Chrissie Barrass, 2011, cover of Mol. Cell
RNA PROCESSING
Co- or post- transcriptional?
Major co-transcriptional mRNA processing steps
Bentley, Nat. Rev. Genetics, 2014
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
Lenasi and Barboric, WIREsRNA, 2013
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)
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
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)
CAPPING
DNA METHYLATION
DNA methylation in mammals occurs mainly at CpG.
Methylation of the promoter suppresses gene expression.
Gene-body DNA methylation protects the gene body from spurious RNA polymerase II entry and cryptic transcription initiation (shown in mouse embryonic stem cells).
Such spurious transcripts can either be degraded by the exosome or capped, polyadenylated, and delivered to the ribosome to produce aberrant proteins.
Elongating Pol II triggers DNA methylation to ensure the fidelity of transcription initiation.
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
Pre-mRNA SPLICING
Alberts B, Bray D, Lewis J, et al., Molecular Biology of the Cell. 3rd edition;
de Almeida and Carmo-Fonseca, FEBS Lett, 2008
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
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
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
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
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
Chemistry of pre-mRNA splicing and U2/U6 model
C complex yeast
Galej et al, Nature, 2016
U1 snRNP
Krummel et al, Nature, 2009
C* complex human second step
Bertramet al, Nature, 2017
SPLICEOSOME
Cryo- EM
U4/U6.U5 tri-snRNP
Nguyen1*, Galej et al, Nature, 2016
C* complex yeast
Fica et al, Nature, 2017
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
SPLICEOSOMAL COMPLEXES
Jurica, Curr.Op.Str.Biol., 2008
Step-wise assembly of the spliceosome and catalytic steps of splicing
TRANSCRIPTION AND SPLICING
Matera and Wang, Nat Rev Mol Cell Biol, 2014
SPLICING FACTORS
Jurica and Moore, Mol.Cell, 2003
5 snRNAs
41 snRNP proteins
> 70 splicing factors
> 30 other proteins
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
Reddy, Ann.Rev.PlantBiol., 2007; Chen and Manley, Nat.Rev.Mol.CellBiol., 2009
ALERNATIVE SPLICING (AS)
communication between the 3’
and 5’ splice sites
Reddy, Annu. Rev. Plant Biol., 2007
ESR – exonic splicing regulatory elements ISR – intronic 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.
Luco and Misteli, Curr Op Gene Dev, 2011
AS – splicing CODE: chromatin, ncRNAs, SF
ALERNATIVE SPLICING (AS)
McManus and Graveley, Cur.Op.Gene.Dev., 2011
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.
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
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
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
AS AND DISEASE
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
AS in YEAST S. cerevisiae
Grund et al, JCB, 2008
2 genuine AS events for
SRC1and
PTC7that 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
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
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
CO-TRANSCRIPTIONAL PROCESSES
SPLICING
Wong et al., TiG, 2014
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
Lenasi and Barboric, WIREsRNA, 2013
TRANSCRIPTION AND SPLICING
CTD Ser-P and splicing Elongation rate and splicing
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
Cleavage by CPSF-73 (human), Brr5/Ysh1 (yeast)
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
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)
Lykke-Andersen and Jensen, Biochemie, 2007
Trx terminaton by Pol II for different transcripts
RNA STRUCTURE-DEPENDENT TERMINATION
Zenkin, Cell Cycle, 2014
U1 snRNA :
transcription directionality,
premature termination and
non-coding transcription
TRANSCRIPTION TERMINATION-
Polymerase pausing
Proudfoot, Science, 2016
POLYMERASE BACKTRACKING
Proudfoot, Science, 2016
The EXOSOME in trx termination
Kilchert et al, Nat Rev Mol Cell Biol, 2016
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 (?)
Alternative cleavage and polyadenylation (APA)
Tian and Manley, TiBS, 2013
Bentley, Nat. Rev. Genetics, 2014
Kinetic model of coupling
transcription and processing
(splicing/AS and
polyadenylation/APA
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
histone mRNA synthesis
histone mRNA decay
Scheer et al, TiG., 2016
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 (yeast)
Baillat and Wagner., TiBS., 2015
Integrator (INT) complex - snRNA 3’ end processing
snRNA-type promoter: DSE recruits transcription factors PSE bound by SNAPc (snRNA activating complex)
CTD of Pol II
3’-box (GTTTN– AAARNNAGA), located 9–19 nt downstream of the snRNA 3’-end INT - recruited contransctiptionaly to snRNA promoter
- interacts with Pol II CTD (Ser7-P/Ser2-P dyad) - cleaves pre-snRNA at 3’box
INTS11 – CPSF73 paralog
snRNA BIOGENESIS (metazoa)
, JCS, 2004
snRNA biogenesis (metazoa)
CRM1 - export receptor
PHAX(-P) - export adaptor, binds to CBC
SMN - survival of motor neuron, binds snRNA and core Sm proteins to assemble mature snRNP TGS1 - trimethylguanosine synthase, hypermethylates m7G cap to 2,2,7-trimethylguanosine cap SPN - import adaptor snurportin; Imp-β - import receptor importin-β
Matera et al, Nat.Rev.Mol.Cel.Biol, 2007
DIS3L2 ?
snRNA biogenesis
(metazoa)
Matera and Zhang
Nat. Rev. Mol. Cell Biol, 2014
DIS3L2 ?
miRNA biogenesis (animals)
Krol et al., Nat.Rev.Genet., 2010
Ha and Kim Nat. Rev. Mol. Cell Biol, 2014
miRNA biogenesis regulation
nucleus cytoplasm
PolI transcripts processing
Müller-McNicoll and Neugebauer Nat. Struct. Mol. Biol, 2014
snRNAs
histone mRNAs
ncRNAs
mRNAs
Matera and Zhang Nat. Rev. Mol. Cell Biol, 2014
mRNA and snRNA processing
Andersen et al, WIREsRNA, 2013
Pol I and Pol III
Vanini, BBA, 2013
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
Landrieux et al., EMBO J., 2006
Pol III TRANSCRIPTION TERMINATION
C1, C2 core subunits (Pol pausing)
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
Trx termination Pol I
Pol II Pol III
Arimbasseri et al, BBA, 2013
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
Pre-rRNA PROCESSING
Phipps et al, WIRERNA., 2010
Pre-rRNA PROCESSING
Henry et al, CMLS, 2008
Pre-rRNA processing requires snoRNAs (small nucleolar
RNAs)U3 snoRNP +
tUTP complex
Interaction of U3 snoRNA with pre-rRNA
Phipps et al, WIRERNA., 2010
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
RNA MODIFICATIONS
tRNAs, rRNAs, snRNAs, and snoRNAs
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 %)
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.
rRNA cotranscriptional PROCESSING and MODIFICATION
Phipps et al, WIRERNA., 2010
• 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
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
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)
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
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
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
tRNA SPLICING
Weitzer et al, WIREs RNA,2015 endonuclease complex
kinase
direct ligation
ligase cofactor
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)
tRNAs and DISEASE
Kirchner and Ignatova, NatRevGenet, 2015
Kirchner and Ignatova, NatRevGenet, 2015
tRNAs and STRESS
tRNA BIOGENESIS: OVERVIEW
Kirchner and Ignatova, NatRevGenet, 2015
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