RNA MISHMASH
RNA MODIFICATIONS
Sibbrrit et al, WIREsRNA 2013
FUNCTIONS
RNA MODIFICATION: mRNA m 6 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 m6A sites per mRNA)• Readers: YTHDF2
methyltransferasesdemethylases
FUNCTIONS of mRNA m 6 A
Dominissini at al, Nat.Rev.Genet., 2014
Readers (or anti-readers): YTHDF2 family preferentially recognize m
6A RNA m
6A 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 6 A
Dominissini at al, Nat.Rev.Genet., 2014;
Pan, TiBS, 2013
Chen and Shyu, TiBS 2016
m 6 A and mRNA STABILITY
promoting deadenylation
affecting local secondary structure
inhibiting deadenylation
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 structure SL
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
Wethmar WIREsRNA, 2014
uORFs = upstream ORFs
Puyeo et al, TiBS, 2016
sORFs, sPEPs, smORF = small ORFs
Andrews and Rothnagel, Nat Rev Genet, 2014
Functional sPEPs
• no endogenous RNAi in Saccharomyces cerevisiae but:
• siRNAs exist in other budding yeast, e.g. Saccharomyces castelli and Candida albicans ( transposable elements, Y’ subtelomeric repeats)
• siRNA generated by non-canonical Dicer (RNase III and two dsRBD domains) , form RNAi complex with Argonaute protein
• S. castelli Dcr1 and Ago1 introduced into S. cerevisiae restore RNAi
RNAi in Saccharomyces cerevisiae?
Drinnenberg et al., Science, 2009
+ has canonical RNAi enzymes
* has second RNaseIII (Dcr) in addition to Rnt1 S. bayanus has Dcr but no Ago – no siRNA
siRNAs produced in S.cerevisiae with S.castelli DCR1 and GFP transgene silenced with DCR1+AGO1
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
