RNA MISHMASH

RNA MISHMASH

RNA MODIFICATIONS

Sibbrrit et al, WIREsRNA 2013

FUNCTIONS

m A RNA-seq

Li at al, Nat Methods, 2017

m C RNA-seq

Li at al, Nat Methods, 2017

RNA MODIFICATION: mRNA m A

Dominissini at al, Nat.Rev.Genet., 2014

N ⁶ -methyladenosine:

• in eukaryotic mRNAs lncRNAs (discovered in 1970s)

• reversible, conserved

• methyltransferase METTL3 or METTL4-METTL14 complex with WTAP (yeast Mum2) in a [G/A/U][G>A]m6AC[U>A>C] context

• demethylases FTO and ALKBH5

• occurrence 0.1–0.4% of As in mammals (~3–5 m

A sites per mRNA)

• Readers: YTHDF2 methyltransferases

demethylases

FUNCTIONS of mRNA m A

Dominissini at al, Nat.Rev.Genet., 2014

Readers (or anti-readers): YTHDF2 family preferentially recognize m

A RNA m

A can be also read by hnRNPs

• Regulation of mRNA stability and localization

• circadian clock

- inhibition of m6A leads to prolonged nuclear retention of circadian mRNAs and delays their nuclear exit

• cell cycle

- meiosis in yeast in nitrogen starvation

• development and differentiation

- in embryonic stem cells (mESCs)

FUNCTIONS of m A

Dominissini at al, Nat.Rev.Genet., 2014;

Pan, TiBS, 2013

Chen and Shyu, TiBS 2016

m A and mRNA STABILITY

promoting deadenylation

affecting local secondary structure

inhibiting deadenylation

Chen and Shyu, TiBS 2016

m A and mRNA STABILITY

promoting deadenylation

affecting local secondary structure

inhibiting deadenylation

FUNCTIONS of m A: pri-miRNA PROCESSING

• m

A is present in pri-miRNA regions

• METTL3 modulates miRNA expression level

• METTL3 targets pri-miRNAs for m

A methylation

• m

A in pri-miRNA is required for normal processing by DGCR8

• HNRNPA2B1 RNA-binding protein recognizes m

A sites

• HNRNPA2B1 nuclear reader recruits Microprocessor

Alarcon at al, Nature, 2015

FUNCTIONS of m A: mRNA SPLICING

Xiao et al, Mol. Cell, 2016

nuclear YTHDC1 m

A reader

• interacts with SR proteins SRSF3 and SRSF10

• facilitates/blocks binding of SRSF3/SRSF10 to pre- mRNAs

• promotes exon inclusion

of targeted mRNAs

FUNCTIONS of m A: TRANSLATION

• m

A in 5’ UTR promotes cap-independent translation

• m

A in 5’ UTR upregulates translation

• cellular stresses increase m

A in 5’ UTRs

• YTHDF2 in heat shock induces m

A-dependent translation of HS mRNAs

• m

A in mRNA body disrupts tRNA selection and translation elongation dynamics

Meyer et al, Cell, 2015;

Zhou et al, Nature, 2015;

Choi et al, Nat. Struct. Mol. Biol.’16

FUNCTIONS of m A: RNAPII and TRANSLATION

Slobodin et al, Cell, 2017

• mRNA transcription rates correlate with translation

• slow PolII results in higher level of m

A in mRNAs

• high level m

A reduces translation rate

• nuclear control on protein

abundance

OTHER FUNCTIONS of m A

X-chromosome inactivation

m

A facilitates gene silencing via nuclear reader DC1 that binds to m

A on Xist.

DC1 may recruit Polycomb and/or other silencing factors (SHARP, LBR, hnRNPU, hnRNPK)

Huisman et al, 2017, TiBS

Epitranscriptomics, Meiosis, Sex determination, Cellular differentiation,

Development, Pluripotency and Reprogramming, Disease, Cancer

m A

MULTIPLE FUNCTIONS

Patil et al, 2017, TiCB

RNA MODIFICATION: mRNA m A

N ¹ -methyladenosine m ¹ A:

• in eukaryotic mRNAs (from yeasts to mammals)

• modified by TRMT6/TRMT61A (nuclear) or TRMT61B, TRMT10C (mitochondrial)

• at mRNA cap and 5’ UTR increases translation

• prevalent in mitochondrial-encoded transcripts inhibits translation

• in different mRNA regions differentially impacts translation

Li at al, Mol Cell 2017

Dominissini et al, Nature 2017

• widespread (20% in humans)

• enriched around the start codon upstream of the first splice site

• preferentially in more structured regions around translation initiation sites

• is dynamic in response to different conditions

• promotes translation

• in cytosol low in few mRNAs

• in tRNA T-loop like structures

• present also in mitochondria

• leads to translational repression

• is disruptive to W-C basepairing

• generally avoided by cells

Safra et al, Nature 2017

FUNCTIONS of m C

• synthesized by TRM4B methyltransferase

• enriched in the CG context and in downstream of translation initiation sites

• present in mRNAs and ncRNAs, also tRNAs

• tissue specific, acts as a epitranscriptome marker

• mRNA export: NSUN2 as the methyltransferase and ALYREF as an m

C reader

Yang et al, 2017, Cell Res

Simms and Zaher, CellMolLifeSci 2016

damaged nucleobases exhibit altered base pairing

RNA REPAIR

oxidative and alkylative

damage on RNA

Simms and Zaher, CellMolLifeSci 2016

RNA REPAIR

R-LOOPs = DNA::RNA hybrid

Aguilera and Garcıa-Muse, Mol Cell, 2012

R-LOOPs in TRANSCRIPTION

Aguilera and Garcıa-Muse, Mol Cell, 2012

Yeast

Metazoans

Preventing R-loops

• DNA::RNA hybrids forming during

transcription before RNP packaging into RNP

• negative effect, may result in

- polymerase stalling, termination defects - replication fork stalling

- DNA damage

- genetic instability

R-LOOPs

DIP:

DNA IP

Le and Manley, Gene Dev, 2005

R-LOOPs

Aguilera and Garcıa-Muse Mol Cell, 2012

collision with: DNA lesions RNA::DNA hybrid

RNAP fork reversal by torsional stress

by template switching

homologous recombination

R-LOOPs accumulate in RNP biogenesis mutants

elongation impairment replication blockage DNA damage

genotoxic agents nucleases

tho and export mutants

WT with PolII stalled on damage Transcription-Coupled Repair (TCR) activated or PolII degraded

mut with PolII stalled on damage TCR not activated, only PolII degradation and global genome repair (GGR)

ssDNA

Sen1 and R-loop degradation by RNaseH prevent genome instability

R-loop accumulate in sen1 mut and may result in homologous recombination via:

- nicks in ssDNA

- ssDNA recognition by proteins

- collapse of colliding replication forks

sen1 mutant

Huertas and Aguillera, Mol Cell, 2004; Gaillard et al, NAR, 2007; Mischo et al., Mol Cell, 2011

ChIP

DIP= IP with DNA/RNA Abs

The level of R-loops and Pol I pileups depend on topoisomerase I and RNase H

Topoisomerases release positive supercoiling built in front of Pol I by rotating DNA during transcripton

This pauses Pol I (pileups) but opens DNA behind, stimulates

R-loops which slow down Pol I

RNase H cleave DNA/RNA hybrids releasing truncated pre-rRNA fragments degraded by TRAMP/exosome

Lack of Top1/2 and RNaseH massive R-loops cause severe Pol I arrest and pileups

El Hage et al., Gene Dev, 2011

R-LOOPs block rRNA transcription

ALTERNATIVE POLYMERASES

Scheer et al, TiG., 2016

OLIGO-URIDYLATION

PUP Poly(U) Polymerases

TUTase Terminal Uridylyl Transferase 3’ oligouridylation 1. Histone mRNA degradation (metazoans)

Mullen and Marzluff, Genes Dev., 2008

DIS3L2

Scheer et al, TiG., 2016

1. Histone mRNA degradation (metazoans)

2. miRNA degradation

precursors C. elegans

Krol et al., Nat Rev Genet, 2010; Kim et al., Cell, 2010

OLIGO-URIDYLATION

mature

Arabidopsis

Chlamydomonas

3. mRNA degradation? (plants)

Lsm1-7

DIS3L2

OLIGO-URIDYLATION

3. mRNA degradation

Scheer et al, TiG., 2016

OLIGO-URIDYLATION

Scheer et al, TiG., 2016

3. other

HISTONE mRNA 3’ end FORMATION

(nonpolyadenylated, metazoa, unique)

Dominski and Marzluff, Gene, 2007

U7 snRNP unique

Sm/Lsm10/11 SL

structure

endonuclease

• Histone pre-mRNA contains conserved stem-loop (SL) structure, recognized by the SLBP (SL-binding protein)

• SLBP, ZFP100 and HDE (histone downstream element) stabilize the binding of U7

• U7 snRNP, specificaly Lsm11, recruits cleavage factors and the cleavage by

endonuclease CPSF-73 generates mature 3’ end of histone mRNA

URIDYLATION

Lee et al, Cell, 2014

Uridylation-dependet mRNA decay

Uridylation of pre-miRNAs and miRNAs

Degradation of histone mRNAs

TUTases

Wethmar WIREsRNA, 2014

uORFs = upstream ORFs

Puyeo et al, TiBS, 2016

small ORFs = sORFs, sPEPs, smORF

Methods to identify sORFs and sPEPs

Plaza et al, Annu Rev Cell Dev Biol, 2017

sORFs

Couso and Patraquim Nat Rev Mol Cell Biol, 2017

Functions of sPEPs

Couso and Patraquim Nat Rev Mol Cell Biol, 2017

Andrews and Rothnagel, Nat Rev Genet, 2014

Functional sPEPs

POLYMERASE BACKTRACKING

Nudlerr, Cell, 2013

Polymerase backtracking in genome stability

Double-strand break (DSB) formation as a result of

codirectional collisions between the replisome and

backtracked RNA polymerase in bacteria. Transcript

cleavage factor (Gre) prevents polymerase backtracking

and R loop formation, preserving genome integrity .

SUMMARY or HOW TO PASS THE EXAM?

- THEORY - METHODS

- GENERAL IDEAS, CONCEPTS, SOLUTIONS - PATHWAYS

- MECHANISMS