RNA MISHMASH

RNA MISHMASH

UNUSUAL ncRNAs: tRFs tRNA-derived RNA fragments

Thompson and Parker, Cell, 2009

Stress-induced enzymatic tRNA cleavage

(S. cerevisiae, D. melanogaster, A. thaliana, A. nidulans, human cell lines)

UNUSUAL ncRNAs: tRFs tRNA-derived RNA fragments

• > 17 short abundant tRFs (13-26 nts), generated by RNaseZ from mature (5’ and 3’ ends) and precursor (3’ trailer) tRNAs identified in the cytoplasm in prostate cancer cells. Lack of tRF1001 impairs cell proliferation.

• Abundant Dicer-dependent tRFs (class I, from mature 3’ and 5’ ends) in HeLa moderately downregulate target genes.

• Class II tRFs (from RNAseZ 3’ cleavage to Pol III termination, cytoplasmic) associate with Ago2-3. Function- regulation of silencing via differential

association with Ago proteins?

Haussecker et al., RNA, 2010 Cole et al., RNA, 2009

Lee et al., Gene Dev., 2009

Ivanov et al, Mol Cell, 2011

Selected 5’-tiRNAs

:

• repress translation in vitro and in vivo

• displace eIF4G/eIF4A from uncapped transcripts

• displace eIF4F from m7G cap

• trigger formation of stress granules (SGs)

• terminal oligoG at the 5’ end important for repression

• translational repressor YB-1 contributes to tiRNA mediated repression

• 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

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

GENE LOOPING (long range) 3C

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)

3C 4C 5C …..

3C

4C

5C

GCC

ChIP Loop

6C

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 PolI 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

OLIGO-URIDYLATION

PUP

TUTase

3’ oligouridylation 1. Histone mRNA degradation

Mullen and Marzluff, Genes Dev., 2008

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?

Lsm1-7

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

RNA MODIFICATION: mRNA m ⁶ A

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

N

-methyladenosine:

•

• 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 m6}A sites per mRNA)

• Readers:

YTHDF2

demethylases

FUNCTIONS of mRNA m ⁶ 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 ⁶ A

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

Pan, TiBS, 2013

RNA DECAPPING

Wang et al. PNAS, 2002 She et al. Nat.Struct. Mol. Biol, 2004

Dcp1p

Dcp2p

• Dcp1/Dcp2 complex participates in mRNA 5’ decay

• catalyses the reaction m⁷GpppX-mRNA  m⁷GDP + 5’p-mRNA

• Dcp2 is the catalytic subunit (pyrophosphatase Nudix domain)

• Dcp1 is required for activity in vivo, interacts with other proteins

• Dcp1/Dcp2p is regulated by Pab1 and activating factors (yeast Lsm1-7, Dhh1, Pat1, Edc1-3, Upf1-3)

Gu et al., M.Cell, 2004

DcpS

• DcpS: HIT pyrophosphatase („histidine triad” on the C-terminus)

• catalyses the cleavage of m⁷GDP  m⁷GMP + Pi remaining after decapping during mRNA 5’ decay

• cooperates with the exosome during mRNA 3’ decay (m⁷GpppX-oligoRNA  m⁷GMP+ pp-oligoRNA)

• functions as an asymmetric dimer

RNA DECAPPING

Rai1 TLC

Rai1 -

- 5’-ppp pyrophosphohydrolase

-

Rai1

decapping endonuclease phosphodiesterase activity

- in vivo acts in RNA surveillance during stress (starvation) to remove aberrantly capped transcripts

Rai1

Δ

Jiao et al, 2010, NatureXiang et al, 2009, Nature

RNA DECAPPING

Chang et al, 2009, Nat Struc Mol Biol 2012

Dxo1

- has

decapping endonuclease but no phosphohydrolase activity - has distributive 5’-3’ exoRNase activity

- in vivo acts in RNA 5’ capping quality control hDXO - has three activities: pirophosphohydrolase,

decapping endonuclease (phosphodiesterase), 5’-3’ exonuclease

Dxo1 Rai1

5’- CAP SURVEILLANCE RNA

Tuttuci and Stutz., NatRevMolCelBiol 2011

SUMMARY or HOW TO PASS THE EXAM?

- THEORY

- PRACTICE/METHODS

- GENERAL IDEAS, CONCEPTS, SOLUTIONS - PATHWAYS

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