OTHER RNA PROCESSING

OTHER RNA PROCESSING

RNA MODIFICATION: mRNA m A

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 m⁶A sites per mRNA)

• Readers: YTHDF2

methyltransferases

demethylases

RNA MODIFICATION: mRNA m A

Patil at al., TiCB, 2018

FUNCTIONS of mRNA m A

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

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

⁶

A RNA m

⁶

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

FUNCTIONS of m A: pri-miRNA PROCESSING

• m

⁶

A is present in pri-miRNA regions

• METTL3 modulates miRNA expression level

• METTL3 targets pri-miRNAs for m

⁶

A methylation

• m

⁶

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

• HNRNPA2B1 RNA-binding protein recognizes m

⁶

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

⁶

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

⁶

A in 5’ UTR promotes cap-independent translation

• m

⁶

A in 5’ UTR upregulates translation

• cellular stresses increase m

⁶

A in 5’ UTRs

• YTHDF2 in heat shock induces m

⁶

A-dependent translation of HS mRNAs

• m

⁶

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

⁶

A in mRNAs

• high level m

⁶

A reduces translation rate

• nuclear control on protein

abundance

FUNCTIONS of m A: A-to-I RNA editing

Xianget al, 2018, Mol Cell

ADAR binding to RNA

is disfavored by m6A

OTHER FUNCTIONS of m A

OTHER FUNCTIONS of m A

X-chromosome inactivation

m⁶A facilitates gene silencing via nuclear reader DC1 that binds to m⁶A 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, 2018, 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

URIDYLATION

Lee et al, Cell, 2014

Uridylation-dependent mRNA decay

Uridylation of pre-miRNAs and miRNAs

Degradation of histone mRNAs TUTases

RNA MODIFICATION: mRNA EDITING

RNA editing: post-transcriptional modification of RNA sequence

- up to 50-60% in Trypanosomes!

- insertions and deletions - C to U or A to I exchange

guide RNA

Guide RNA (gRNA) present in 20S RNP

(editosome) is partially complementary

to the mRNA in vicinity of editing

mRNA EDITING: A to I by ADAR

ADAR

adenosine deaminase acting on RNA

Nishikura, Nat.Rev.Mol.Cel.Biol., 2006

Editing of Apolipoprotein B mRNA

CAA codon

2153

UAA

Apo-B100 4563 aa function: transport of cholesterol in the blood

liver intestine

Apo-B48 2152 aa

function: absorption of lipids from the intestine

TAA

CAA UAA UAA

apoB gene

apoB mRNA

no editing CAA to UAA

A::U I::C

Nishikura, Nat.Rev.Mol.Cel.Biol., 2006

the

siRNA pathway heterochromatic silencing

the

EDITING of ncRNAs

RNAi and A to I editing

the

miRNA processing

(nuclease)

RNA EDITING and RNAi

Nishikura,Ann. Rev. Biochem., 2010

Ota et al., Cell 2013

Additional function of ADAR1:

ADAR1 increases the rate of pre-miRNA cleavage by Dicer and facilitates loading of miRNA onto RISC complex

RNA EDITING and RNAi

Serganov and Patel, Nat. Rev. Genet., 2007

organelles (fungi, plants), bacteria,

mitochondria (animals) viroids, eukaryotes plant satellite RNA, viruses

Hammerhead, Hairpin, HDV

mRNA splicing-like

organelles (fungi, plants), bacteria, archea

Mechanism:

nucleophilic attack of the ribose -OH group (H₂O, Me²⁺) on the phosphate

SELF-SPLICING RIBOZYMES

Serganov and Patel, Nat. Rev. Genet., 2007

SELF-SPLICING RIBOZYMES

Group I intron Group II intron

Hammerhead Hairpin HDV

Serganov and Patel, Nat. Rev. Genet., 2007; Walter, Mol. Cell, 2007

SELF-SPLICING RIBOZYMES: MECHANISM

Small ribozymes (hammerhead)

2’-OH activated by G12 attacks the scissile phosphate; the leaving group is protonated by a general acid catalyst leading to 2’3’-cyclic phosphate and 5’ OH termini

Large ribozymes (group I intron)

Two divalent metal ions coordinate the scissile phosphate and adjacent ribose; distal 3’-OH activated by a base catalyst attacks the phosphodiester,

E. coli

RNase P RNA

Serganov and Patel, Nat Rev Genet, 2007; Evans et al, TiBS, 2006

RNase P RNA – a true enzyme

tRNA processing, multiple turnover

TRANS-SPLICING

Lasda and Blumenthal, WIRERNA., 2011

- SL (spliced leader) trans-splicing joins short 5’ exon from the

specialized SL RNA

- Genic trans-splicing joins exons of different pre-mRNA transcripts

-Both utilize the basic splicing

machinery with SL snRNP (U2, U4, U5, U6, no U1)

- used by protozoa (Kinetoplastae) to produce variable surface

antigens and when changing life- stages

Caenorhabditis elegans Drosophila melanogaster mammals

Nematodes Trypansomes Chordates

5’ splice site

Sm

Spliced leader 2 (SL2) has features of a 5’ splice site and an snRNA

SL LEADER RNA

SL LEADER RNAs

Lasda and Blumenthal, WIRERNA., 2011 5’ trans splice

site (tss)

SL TRANS-SPLICING

Blumenthal, Trans-splicing and operons, 2005

~70% of C. elegans genes begin with a 22 nucleotide SL sequence

C. elegans OPERONS

most a few

Blumenthal, Trans-splicing and operons, 2005

13-15% of all genes in C. elegans are expressed as part of an operon

Trans-splicing is not limited to lower eukaryotes, examples are found in humans, rat and Drosophila

Dorn et al. PNAS, 2001

mod(mdg4) locus

TGCCCACTAaACCCCATGCTTTCGGTTTTCCTCGACTCTCGAG ATACGGAGATCAGTT 5’ splice

site branchpoint polypyrimidine tract

GENIC TRANS-SPLICING

GENIC TRANS-SPLICING

Dorn et al. PNAS, 2001

Sequence comparison of new Mod(mdg4) protein isoforms

HOMING PROCESS

Barzel et al. NAR, 2011

Copying and propagation of the gene into the allele initially lacking this gene (selfish genetic elements?)

I-AniI LAGLIDADG homing endonuclease

TYPES:

LAGLIDADG GIY-YIG

His-Cys box H-N-H

PD-(D/E)xK Vsr-like

Stoddard, Structure, 2011

HOMING PROCESS

RNA

RNA

RNA

DNA

DNA DNA

DNA

Nemeth and Langst. Trends in Genet, 2011

NUCLEAR STRUCTURES/BODIES

Zhao at al. Curr. Op. Gen. Dev., 2009

NUCLEAR STRUCTURES/BODIES

http://bellsouthpwp2.net/b/h/bhagavathula4745/The%20nucleus.htm

NUCLEAR STRUCTURES/BODIES

NUCLEAR STRUCTURES/BODIES

Mao at al TiG., 2011

NUCLEAR RNP STRUCTURES

Cajal Bodies

(Ramon y Cajal, 1903)

- RNP assembly factory: sn/snoRNA, telomerase RNA processing, modification and assembly - contain specific scaRNAs (RNA modification)

- associate with telomeres and histone and snRNA gene loci - different composition (coilin, SMN protein,

Nuclear Speckles

(Interchromatin Granule Clusters, IGCs) - enriched in splicing related factors (snRNP, SR proteins)

- usually not transcriptionally active but transcriptionally active genes also associate with NS - role in RNA processing or storage of RNA processing factors

Paraspeckles

- regulate the expression of some genes by nuclear retention of RNA - contain PSF/SFPQ, P54NRB/NONO and PSPC1 proteins

- organized around ncRNA (eg. NEAT1)

PML Bodies

(Promyelocytic Leukemia Nuclear Bodies)

- role in tumor suppression, viral defense, DNA repair, transcriptional regulation - localize to gene-rich and transcriptionally active regions of chromatin

- composition might be heterogeneous and functionally different - found also in the cytoplasm

PcG Bodies

– Polycomb Bodies

- a subset of RNAi factors (AGO, Dicer, Piwi) localize to PcG Bodies - located close to heterochromatin, centers of gene repression

OPT Domain

appear in G1 phase, represent sites of DNA damage

plant Dicing bodies

contain DCL1, HYL1 and SE proteins, function in pri-miRNA processing

CB NS

PS

PML

PcG

NUCLEAR STRUCTURES/BODIES

Mao at al TiG., 2011

Reaction sites

-

pre-RNA processing in nucleoli - modification and assembly of snRNAs in CB

Hot spots

- gene activation or repression - epigenetic reactions

- stabilization of interactions between gene loci

Storage/modification sites of proteins and RNAs

-

some A-to-I hyperedited mRNAs are retained in paraspecles

- phosphorylation of SR proteins in nuclear speckles

- sumoylation of nuclear proteins in

PcG bodies

NUCLEAR RNP STRUCTURES

Sleeman and Trinkle-Mulcahy, Curr Op Cell Biol, 2014

PARASPECKLE FUNCTION

Bond and Fox JCB, 2009

CAJAL BODY FUNCTION

Matera and Shpargel, Curr. Op. Cel. Biol., 2006

DIFFERENT CBs?

Matera and Shpargel, Curr. Op. Cel. Biol., 2006

TAKE-HOME MESSAGE

• There are strange and unusual RNA processing reactions like editing, self-splicing, trans-splicing, retrohoming....

• They contribute to the gene expression versatility

• RNA processing, modification, assembly of RNPs occur in

different nuclear RNP substructures