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(1)

Chrissie Barrass, 2011, cover of Mol. Cell

RNA PROCESSING

Co- or post- transcriptional?

(2)

Major co-transcriptional mRNA processing steps

Bentley, Nat. Rev. Genetics, 2014

(3)

Co-transcriptional mRNA processing

Phatnani and Greenleaf, 2006

CTD posphorylation status

Phospho-CTD

Associated Proteins

- transcription

- chromatin structure - RNA processing

(splicing, 3’ end formation) - RNA export

- RNA degradation - snRNA modification - snoRNP biogenesis - DNA metabolism

- protein synthesis and degradation

(4)

Lenasi and Barboric, WIREsRNA, 2013

(5)

Co-transcriptional mRNA processing

Bentley, Nat. Rev. Genetics, 2014

“Miller spread” electron micrograph

(D. melanogaster)

DNA template + engaged Pol II + nascent RNA transcripts + bound proteins (blobs) + co-transcriptionally spliced out introns (arrows)

(6)

Co-transcriptional mRNA processing

Bentley, Nat. Rev. Genetics, 2014

• DNA template - modified histones affect trx and recruitment of processing factors

Engaged Pol II with CTD (P-status in blue and green) binding processing factors

(capping factors, spliceosome, termination and 3’ cleavage and polyadenylation machinery)

• Nascent RNA – capped with 3’ polyA AAUAAA signal

Proteins bound to CTD and/or RNA

(7)

CAPPING

Guo and Lima, Cur. Op.Str.Biol., 2005

GT/Ceg1-guanylyltransferase MT/Abd1-methyltransferase (promote early elongation) Cet1-RNA triphopshatase (inhibits re-initiation)

CBC-cap binding complex

Co-transcriptional capping

- occurs after the synthesis of 10- 15 nt of RNA

- CE recruitment to CTD requires high Ser5-P

NH N N

N O

O

OH OH O P O

O- O

N N

N+ O- H2N N

O OH OH

O P O- O

P O- O O

NH2

CH3

γ β α

7-methylguanosine 5’-5’-triphosphate bridge (m7G)

(8)

CAPPING

(9)

Pre-mRNA SPLICING

Two step mechanism: nucleophilic attack of the ribose 2’-OH group (branch point Adenosine, H2O, Me2+) on the phosphate

Eukaryotes

organelles (fungi, plants), bacteria,

mitochondria (animals)

mRNA splicing-like

organelles (fungi, plants), bacteria, archea

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

RIBOZYMES

Exon 1 Exon 2

(10)

Pre-mRNA SPLICING

Alberts B, Bray D, Lewis J, et al., Molecular Biology of the Cell. 3rd edition;

(11)

de Almeida and Carmo-Fonseca, FEBS Lett, 2008

(12)

1. Average human pre-mRNA contain 27,000 nucleotides and 9 exons 2. Average exon contain 145 nucleotides

3. There are exons with only 3 nucleotides (one amino acid) 4. Average intron contain 3500 nucleotides

5. Average mRNA contains 1340 nucleotides, so only 5% of pre-mRNA ends up in mRNA

6. Dystrophin contains 3684 amino acids and is encoded by the largest human gene of 2.5 million nucleotides and 79 exons

Do you know that:

Zbigniew Dominski, lectures 2008

(13)

Pre-mRNA SPLICING: CIS ELEMENTS

yeast

human

Warf and Berglund, 2010, TiBS; Reddy, Ann.Rev.PlantBiol., 2007

5’ Splice Site 3’ Splice Site

3’ Exon

A

Branch Point “A”

5’ Exon

5’ Splice Site 3’ Splice Site

Zbigniew Dominski, lectures 2008

The consensus splicing sequences are not so conserved after all

(14)

Pre-mRNA SPLICING: TRANS ELEMENTS

active center

5 snRNAs U1, U2, U4, U5, U6

snRNAs

D1 G D3 B

F D2 E

Sm/Lsm

pre-mRNA::snRNA base-pairing

Warf and Berglund, 2010, TiBS; Reddy, Ann.Rev.PlantBiol., 2007

(15)

STRUCTURAL REARRANGEMENTS

5’ Exon AG GUAAGU CUR CU Yn YAG G 3’ Exon

U2 snRNP

U4 snRNP U6 snRNP

A

65 35 U1 snRNP

snRNP U5 U4/U6.U5 tri-snRNP

snRNP specific proteins

Zbigniew Dominski, lectures 2008

(16)

STRUCTURAL REARRANGEMENTS

Zbigniew Dominski, lectures 2008

U2 snRNP

U1 snRNP

U5 snRNP

A

U4 snRNP U6 snRNP

U1 and U4 snRNPs leave the complex, the U6 snRNP is involved in catalysis

3’ Exon 5’ Exon

A

U2 snRNP

U6 snRNP U5 snRNP

recycling

(17)

Chemistry of pre-mRNA splicing and U2/U6 model

(18)

Cryo EM

C complex native spliceosome

SPLICEOSOME

Azubel et al, Mol. Cell, 2004;

Jurica et al, Nat. Str. Mol. Biol, 2009

U1 snRNP

Krummel et al, Nature, 2009

(19)

SPLICEOSOMAL COMPLEXES

0 20 40 time (min)

Z A B C

native gel, labeled pre- mRNA

substrate

Crucial components of the spliceosome:

Prp8 (U5 specific, contact 5’ ss, BP, 3’ss)

Prp19 and NTC (the nineteen complex) important for catalytic activation SF3a/SF3b stabilize U2-BP interaction

(20)

SPLICEOSOMAL COMPLEXES

Jurica, Curr.Op.Str.Biol., 2008

(21)

Step-wise assembly of the spliceosome and catalytic steps of splicing

TRANSCRIPTION AND SPLICING

Matera and Wang, Nat Rev Mol Cell Biol, 2014

(22)

SPLICING FACTORS

Jurica and Moore, Mol.Cell, 2003

5 snRNAs

41 snRNP proteins

> 70 splicing factors

> 30 other proteins

(23)

5’ Exon AG GUAAGU CURACU Yn YAG G 3’ Exon 5’ splice site branch site 3’ splice site

PolyY

MINOR SPLICEOSOME (U12-type)

Minor type of splicing depends on U4atac, U6atac, U11 and U12 snRNPs

Zbigniew Dominski, lectures 2008

Konig et al, 2007, Cell

Slower splicing of U12-type introns often leads to an aberrant mRNA with single unspliced U12-type intron, which leaves the nucleus and is spliced by the cytoplasmic minor spliceosome.

Lack of minor cytoplasmic splicing results in degradation of aberrant transcripts by NMD.

(vertebrates)

UCCUURAY

5’ Exon GUAUCCUUY YAG 3’ Exon

~1% of pre-mRNAs have U12 type atac introns

AUAUCCUUY YAC

(24)

Reddy, Ann.Rev.PlantBiol., 2007; Chen and Manley, Nat.Rev.Mol.CellBiol., 2009

ALERNATIVE SPLICING (AS)

communication between the 3’

and 5’ splice sites

(25)

Reddy, Annu. Rev. Plant Biol., 2007

ESR – exonic splicing regulatory elements ISRintronic splicing regulatory elements ESS/ISS exonic/intronic splicing silencers ESE/ISE - exonic/intronic splicing

enhancers

SR – Ser/Arg rich proteins

PTB – polypyrimidine tract-binding proteins hnRNP – heterogenous nuclear RNP

AS occurs at the level of recognition of splice sites and other regulatory elements by RNA-binding proteins

ALERNATIVE SPLICING (AS)

Exons and introns often contain sequences that facilitate or inhibit splice site usage.

These elements bind splicing activators or repressors.

(26)

Luco and Misteli, Curr Op Gene Dev, 2011

AS – splicing CODE: chromatin, ncRNAs, SF

(27)

ALERNATIVE SPLICING (AS)

McManus and Graveley, Cur.Op.Gene.Dev., 2011

(28)

ALERNATIVE SPLICING (AS)

Keren et al, Nat.Rev.Genet., 2010

SR proteins bind to ESEs to stimulate the binding of U2AF to the upstream 3′ splice site (ss) or the binding of the U1 snRNP to the downstream 5′ ss.

SR proteins function with other splicing co-activators (TRA2) and the SR- related nuclear matrix proteins SRm160–SRm300.

(29)

RRM: RNA recognition motif RRMH: RRM homolog

second RNA-binding domain with a poor match to the RRM consensus

Human SR PROTEINS

Zbigniew Dominski, lectures 2008

(30)

Female embryo

XX plus 2A X:A = 1

Male embryo

XY plus 2A X:A = 0.5 Sex-lethal

pre-mRNA Sex-lethal

mRNA Sex-lethal

protein

No Sex-lethal protein SL -

STOP

=

AS: Drosophila sex determination

Zbigniew Dominski, lectures 2008

Sex Lethal controls AS of “Male-specific lethal 2” (Msl2) produced in males

Msl2 pre-mRNA

Msl2 mRNA

Msl2 protein Msl2 translation inhibited Msl2 SL - SL

- SL - SL

-

AS generates “Sex Lethal” protein female embryos, a splicing inhibitor

(31)

Transformer pre-mRNA Transformer

mRNA Transformer

protein No Tra

SL -

Tra +

AS: Drosophila sex cont.

Sex Lethal modifies AS of Transformer pre-mRNA

Zbigniew Dominski, lectures 2008

Tra – splicing activator - affects production of F/M Double-sex proteins:

transcriptional factors controlling expression of female/male genes

Transformer protein

Double-sex pre-mRNA Double-sex

mRNA Double-sex

protein

3 4 5

3 5

F Double-sex

4

Tra

+ No Tra

3 4 5

3 5

M Double-sex

(32)

AS AND DISEASE

(33)

AS in YEAST S. cerevisiae

290 intron-containing genes (5%), most are single introns

introns are enriched in highly expressed genes

yeast has probably lost introns in many genes

• 45 intron-containing genes are inefficiently spliced during

vegetative growth

• regulated splicing of 13 of the 20 intron-containing meiotic genes

+ RPL30, YRA1, MTR2

• regulated splicing/AS in most cases – intron retention

(34)

AS in YEAST S. cerevisiae

Grund et al, JCB, 2008

2 genuine AS events for

SRC1

and

PTC7

that generate 2 proteins:

- SRC1

splice variants (different 5’ ss) give products of full and reduced activity

- PTC7

AS- different localization of proteins

product of unspliced mRNA localizes to the nuclear envelope, product of PTC7 spliced mRNA to mitochondria

SRC1

(35)

Some facts on AS

• AS, widespread in higher eukaryotes, increases protein complexity (expression dependent on tissue type, cell cycle phase or stage of development;

different level of biochemical activity; the presence of important regulatory domains)

• 75% of human and 50% of plant genes are estimated to produce AS events

Average human pre-mRNA generates 3 different mRNAs

AS is most common in neurons

AS is linked with transcription

- promoter structure contributes to AS - transcription activators affect AS

- elongation rate: slow trx may favor inclusion of alternative exons, fast trx promote exclusion of these exons

• AS can affect mRNA stability and turnover:

many alternatively spliced transcripts (> 30%) contain premature

termination codons (PTC) that generate Nonsense Mediated Decay (NMD) substrates

(36)

Co-transcriptional mRNA processing:

SPLICING

pre-mRNA:

- m7G cap synthesis (Ser5-P) - spliceosome assembly

- splicing, at least partially

Exon1 Exon2

Intron

Spliceosome

Munoz et al., TiBS, 2009

Spliceosome step-wise co-trx assembly

- U1 recruited by interaction with the 5’ss and BP

- U2 and U4/U5/U6 tri-snRNPs

- structural re-arrangements within the spliceosome

Lacadie and Rosbash, Mol.Cell, 2005

(37)

Kim and Kim, EMBO, 2007

Alexander et al, RNA, 2010

Kinetic analysis of transcription by high-resolution qRT-PCR

nascent (uncleaved) spliced transcript

Co-transcriptional mRNA processing:

SPLICING

(38)

Lenasi and Barboric, WIREsRNA, 2013

TRANSCRIPTION AND SPLICING

CTD Ser-P and splicing Elongation rate and splicing

(39)

CLEAVAGE AND POLYADENYLATION

mRNA

ncRNA

Cleavage and polyadenylation complex (CP) (recruited at Ser2-P CTD)

Jacquier, Nat. Rev. Genet, 2009

Millevoi and Vagner, NAR, 2008 metazoan

the

yeast

the

(40)

TRANSCRIPTION TERMINATION:

hybrid allosteric- torpedo model

Luo and Bentley, Gene Dev, 2006

Pap1 CP

3’-end processing factors are recruited to Ser2-P CTD at 3’ end of genes via CID (CTD-interacting domain) of Pcf11 for CP and Rtt103 for Rat1 5’-3’

exonuclease and its activator Rai1.

Pcf11 and Rat1 coordinately contribute to the recruitment of 3’- end processing factors

(41)

Jacquier, Nat. Rev. Genet 2009

Pol II

CTD

Nrd1/Nab3/Sen1-dependent TERMINATION

mRNA

ncRNA

Nrd1/Nab3/Sen1 complex

sn/snoRNAs

CUTs

short mRNAs (< 600 nt)

(Ser5-P)

(42)

RNA STRUCTURE-DEPENDENT TERMINATION

Zenkin, Cell Cycle, 2014

(43)

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.

(44)

Alternative cleavage and polyadenylation (APA)

Tian and Manley, TiBS, 2013

APA impacts the cellular transcriptome and proteome - Alternative 3’UTRs regulate mRNA metabolism (stability) - APA affects protein isoforms (pA signals located in exons) - APA contributes to the variety of lncRNAs

APA dynamics under different biological conditions - Tissue specificity

- Controls response to extracellular signals

- Responds to growth and developmental conditions

APA is modulated by different factors: CP, RBPs, splicing and snRNPs, transcription, chromatin structure and histone modification (?)

(45)

Alternative cleavage and polyadenylation (APA)

Tian and Manley, TiBS, 2013

(46)

Bentley, Nat. Rev. Genetics, 2014

Kinetic model of coupling

transcription and processing

(splicing/AS and

polyadenylation/APA

(47)

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

(48)

Nsi1/

Reb1

Rnt1

Fob1

25S

5’

Pol I

Rnt1 Nsi1/Re

b1

Rat1/

Rai1

Pol I termination factors:

DNA-binding protein Nsi1/Reb1

• Pol I subunit Rpa12

endonuclease Rnt1

RFB binding protein Fob1

• 5’-3’ exonuclease Rat1/Rai1 (torpedo mechanism)

RNA helicase Sen1

Nrd1/Nab3 complex (??)

Pol I TRANSCRIPTION TERMINATION

yeast

mammalian

PTRF – release factor

SETX – helicase, Sen1 homolog

TTF-I – transcription termination factor I

Richard and Manley, GeneDev., 2009

transcript release element T-stretch + TTF-I pause site

(49)

Landrieux et al., EMBO J., 2006

Pol III TRANSCRIPTION TERMINATION

C37-C53 subcomplex is situated across the cleft near RNA exit C11 (TFIIS) subunit of Pol III has intrinsic RNA cleavage activity important for Pol lll termination

Fernadez-Tornero et al., Mol. Cell, 2007

Pol III EM structure

Richard and Manley, Gene Dev., 2009

(50)

Making the ribosome takes approximately 200 non-ribosomal proteins, 100 snoRNAs and 80 ribosomal proteins!

Nob1 Utp24

Fatica and Tollervey, Cur. Op. Cel. Biol., 2002

rRNA PROCESSING and MODIFICATION

Rrp17

Rrp17 Ngl2

(51)

Pre-rRNA PROCESSING

Phipps et al, WIRERNA., 2010

(52)

Pre-rRNA PROCESSING

Henry et al, CMLS, 2008

Pre-rRNA processing requires snoRNAs (small nucleolar

RNAs)

U3 snoRNP +

tUTP complex

(53)

Interaction of U3 snoRNA with pre-rRNA

Phipps et al, WIRERNA., 2010

(54)

Early cleavages (A0-A2) in the pre-rRNA and modification of riboses (2’-OMe) and bases (pseudo-U) are carried by snoRNP complexes

rRNA PROCESSING and MODIFICATION

Reichow et al., NAR, 20027

Cbf5

boxH/ACA: pseudouridylation

Nop1/

fibrillarin

boxC/D: 2’-O-methylation

(55)

RNA MODIFICATIONS

tRNAs, rRNAs, snRNAs, and snoRNAs

(56)

CH

3

S-adenosylmethionine (SAM) 2’-O-methyl transferase

1. 2’-O-methylation (modification of the ribose sugar)

2. Conversion of uridine to pseudouridine by pseudouridine synthase

RNA MODIFICATIONS

tRNAs, rRNAs, snRNAs, and snoRNAs

U

Common in rRNA, tRNA (up to > 1 %)

(57)

rRNA cotranscriptional PROCESSING and MODIFICATION

Granemman and Baserga, Curr.Op.CellBiol., 2005;

Kos and Tollervey, Mol.Cell’10 coupled co-trx

Rnt1 cleavage and termination

co-trx cleavage

Cleavage dividing small and large subunits is largely co-transcriptional (70%) Also rRNA modification (ribose methylation) is co-transcriptional and occurs on the nascent transcript, predominantly for the small subunit and partially for the large subunit.

(58)

rRNA cotranscriptional PROCESSING and MODIFICATION

Phipps et al, WIRERNA., 2010

(59)

• All tRNAs share a common cloverleaf secondary structure and a common tertiary structure which resembles an inverted L.

• The L shape maximizes stability by lining up base pairs in the D stem with those in the anticodon stem, and the base pairs in the T stem with those in the acceptor stem.

tRNA PROCESSING: 3D STRUCTURE

(60)

tRNA precursors:

- 5’ end by RNAse P - 3’ end by tRNase Z - alternative 3’ pathway:

exonucleolytic 3’-end processing by Rex1 and Rrp6

tRNA PROCESSING

Zbigniew Dominski, lectures 2008

D

Anticodon acceptor stem RNase P

tRNase Z

5’ leader 3’ leader

Rex1

Rrp6

(61)

tRNA 5’ and 3’ end PROCESSING PATHWAYS

Schurer et al. Biol. Chem. 2001 5-15 nt

CCA adding enzyme CCase

Ntase tRNase Z

RNase E RNases

PH, D T, II, PNPase E BN/Z (exo/endo)

(62)

tRNA MATURATION

CCA addition by tRNA nucleotidyl-transferase (collaborative templating)

Schurer et al, Biol.Chem., 2001

Aminoacylation by tRNA aminoacyl synthetases

two classes: class I and class II (aminoacylate 2’-OH and 3’-OH of A, respectively)

can occur in the nucleus and in the cytoplasm

(63)

Abelson et al. J. Biol. Chem. 1998

tRNA SPLICING

Hopper and Shaheen, TiBS,2008

nucleus processing aminoacylation

cytoplasm splicing aminoacylation

tRNA splicing occurs in the cytoplasm

• tRNA travels between nucleus and cytoplasm during processing steps

(64)

MECHANISM of tRNA SPLICING

adenylyl synthetase

ligase cyclic

phosphodiesterase

intron

Abelson et al. J. Biol. Chem. 1998

(2’ phosphotransferase)

1

2

3

red-intron

green-anticodon

YEAST:

272 tRNA genes 59 contain introns

(65)

RNA MODIFICATION: tRNA

Hopper and Phizicky, GeneGev. 2003

Functions of modifications:

• contribute to folding

reinforce 3D structure

provide stability

• facilitate alternative structures

affect codon recognition (wobble bp)

• contribute to translation (frameshifting)

(66)

Fatica et al., EMBO, 2000

sn/snoRNA processing

(small nuclear and nucleolar RNAs)

3’ and 5’ processing

RNA precursor RNP proteins

termination RNA precursor

Rnt1 Rnt1

Rat1/Xrn1

RNP proteins

(67)

AAAAAAAAAAA

AAAAAAAAAAA Exosome: 3’- 5’ exo/endo-nuclease

• complex; 10 core components (RNA BP)

• catalytically active hydrolytic Dis3/Rrp44 (RNase II)

• nuclear cofactors- RNA BP Rrp47, nuclease Rrp6 (RNase D),

RNA helicase Mtr4

• cytoplasmic cofactors- Ski2-3-8 complex (RNA helicase Ski2), GTPase Ski7

• subtrates- processing and/or degradation of almost all RNAs

TRAMP: nuclear surveillance

Trf4/5 + Air1/2 + Mtr4 poly(A)

polymerase RNA binding

proteins RNA DEVH helicase

sn/snoRNA processing

(68)

TAKE-HOME MESSAGE

RNA capping, splicing, 3’ end formation, export occur, entirely or partly, cotranscriptionally

Splicing is carried out by a large complex, spliceosom, with a catalytic heart made of snRNAs

(+ several protein components)

Alternative splicing, a highly regulated process (SR proteins) , increases protein complexity but often generates NDM substrates

Transcript 3’ end formation is linked to transcription

termination, both depend on Cleavage and Polyadenylation or Nrd1/Nab3 complexes

RNA modification is largely post-transcriptional, but co-trx

cases (rRNA) also occur

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