent Pol V ncRNAs IGS

serve as scaffold for AGO4/siRNAs and other factors and target DRM2

(SUV2, SUV9 that bind meDNA)

(17)

Methylated DNA bind MBD (methyl-CpG binding domain) proteins which recruit histone deacetylase complex and histone methyltransferase. This leads to chromatin condensation and gene repression.

DNA methylation is affected by nucleosme positioning, methylases are targeted to nucleosomes.

Only a subset of methylated TEs are targeted by RNAi

of repetitive sequences and transposons (TE) by RNAi

Texeira and Colot, 2009, EMBO J.

DNA METHYLATION: SILENCING

A. thaliana

KYP (SUVH4) - H3K9 methyltransferase DDM1 - SNF2 family ATPase, chromatin remodeler

RDR2 - RNA-dep RNA polymerase MET1, CMT3, DRM2 - DNA

methyltransferases

IBM1- histone demethylase

(18)

DNA METHYLATION

DNA methylation in mammals occurs mainly at CpG.

Methylation of the promoter suppresses gene expression.

Gene-body DNA methylation protects the gene body from spurious RNA polymerase II entry and cryptic transcription initiation (shown in mouse embryonic stem cells).

Such spurious transcripts can either be degraded by the exosome or capped, polyadenylated, and delivered to the ribosome to produce aberrant proteins.

Elongating Pol II triggers DNA methylation to ensure the fidelity of

transcription initiation.

(19)

HISTONE MODIFICATIONS - chromatin structure

Histones N tails outside the nucleosome are accessible to modifying enzymes

Bannister and Kouzarides, 2011, Cell Res.

Proteins binding modified histones

histone code:

phosphorylation (P) acetylation (Ac)

methylation (Me)

S/T phosphatases kinases -P S/T

K

histone

acetylotransferases (HAT) histone deacetylases (HDAC)

K

-COCH3

K/R methylases

demethylases

K/R

-CH3

ubiquitination (Ub)

sumoylation (Su)

(20)

-COCH3 Lysine-Ac

-CH3

N CH3 O

Lysine-Me Kme1 Kme2 Kme3

+

NH2 CH3

+

NH CH3

CH3

+

N CH3

CH3

CH3

-P Serine-P S-P on H3S10 and H3S28 activate

transcription by inhibiting H3K9-Me and promoting K-Ac

K-Me increases hydrophobic and cationic character of aa K-Ac more neutral than K

reduces DNA-histone interaction loosens chromatin structure

Histone modifications affect chromatin structure or regulate binding of chromatin factors

K-Ac and S-P reduce the positive charge of histones, loosen chromatin and activate transcription

K-Me (K-Ac) act mainly via protein binding, may inhibit or activate transcription:

H3K4me3 – active transcription mark (recognized by PD finger proteins, can recruit DNA modifying enzymes)

H3K9me3 - repressive chromatin mark (recognized by HP1)

HISTONE MODIFICATIONS - chromatin structure

(21)

Me P Ac K4 S10 K14

H3 Me Me P

K9 K27 S28 H3

Histone modifications in coding regions and transposons differ

red = strong correlation green= weak correlation

H3K9 methylation correlated with methylated DNA (meC) and trasposons

H3K4 methylation in actively transcribed genes

mRNA H3K4me

gene

H3K9me Me-C

transposon

Lippman et al., 2004, Nature

HISTONE MODIFICATIONS - chromatin structure

(22)

Polycomb Repressive Complex 2

NURF55

Extra sex comb (ESC)

PRC2 D. melanogaster A. thaliana

Enhancer of zeste (E(Z), methylase)

Suppressor of zeste 12 (SU(Z)12)

CURLY LEAF (CLF) MEDEA (MEA)

SWINGER (SWN)

FERTILIZATION INDEPENDENT ENDOSPERM (FIE)

FERTILIZATION-INDEPENDENT SEED 2 (FIS2)

EMBRYONIC FLOWER 2 (EMF2) VERNALIZATION 2 (VRN2)

MULTICOPY SUPPRESSOR OF IRA1 (MSI1,2,3,4,5)

MEA + FIS2

CLF/SWN + VRN2

CLF/SWN + EMF2 germination

induction of flowering

flower development

H3K27me3 – methylating complexes

PRC 1 PRC1-like

LHP1

H3K27me3 methylation maintenance

H3K27me3

active genes silenced genes stably silenced genes

PRC 2 PRC 1

H3K27me3

(23)

HISTONE CODE

(24)

HISTONE CODE

(25)

HISTONE CODE, HISTONE READERS

(26)

CHROMATIN REMODELING

Clapier and Cairns, 2009, Annu Rev Biochem

(27)

SWI2:

contain bromodomain all remodelling types

INO80/SWR:

histone exchange ISWI:

nucleosome repositioning CHD:

contain chromodomain rtanscription regulation

Clapier and Cairns, 2009, Annu Rev Biochem

CHROMATIN REMODELLERS

EXAMPLE

Arabidopsis DDM1, ATPase from the SWI/SFN family - involved in transposon methylation

- links chromatin remodeling and DNA methylation

(28)

HISTONE VARIANTS

Histones are exchanged by INO80/SWR remodeling complexes Ex: H2 variant H2A.Z activates transcription

Talbert and Henikoff, 2010, Nat Rev Mol Cell Biol

(29)

Some ncRNA (PolII transcripts), such as siRNAs or lncRNAs, recruit silencing complexes to specific genomic loci

Chen and Carmichael, 2010, WIREs RNA

EPIGENETIC CHROMATIN MODIFICATION by ncRNAs

(30)

A135 A190

AC40 AC 19

Common subunits (same in all) Core subunits (similar in all)

Rpb2 Rpb1

Rpb3 Rpb 11

C128 C160

+ 4 others + 2 others + 5 others

AC40 AC 19

6 5 10

8 9 6 5 10

8 9

6 5 10 8 9

RNA Pol I RNA Pol II RNA Pol III

Zbigniew Dominski, lectures 2008

RNA POLYMERASES

ribosomal RNA

35S precursor contains 18S, 5.8S and 25S subunits

mRNA , most snRNAs (U1, U2, U3, U4, U5, U11, U12, U4atac),

snoRNAs, microRNAs, telomerase RNA

tRNA, 5S rRNA, U6 snRNA, U6atac snRNA, 7SK RNA, 7SL RNA, RNase P RNA,

RNase MRP RNA

(31)

Porrua and Libri, BBA, 2013

TRANSCRIPTION

(32)

Pol II

Yeast RNA Pol II

Berneckyet al, 2016, Nature

Mammalian RNA Pol II cryoEM structure

(33)

Gnatt et al, Science, 2001 (Kornberg’s lab)

• 12 subunits

• core by specific Rpb1-3, 9 and 11

Rpb5-6, 8, 10 and 12 - shared by Pol I-III

• specific subcomplex Rpb4/7 not essential

• associated factors RAP74, RAP30 (TFIIF)

YEAST Pol II

Armache et al., Curr Opin Struct Biol, 2005

(34)

Pol II factors

http://cats.med.uvm.edu/cats_teachingmod/microbiology/cats_mmg_courses_new.htm

(35)

Pol II factors

(36)

Pol II – network of factors

Distant and proximal factors interact via DNA looping.

Trx factors recruit chromatin-modifying factors and trx coactivators near TSS, these in turn recruit PIC components. PIC assembly is modulated by repressors and

negative cofactors. On initiation Pol II CTD is P at Ser5 by CDK7. Trx elongation can be blocked by NELF and DSIF (Spt4/5) resulting in paused polymerase. Elongation is promoted by CDK9 P CTD at Ser2. Ser2-P CTD recruits chromatin, trx elongation, RNA processing and export factors.

CDK7/9 - cyclin-dependent kinase 7/9

NELF - negative trx elongation factor

DSIF - DRB sensitivity-inducing factor

P-TEFb= CDK9+ cyclin T1

(37)

Fuda et al, 2009, NatureKoch et al,,2008, TiBS

Pol II – network of factors

(38)

Pol II – network of factors

Reiter et al, 2017, Curr Op Genet Dev

Cooperation of TF and co-factors via chromatin modification

Model of more flexible and dynamic

(also transient) interactions of TFs

and co-factors

(39)

Goodrich and Kugel, Nat. Rev. Mol. Biol., 2006

Pol II C-terminal domain (CTD)

Tyr

1

Ser

2

Pro

3

Thr

4

Ser

5

Pro

6

Ser

7

26 (yeast) - 52 (human) repeats

Meinhart and Cramer, 2004 Saunders et al, 2006, Nat.Rev.Mol.Cel.Biol

(40)

CTD CODE

Saunders et al, 2006, Nat.Rev.Mol.Cel.Biol

Ser5-P

Cyclin-dependent kinase-7 (CDK7) of TFIIH and CDK8 Phosphatases SSU72, FCP1

SCPs small CTD phosphatases Ser2-P

CDK8 and CDK9 of P-TEFb FCP1

ESS1/PIN1 Peptidyl-prolyl isomerases

Egloffs et al, 2012, TiG

mammalian CTD non-consensus repeats

Zaborowska et al, 2015, NatStrMolBiol

(41)

CTD CODE

Srivastava and Ahn, Biotechnol Adv 2015

(42)

CTD CODE

Zaborowska et al, 2015, NatStrMolBiol

(43)

CTD CODE

Srivastava and Ahn, Biotechnol Adv 2015

(44)

Srivastava and Ahn, Biotechnol Adv 2015

CTD CODE vs HISTONE CODE

Interactions between RNAPII CTD modifications

and histone-modifying enzymes

(45)

CONTROVERSY in CTD CODE

Srivastava and Ahn, Biotechnol Adv 2015

Noijma et al (Proudfoot lab), Cell 2015

Pol II ChIP (summary )

NET-Seq

(46)

CTD CODE

(47)

CTD MODIFYING ENZYMES and CYCLES

CDK: Cyclin-dependent

SCPs: Small CTD Phosphatases Peptidyl-prolyl isomerases

Egloffs et al, 2012, TiG

(48)

CTD MODIFYING ENZYMES

Zaborowska et al, 2015, NatStrMolBiol

(49)

Andersen et al, WIREsRNA, 2013

PolII and CTD TRASNCRIPTION CYCLES

(50)

CTD and FUNCTIONS

Egloffs et al, 2012, TiG

(51)

Pol II – INITIATION

Sainsbury, Bernecky and Cramer, Nat Rev Mol Cell Biol’15

Most regulatory steps occur

during intiation

(52)

Sikorski and Buratowski, 2009, Cur. Op. Cell Biol.

TBP assembles via TFIID

(TATA-less genes) stepwise recruitment of basal initiation factors Mediator bridges interactions between activators and the basal initiation machinery

TBP assembles via the SAGA complex (TATA-containing genes)

Mot2/bTAF1 and NC2 repress trx by removing unproductive TBP from DNA

PIC

TFIID TBP

(TATA binding protein) TAFs

(TBP-associated factors)

PIC - PRE-INITIATION COMPLEX ASSEMBLY

(53)

Pol II – initiation to elongation transition

Jonkers and Lis, Nat Rev Mol Cell Biol’15

Enhancers and Promoters

TF assembly

PIC assembly Initiation

Pol II pausing

(54)

Pol II – initiation to elongation transition, cont .

Key role of P-TEFb

in pause release

(55)

TRANSCRIPTION ELONGATION

Koch et al,,2008, TiBS

Promoter proximal pausing involves abortive transcription. While waiting for Ser2-P, PolII transcribes short (20-40 nt) nascent RNA cleaved by elongation

factor TFIIS, which allows PolII backtracking to resume transcription after arrest.

Ser5-P Ser2-P CTD

(56)

Pol II – initiation to elongation transition

7SK snRNP and P-TEFb

Quaresma et al, 2016, NAR; McNamara et al, 2016, Cell Cycle

P-TEFb (positive trx elongation factor: Cdk1, CycT1) controls Pol II transition from promoter- proximal pausing to effective elongation by phosphorylating CTD at S2, NELF and DSIF, followed by recruitment of PAF1, Cdk12 and CycK

Key role of P-TEFb in pause release

(57)

Pol II – initiation to elongation transition

7SK snRNP and P-TEFb

Quaresma et al, 2016, NAR; McNamara et al, 2016, Cell Cycle

7SK snRNP complex is present at the promoters of most Pol II genes.

Following stimuli P-TEFb is released by TF, PPMG1, DDX21 helicase and SF2 7SK snRNP:

PolI II RNA subunit, two core proteins

(MePCE, LARP7)

(58)

POLYMERASE PAUSING

shortly after trx initiation

Adelman and Lis, 2012, Nat Rev Genet

PIC

early

elongation complex

stable, backtracked PolII, restart requires TFIIS cleavage

unstable,

dissociating PolII

(59)

POLYMERASE PAUSING

Release from polymerase pausing by different P-TEFb containing complexes

Chen et al. Nat Rev Mol Cell Biol 2018

7SK snRNP

represses transcription by sequestering and inhibiting up to 90% of cellular P- TEFb

BRD4

Bromodomain containing protein 4

SEC super elongation complex

SEC and BRD4

active forms of P- TEFb that

promote Pol II release from

pausing

(60)

POLYMERASE PAUSING

Adelman and Lis, 2012, Nat Rev Genet

negative NELF-DSIF trx elongation factors

recruitment of P-TEFb kinase NELF-DSIF phosphorylation NELF release, DSIF becomes a positive elongation factor

PolII escapes pausing

new PolII enters the pause region

(61)

POLYMERASE PAUSING

Chen et al. Nat Rev Mol Cell Biol 2018

(62)

POLYMERASE PAUSING

Adelman and Lis, 2012, Nat Rev Genet

(63)

POLYMERASE PAUSING

Integrator (INT) contributes to

promoter-proximal pause-release

Baillat and Wagner, TiBS 2015

Pol II pausing after trx initiation 40-60 nts

downstream of TSS, with NELF and DSIF

On activation, INT enriched at pause site rectruits p-TEFb and SEC which phosphorylate NELF and Ser2

NELF-P is displaced, DSIF-P

becomes a positive elonation

factor and Pol II is converted

into an elongation-competent

state

(64)

POLYMERASE PAUSING

Chen et al. Nat Rev Mol Cell Biol 2018

(65)

POLYMERASE PAUSING - structural bases

Young Kang et al, 2018, Mol Cell

CryoEM structure of elemental and hairpin- stabilized paused Pol II

• uncoupling of DNA and RNA translocation (RNA

translocates but DNA does not)

• Pol II in half-translocated state is unable to bind NTP

• hairpin-stabilized

allosteric changes inhibit

trigger loop (TL) folding

and pause Pol II

(66)

POLYMERASE BACKTRACKING

Nudlerr, Cell, 2013 Sheridan et al, MCell, 20138

- Rescue from backtracking is a major stimulus of rapid transcriptional elongation

- Rescue from backtracking is important for escape from promoter-proximal pause sites - RNA cleavage by Pol II (TFIIS) is essential for rapid activation of stress-inducible genes

Pol II arrest and backtracking occur at

roadblocks (DNA binding factor or a nucleosome)

positions of base mis-incorporation

(67)

POLYMERASE BACKTRACKING

Polymerase backtracking in genome stability

Double-strand breaks (DSB) are formed due to collisions between the replisome and backtracked RNA polymerase in bacteria.

Transcript cleavage factor (Gre) prevents polymerase backtracking and R loop formation, preserving genome integrity .

Nudlerr, Cell, 2013

(68)

Pol II ELONGATION

Jonkers and Lis, Nat Rev Mol Cell Biol’15

Pol II density and elongation rates are not

constant but vary throughout the gene

(69)

Chen et al. Nat Rev Mol Cell Biol 2018

Pol II ELONGATION

is regulated by enhancers

(70)

Malik and Roeder, 2010, Nat Rev Genet

evolutionarily conserved, multiprotein complex

transcriptional co-activator, sensor, integrator of signals

involved also in - chromatin structure

- formation of gene loops - gene silencing

- development Metazoa

Yeast, 25 subunits, 1.4 MDa

ACT - trx activator

II B D E H F – trx factors

inhibitory Mediator subunits

Bjorklund and Gustafsson, 2005, TiBS

MEDIATOR - a central integrator of trx

(71)

MEDIATOR - a central integrator of trx

Allen and Taatjes, 2015, Nat Rev Mol Cell Biol

Structural changes in

Mediator control its

interactions with other

transcription regulators

(72)

Malik and Roeder, 2010, Nat Rev Genet

activated chromatin Ac-, Me- histones

Mediator recruitment Mediator restructuring, loss of the kinase module PIC formation

General transcription factors + PolII

MEDIATOR - a central integrator of trx

Mediator key role in chromatin architecture, PIC assembly, trx pausing and

elongation

(73)

SAGA

Spt–Ada–Gcn5 acetyltransferase

multisubunit histone

modifying complex (2 MDa)

contains two modules - HAT acetylating

- DUB deubiquitinating

transcriptional activator

involved also in

- transcript elongation - regulation of protein stability

- telomere maintenance

K

outelou, et al., 2010, Cur. Op. Cell Biol.

(74)

SAGA in

TRANSCRIPTION

a) recruitment via activator HAT (histone acetyltransferase)

module acetylates histones b) TBP module anchors SAGA via

TBP (TATA binding protein), CTD Ser5-P promotes initiation c) SAGA recruited to PolII assists

elongation, HAT acetylates and DUB deubiquitinates H2B

d) H2B-Ub recruits Ctk1 which

promotes CTD Ser2-P and in

turn elongation

(75)

Nucleosome positioning

The length of typical internal exons (grey boxes) is comparable to the

DNA wrapped around a nucleosome. Nucleosome positioning relative to

the transcription start site (TSS), transcription termination site (TTS) and, to

a lesser extent, exons helps to define the boundaries of these elements,

providing a platform for crosstalk between chromatin, transcription and

splicing. Nucleosomes at introns are less stable (dased lines). A sleeping

Pol II represents pausing events at splice sites (AG and GT). Consistent

with nucleosome phasing over exons, slower transcription elongation

has been measured over exonic sequences

(76)

Nucleosome dynamics during transcription.

INITIATION: DNA-binding activators at UAS recruit SAGA (acetylates nucleosomes) and SWI/SNF (displaces nucleosomes), histones are transferred to histone chaperones. PIC and Pol II form at the new nucleosome-free region.

ELONGATION: Nucleosomes in front of the Pol II are acetylated and displaced to Spt6/FACT chaperones, which reassemble nucleosomes behind PolII. H3 is methylated by Set2

methyltransferase, this promotes nucleosome deacetylation by Rpd3S, which restores nucleosome stability.

Multiple elongating polymerases displace histones and overcome nucleosomal barrier.

NUCLEOSOME POSITIONING - ELONGATION

nucleosome- free region

H2A.Z

UAS

H2A.Z

Workman, 2006, Gene Dev

(77)

Pol II TRANSCRIPTION

THROUGH NUCLEOSOMES

Selth et al, 2010, Ann. Rev. Biochem.

Transcription through nucleosomes dislocates histone proteins to histone chaperones.

Progression of PolII may occur without complete displacement of histone proteins.

Only H2A/H2B is reloaded by FACT (facilitates chromatin transcription) downstream of PolII.

Nucleosomes in front of the Pol II are acetylated by HATs and displaced to Spt6/FACT

chaperones, which reassemble nucleosomes behind PolII. H3 is methylated by Set2

methyltransferase, this promotes nucleosome deacetylation by Rpd3S, which restores

nucleosome stability.

(78)

CONSTITUTIVE vs ACTIVATED TRANSCRIPTION

single mRNA–sensitivity FISH using 50- nt DNA probe with five fluoresent dyes

Modeling kinetics

constitutively

active genes cell-cycle

regulated genes SAGA-regulated genes

Constitutively expressed genes are transcribed by single events separated in time; regulated genes (e.g. by SAGA) are expressed by transcriptional bursts

Zenklusen et al., 2008, Nat.Str.Mol.Biol

(79)

GENE LOOPING - Pol II (long range), also Pol I

Ansari and Hampsay, GDev, 2005; El Kaderi et al., JBC, 2009

Loop formation requires interaction between factors at the promoter (THIIB) and terminator (Rna15 from CF1) /in mammals: transcription factors, nuclear receptors, insulators, chromatin remodellers, Polycomb, architectural proteins/

Loop function: facilitation of transcription reinitiation of PolII, but also repression of gene expression (PcG, DNA methylation)

activated transcription

Scaffold = transcription factors

(TFIID, A, E, H)

(80)

Aguilera and Garcıa-Muse, Mol Cell, 2012

R-LOOPs in TRANSCRIPTION

DNA::RNA hybrids forming during transcription before RNP packaging.

Accumulate in topoisomerase or RNA

biogenesis mutants (tho, sen1, mRNA export)

(81)

R-LOOPs in TRANSCRIPTION

ChedinTiG, 2016

R-loops in targeting

ncRNAs?

(82)

Le and Manley, Gene Dev, 2005

R-LOOP negative effects

- polymerase stalling - termination defects

- replication fork stalling - DNA damage

- genetic instability

Yeast

Metazoans

Preventing R-loops:

helicase Sen1; THO complex;

Topoisomerases

degradation by RNaseH

R-loop effects repaired by template switching or homologous

recombination

(83)

Regulation of Pol II trx by ncRNAs

1. RNA: siRNA-mediated TGS (transcriptional gene silencing) 2. lncRNA-mediated TGS via transcription itself

LncRNA transcription causes increased nucleosome density (yeast)

LncRNA transcription causes repressive histone modifications (yeast)

LncRNA transcription recruits DNA methylation at promoter (humans)

Kornienko et al, BMC Biol., 2013

(84)

lncRNA transcripts acting in cis or in trans:

Regulation of Pol II trx by ncRNAs

Mercer et al., Nat.Rev.Genet., 2007

recruit chromatin modifying complexes resulting in

heterochromatin formation

ncRNAs act as repressors or

activators of transcription by

binding to proteins or DNA

TGS

(85)

Regulation of Pol II trx by ncRNAs: eRNAs

eRNAs: enhancer RNAs, short (not always, up to 2 kb) ncRNAs transcribed from enhancer regions

eRNAs synthesized at enhancers are targeted to defined regulatory regions, i.e. promoter (A)

eRNAs mediate chromatin accessibility and recruitment of factors for transcription and stabilization of enhancer-promoter contacts.

Mousavi et al., RNA Biol., 2014

(86)

Quinn and Chang, Nat Rev Genet 2015

Chromosome looping

Functions of eRNAs

Some eRNAs (e.g. LUNAR1 near the IGF1R locus) mediate

chromosome looping between enhancers and nearby genes

via Mediator or MLL protein complexes

(87)

YEAST Pol I

Kuhn et al, Cell, 2007 (Cramers lab)

14 subunits

• core by specific A190, A135, AC40, AC19, A12.2 subunits

• Rpb5-6, 8, 10 and 12 - shared by Pol I-III

• specific subcomplexes A14/A43 and A49/A34.5

• no CTD

• has intrinsic 3’ RNA cleavage activity (A12.2/Rpa12) -

possible roles in proofreading and transcription termination

(88)

rRNA synthesis

40S small subunit

60S large subunit 80S ribosome

nucleolus 70-80% of cellular transcription is by Pol I to make rRNA

50% of Pol II transcription is for ribosomal protein genes

rDNA transcription units are arranged in tandem repeats in 150-200 copies

processing

termination

RNP assembly

• RNA modification

(2’-O methylation, pseudourydylation)

Pre-rRNA

Regulation of rDNA transcription

Grummt, FEBS J., 2010

(89)

rDNA silencing by pRNA and NoRC

Matthews and Olsen, Embo Rep., 2006;

Tucker et al., Cur. Op. Cell. Biol., 2010

NoRC –establishes and maintains heterochromatic state at

promoters of silent rDNA repeats (histone modifications and CpG methylation)

- TIP5 TTF-I-interaction protein5 - SnF2 ATP-dependent chromatin remodeler

other

- TTF-1 transcription factor I - UBF upstream binding factor - DNMT DNA methyltransferase - HDAC1 histone deacetylase

NoRC – mammalian nucleolar remodeling complex

(90)

Crucial step: recruitment of active Pol I to transcription factors by Rrn3/TIF-IA

Moss, Cur. Op. Gen. Dev., 2004

yeast

Grummt, FEBS J., 2010

enancers

TAF-I: Pol I-specific TBP- associated factor

TBP: TATA-binding protein (general factor)

TFIIH: transcription factor SL1: selectivity factor 1 (recruits Pol I)

TTF-I: transcription termination factor I

TTF-IA: recruits Pol I

UBF: upstream binding factor (binds to UCE)

UCE: upstream control element

Pol I TRANSCRIPTION

mammals

(91)

Regulation of rRNA synthesis – TOR

(target of rapamycin)

TOR regulates ribosome synthesis via three

polymerases

Mayer and Grummt, Oncogene, 2006

(92)

Pol III TRANSCRIPTION

Pol III promoters

Dieci et al, TiG, 2007

TTTT - transcription termination signal TATA - TATA box or TATA-like sequence

PSE – proximal sequence element DSE - distal sequence element box A

box B boxC

Vannini et al, Cell, 2010 Flores et al, PNAS, 1999

(93)

Pol III TRANSCRIPTION

tRNA

5S

(94)

Pol III REGULATION by Maf1

Willis and Morris, TiBS, 2007

Maf1 – Pol III inhibitor

NORMAL growth

Maf1 is phosphorylated and remains in the cytoplasm

P-states of Maf1 are regulated by RAS /cAMP and TOR pathways

STRESS (starvation)

- Maf1 is dephosphorylated and imported to the nucleus

- Maf1 inhibits:

de novo assembly of TFIIIB

transcription by binding to Pol III genes

(95)

Pol I and Pol III

Vanini, BBA, 2013

(96)

TAKE-HOME MESSAGE

Transcription of different RNAs eukaryotic is carried out by specialised RNA polymerases, I –III (all) and IV/V (plants)

• Transcription regulation is achieved on several levels:

chromatin structure and modification, recruitment of transcription factors, silencing mechanisms (ncRNAs)

Many RNA processing events occur cotranscriptionally (capping, splicing, 3’ end formation, export)

Transcription is regulated in response to nutrients, stress,

cell cycle, development stage, etc...

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