rna processing dna mature rna protein precursor rnas pre-rrna pre-trna pre-mrna cleavage nucleotide...
TRANSCRIPT
RNA PROCESSING
DNA
MATURERNA
PROTEIN
PRECURSOR RNAs
pre-rRNApre-tRNApre-mRNA
CleavageNucleotide additionNucleotide insertionNucleotide removalSequence additionSequence removalBase modificationSugar modification
Other RNA-related factors affecting expressionabundance (combination of transcription and degradation)localizationrecruitment to ribosomes
RNAs THAT FUNCTION IN RNA PROCESSING
rRNAsnoRNAs form complexes with protein, direct nt modifications
snoRNAs also modify tRNAs, and likely other RNAstRNARNase P has both RNA and protein components
mRNAsnRNPs U1,2,4,5,6 form spliceosomes with many proteinsgRNAs provide sequence information for RNA editingmiRNAs important for regulating gene expressionsiRNAs important for regulating gene expression
RNAs THAT FUNCTION IN RNA PROCESSING
RNA functions in RNA processing
based on complementary basepairing to direct site of action
action is usually catalyzed by protein
some RNAs—ribozymes—are have catalytic activityself-splicing intron in Tetrahymena rRNA‘hammerhead’ ribozymes are self-cleaving
another RNA with catalytic function is LSU rRNA
Telomerase RNA for telomere replicationRNA primer for mitochondrial replication
PROCESSING OF rRNAs
Cleavage: Pre-rRNA is cleaved to 18S, 5.8S, 28S rRNAs;cleavage order is precise (within species).
Modification: Bases and sugars are modified prior to assembly into ribosomes.
5S rRNA encoded separately, elsewhere in genome
ETS ITS1 ITS2 ETS18S 5.8S 28S
ETS ITS1 ITS2 ETS18S 5.8S 28S
ETS ITS1 ITS2 ETS18S 5.8S 28S
ETS ITS1 ITS2 ETS18S 5.8S 28S
ETS ITS1 ITS2 ETS18S 5.8S 28S
Schematic is generic and not to scale;
rRNA PROCESSING IN NUCLEOLUS
Modifications of nucleotides:
rRNAs
~100 riboses are 2’O-methylated10 bases methylated95 Us isomerized to pseudoUs (ψs)
tRNAs~100 kinds of modified nucleotidessome incorporated during transcriptionsome chemically modified post-transcription
RNA MODIFICATION
snoRNAs
modify rRNAs, tRNAs, miRNAs, siRNAs, and mRNAsC/D snoRNAs direct methylationH/ACA snoRNAs direct pseudouridylation
number of snoRNAs variable between organisms; more being found size range ~60 to ~300 ntencoded individually, in polycistronic clusters, or in introns.
Most C/D snoRNAs have 5’ trimethylguanosine (TMG) cap. So do snRNAs. Patients with motor neuron degeneration diseases often develop antibodies that recognize TMG caps.
SnoRNA interactions with RNA
C/D snoRNAsdirect methylation
H/ACA snoRNAsdirect pseudouridylation
snoRNAs can interact with and modify either one or two sites.
PROCESSING OF tRNAs
All tRNAs undergo:Cleavage to form 5’ and 3’ endsNucleotide modification 3’ CCA addition
Some tRNAs undergo:Intron excisionRNA editing
Following processing, tRNAs are charged by amino acyl transferasesbefore trafficking to cytoplasm.
anticodon
5’ leader 3’ trailer
intron
CCA addition
tRNA PROCESSING
Removal of 5’ leader and 3’ trailer; order not absolute
CCA may be encoded (prok.) or added post-transcriptionally (euk.)
Acceptor stem sometimes edited
Some tRNAs have introns in the anticodon loop
Many nucleotide modifications
editingintron
PROCESSING OF mRNAs
Capping
Polyadenylation
Splicing
Editing
mRNA processing - capping
5’ capping
required for translation of eukaryotic mRNAs
mediates initial ribosome binding
7-methylguanosine cap added as RNA exits RNApol II.
G linked via a 5’-5’ pyrophosphate bridge to first nt of mRNA
G methylated post-addition
first bases in mRNA may be methylated
mRNA processing
From birth to death, an mRNA associates with a variety of proteins and other RNAs that modify it directly or affect its abundance and recruitment to ribosomes.
mRNP (messenger ribonucleoprotein particle) – mRNA + associated proteins.
RNA processing - splicing
Removes blocks of non-coding sequence (introns), ligates the surrounding coding sequences (exons).
Catalyzed by an RNA/protein complex, the spliceosome, which is composed of 5 small nuclear RNAs (snRNAs) designated U1, U2, U4, U5, and U6 plus 50+ proteins
Occurs by two transesterification reactions (no energy)
1. Branch point 2’OH attacks 5’ splice junction
2. 3’OH of 5’ fragment attacks 3’ splice site, forming a phosphodiester bond
The intron is released as a lariat in cis-splicing or a Y intermediate in trans-splicing.
2’OH attack
cis-SPLICING
3’OH attack
Pre-mRNA
5’ exon 3’ exonGU A YAG
1st TE
2nd TE
Splicing snRNPs
RNA SPLICING
U1 RNA (snRNP) forms helix with 5’ splice site
U2 RNA (snRNP) forms helix with branch point
U4, U5, U6 RNA (snRNP)
forms helix with 5’ splice site, displacing U1
forms helix with U2, with loss of U4
First step of splicing occurs
Rearrangement occurs
Second step of splicing occurs
trans-SPLICING
cis-splicing: both exons on same RNAtrans-splicing: exons on different RNAs
trans-splicing
first identified in trypanosomatids adds first bases of 5’ UTR (spliced leader) including 5’ cap ALL tryp mRNAs encoded by nucleus are trans-spliced
some helminth mRNAs are trans-spliced, not alladds 5’ end sequences
a few cases of trans-splicing reported in mammals
catalyzed by a spliceosomeoccurs by successive transesterifications.
trans-SPLICING
Y intermediate
Y intron
2’OH attack
3’OH attack
RNA splicing – mechanisms for diversity
Alternate splicingAlternate promotersAlternate polyadenylation sites
Once considered the exception, it now appears that generating more than one mRNA per gene is a common mechanism for increasing diversity without the ‘expense of maintaining additional genes. Based on ESTs, at least 50% of human genes may produce alternatively spliced mRNAs. Drosophila Dscam gene theoretically has 38,016 possible mRNAs!
RNA PROCESSING - EJC
Exon junction complex (EJC)
core set of proteins and a changing cast of other proteins
impacts mRNA splicing, export, localization, translation, and turnover
associates with mRNA 20-25 nt upstream of exon-exon junctions.
binding to mRNA is position-dependent, not sequence dependent.
effect is location-dependent. EJC in ORF enhances translation. EJC in 3’ UTR enhances turnover.
stays associated with mRNA until translation begins.
RNA 3’ end formationDetails for transcription termination and 3’ end cleavage are debated.
3’ ends of (almost all) eukaryotic mRNAs are generated by cleavage.
Same or similar endonuclease used for 3’ end of mRNAs and of snRNAs.Poly(A) tail is added following 3’ end formation and mRNP is exported to the cytoplasm.
RNA export
mRNP export from nucleus:association with adaptorsexit through NPC
RNA utilization
Localization within cell
Storage until needed
Recruitment to ribosomes
RNA turnover
Proteins destined to be associated may be translated on co-localized polysomes. PABP binds eIF-4E, eIF-4G, circularizing polysomes and increases efficiency of protein synthesis.
mRNA localization
a) DAPI stained S. cerevisiae; b) ASH1 mRNA in same cells; c) hairy (green) and even-skipped (red) mRNAs in Drosophila embryo; d) vasa mRNA localizing to division planes in zebrafish embryo, red is β-catenin; e) dpp mRNA (red) at centrosomes in 8 cell embryo. Blue is DAPI, green microtubules; f) β-actin in cultured neurons (red), green is tau, an axonal marker.
RNA utilization
Localization within cell
Storage until needed
Recruitment to ribosomes
RNA turnover
Translational control
RNA turnover
Steady state abundance of any molecule reflects the balance between its rate of synthesis and degradation. Finetuning cell functions thus requires not only transcription but mRNA turnover.
Recently siRNAs and miRNAs have been identified as exerting considerable effect on RNA degradation and translational blocking, respectively.
In addition, the poly(A) tail is an important feature. mRNAs with long poly(A) tails are preferentially translatedmRNAs with short or absent poly(A) tails are stored or degraded.
RNA turnover
a) Deadenylation followed by decapping and degradation from 5’ end. Alternatively, deadenylation is followed by degradation from 3’ end to the cap, followed by cap degradation.
b) Proteins that recognize prematurely terminated translation trigger both deadenylation and decapping
c) Recruitment of proteins to AU-rich elements triggers both deadenylation and decapping.
RNA turnover
RNA turnover is localized to discrete foci in the cytoplasm called processing bodies or P bodies. Enzymes are partially degraded mRNAs have been co-localized to P bodies
P bodies in HeLa cell visualized using anti-hDCP1a. Nucleus stained with DAPI.
siRNA, miRNA
siRNA: small interfering RNAmiRNA: microRNA
Both are processed to 21-23 nt RNAs which associate with proteins in a RISC complex (RNA-induced silencing complex).
Key roles in regulating gene expression in many eukaryotes but not universal.
RNA editing changes the sequence of an RNA from that encoded by DNA, producing a functional transcript.
First considered a bizarre relic; now recognized as widespread
RNA editing has been reported in:
protozoa, plants and mammals, not yet fungi or prokaryotes
nuclear, mitochondrial, chloroplast, and viral RNAs
mRNA, tRNA, rRNA
Two general typesBase modification (deaminase)
A to I double-stranded mechanism, seen in viruses, human genesC to U, U to C seen in chloroplasts, plant mitochondria, human genes
Insertion/deletionU insertion/deletion, seen in kinetoplastid protozoamono/di nucleotide insertion, seen in Physarumnucleotide replacement, seen in Acanthamoeba tRNAs
RNA editing
AAAAAUAAGUAUUUUGAUAUUAUUAAAGUAAAAGAGGAAUUUUGGGCGGAAGAGAAGGA
GACAGGAGAGGAAAUGAAGGAGAAAGGUUUUGAGAGGGGGGUUUUUUGAGGGGAGGAAA
AAGAAUUUUGAAUUUGAACUAUUUGUUUAAGUUAUGGGAGAGAAGCAAGGAGGAGAAAA
GUAGGGGAAUUUUGAGGAGAUUCUUGGGGAGAGGCGGGCGGGCGACGGCGGUUUUGAAA
ACACCCAUUUUUAGGAGGAUAAGAGGGGAGAAAAGGGGAAAUGGAAUUGGGAAUUGCCU
UUGCCAAACUUUUAGAAGAAAGAGCAGGAAAGGUUAGGGGGAGGAGAGAAGAAAGGGAA
AGUUGUGAUUUUGGAGUUAUAGAAUAAGAUCAAAUAAGUUAAUAAUA
M K E K G F E R G V F W G E E
K E F W I W T I C L S Y G R E A R R R
K V G E F W G D S W G E A G G R R R F
W K H P F L G G ter E G R K G E M E L G I
A F A K L L E E R A G K V R G R R E E
R E S C D F G V I E ter D Q I S terter
pre-edited
AAAAAUAAGUAUUUUGAUAUUAUUAAAGUAAAAGAGGAAUUUUGGGCGGAAGAGAAGGA
GACAGGAGAGGAAAUGAAGGAGAAAGGUUUUGAGAGGGGGGUUUUUUGAGGGGAGGAAA
AAGAAUUUUGAAUUUGAACUAUUUGUUUAAGUUAUGGGAGAGAAGCAAGGAGGAGAAAA
GUAGGGGAAUUUUGAGGAGAUUCUUGGGGAGAGGCGGGCGGGCGACGGCGGUUUUGAAA
ACACCCAUUUUUAGGAGGAUAAGAGGGGAGAAAAGGGGAAAUGGAAUUGGGAAUUGCCU
UUGCCAAACUUUUAGAAGAAAGAGCAGGAAAGGUUAGGGGGAGGAGAGAAGAAAGGGAA
AGUUGUGA**UUGGAGUUAUAGAAUAAGAUCAAAUAAGUUAAUAAUA
AAAAAUAAGUAUUUUGAUAUUAUUAAAGUAAAAGAGGAAUUUUGGGCGGAAGAGAAGGA
GACAGGAGAGGAAAUGAAGGAGAAAGGUUUUGAGAGGGGGGUUUUUUGAGGGGAGGAAA
AAGAAUUUUGAAUUUGAACUAUUUGUUUAAGUUAUGGGAGAGAAGCAAGGAGGAGAAAA
GUAGGGGAAUUUUGAGGAGAUUCUUGGGGAGAGGCGGGCGGGCGACGGCGGUUUUGAAA
ACACCCAUUUUUAGGAGGAUAAGAGGGGAGAAAAGGGGAAAUGGAAUUGGGAAUUGCCU
UUGCCAAACUUUUAGAAGAAAGAGCAGGAAAGGUUAGGGGGAGGAGAGAAGAAAGGGAA
AGUUGuuUGuuA**UUGGAGUUAUAGAAUAAGAUCAAAUAAGUUAAUAAUA
AAAAAUAAGUAUUUUGAUAUUAUUAAAGUAAAAGAGGAAUUUUGGGCGGAAGAGAAGGA
GACAGGAGAGGAAAUGAAGGAGAAAGGUUUUGAGAGGGGGGUUUUUUGAGGGGAGGAAA
AAGAAUUUUGAAUUUGAACUAUUUGUUUAAGUUAUGGGAGAGAAGCAAGGAGGAGAAAA
GUAGGGGAAUUUUGAGGAGAUUCUUGGGGAGAGGCGGGCGGGCGACGGCGGUUUUGAAA
ACACCCAUUUUUAGGAGGAUAAGAGGGGAGAAAAGGGGAAAUGGAAUUGGGAAUUGCCU
UUGCCAAACUUUUAGAAGAAAGAGCAGGAAAGGUUAGGGGGAGGAGAGAAGAAAGuuGu
uGAAAuuuG**GuuUGuuA**UUGGAGUUAUAGAAUAAGAUCAAAUAAGUUAAUAAUA
AAAAAUAAGUAUUUUGAUAUUAUUAAAGUAAAAGAGGAAUUUUGGGCGGAAGAGAAGGA
GACAGGAGAGGAAAUGAAGGAGAAAGGUUUUGAGAGGGGGGUUUUUUGAGGGGAGGAAA
AAGAAUUUUGAAUUUGAACUAUUUGUUUAAGUUAUGGGAGAGAAGCAAGGAGGAGAAAA
GUAGGGGAAUUUUGAGGAGAUUCUUGGGGAGAGGCGGGCGGGCGACGGCGGUUUUGAAA
ACACCCAUUUUUAGGAGGAUAAGAGGGGAGAAAAGGGGAAAUGGAAUUGGGAAUUGCCU
UUGCCAAACUUUUAGAAGAAAGAGCAGGAAAGGUUAGGGGGuuuAGuuuuGuAuuuGAu
uuuuGAuAGuuAuuAuAuuGuuGuuGAAAuuuG**GuuUGuuA**UUGGAGUUAUAGAA
UAAGAUCAAAUAAGUUAAUAAUA
AAAAAUAAGUAUUUUGAUAUUAUUAAAGUAAAAGAGGAAUUUUGGGCGGAAGAGAAGGA
GACAGGAGAGGAAAUGAAGGAGAAAGGUUUUGAGAGGGGGGUUUUUUGAGGGGAGGAAA
AAGAAUUUUGAAUUUGAACUAUUUGUUUAAGUUAUGGGAGAGAAGCAAGGAGGAGAAAA
GUAGGGGAAUUUUGAGGAGAUUCUUGGGGAGAGGCGGGCGGGCGACGGCGGUUUUGAAA
ACACCCAUUUUUAGGAGGAUAAGAGGGGAGAAAAGGGGAAAUGGAAUUGGGAAUUGCCU
UUGCCAAACUUUUAGAAGAAAGAGCAGGAAAGG**AuuuuuuGuuGuuuuuGuuGuuuG
uuuAGuuuuGuAuuuGAuuuuuGAuAGuuAuuAuAuuGuuGuuGAAAuuuG**GuuUGu
uA**UUGGAGUUAUAGAAUAAGAUCAAAUAAGUUAAUAAUA
AAAAAUAAGUAUUUUGAUAUUAUUAAAGUAAAAGAGGAAUUUUGGGCGGAAGAGAAGGA
GACAGGAGAGGAAAUGAAGGAGAAAGGUUUUGAGAGGGGGGUUUUUUGAGGGGAGGAAA
AAGAAUUUUGAAUUUGAACUAUUUGUUUAAGUUAUGGGAGAGAAGCAAGGAGGAGAAAA
GUAGGGGAAUUUUGAGGAGAUUCUUGGGGAGAGGCGGGCGGGCGACGGCGGUUUUGAAA
ACACCCAUUUUUAGGAGGAUAAGAGGGGAGAAAAGGGGAAAUGGAAuUUGuuGGAuuuA
UUuGCC***GCCAuAuuAC****AGuuAuuuAuuuuuuGuAAuAuGAuuuuGCAGuuGA
uAAuGG**AuuuuuuGuuGuuuuuGuuGuuuGuuuAGuuuuGuAuuuGAuuuuuGAuAG
uuAuuAuAuuGuuGuuGAAAuuuG**GuuUGuuA**UUGGAGUUAUAGAAUAAGAUCAA
AUAAGUUAAUAAUA
AAAAAUAAGUAUUUUGAUAUUAUUAAAGUAAAAGAGGAAUUUUGGGCGGAAGAGAAGGA
GACAGGAGAGGAAAUGAAGGAGAAAGGUUUUGAGAGGGGGGUUUUUUGAGGGGAGGAAA
AAGAAUUUUGAAUUUGAACUAUUUGUUUAAGUUAUGGGAGAGAAGCAAGGAGGAGAAAA
GUAGGGGAAUUUUGAGGAGAUUCUUGGGGAGAGGCGGGCGGGCGACGGCGGUUUUGAAA
ACACCCAUUUUUAGGAGGAUAAGAGGGGAGAAAAGGGGuuuAuuAuuuuAUGuGuuuuu
AuAuUUGuuGGAuuuAUUuGCC***GCCAuAuuAC****AGuuAuuuAuuuuuuGuAAu
AuGAuuuuGCAGuuGAuAAuGG**AuuuuuuGuuGuuuuuGuuGuuuGuuuAGuuuuGu
AuuuGAuuuuuGAuAGuuAuuAuAuuGuuGuuGAAAuuuG**GuuUGuuA**UUGGAGU
UAUAGAAUAAGAUCAAAUAAGUUAAUAAUA
AAAAAUAAGUAUUUUGAUAUUAUUAAAGUAAAAGAGGAAUUUUGGGCGGAAGAGAAGGA
GACAGGAGAGGAAAUGAAGGAGAAAGGUUUUGAGAGGGGGGUUUUUUGAGGGGAGGAAA
AAGAAUUUUGAAUUUGAACUAUUUGUUUAAGUUAUGGGAGAGAAGCAAGGAGGAGAAAA
GUAGGGGAAUUUUGAGGAGAUUCUUGGGGAGAGGCGGGCGGGCGACGGCGGUUUUGAAA
ACACCCAUUUUUAGGAGGAUAAGAGGGuuuuuuGuuAuuGAuuuAuuuuAuuuAuuuuu
GuGuuuuGuuuuuGuuuAuuAuuuuAUGuGuuuuuAuAuUUGuuGGAuuuAUUuGCC**
*GCCAuAuuAC****AGuuAuuuAuuuuuuGuAAuAuGAuuuuGCAGuuGAuAAuGG**
AuuuuuuGuuGuuuuuGuuGuuuGuuuAGuuuuGuAuuuGAuuuuuGAuAGuuAuuAuA
uuGuuGuuGAAAuuuG**GuuUGuuA**UUGGAGUUAUAGAAUAAGAUCAAAUAAGUUA
AUAAUA
AAAAAUAAGUAUUUUGAUAUUAUUAAAGUAAAAGAGGAAUUUUGGGCGGAAGAGAAGGA
GACAGGAGAGGAAAUGAAGGAGAAAGGUUUUGAGAGGGGGGUUUUUUGAGGGGAGGAAA
AAGAAUUUUGAAUUUGAACUAUUUGUUUAAGUUAUGGGAGAGAAGCAAGGAGGAGAAAA
GUAGGGGAAUUUUGAGGAGAUUCUUGGGGAGAGGCGGGCGGGCGACGGCGGUUUUGAAA
ACACCCAUUUUUAGGAGGAUAuuuuAuuuuuGuuuuuuuuuuuuAGGGuuuuuuGuuAu
uGAuuuAuuuuAuuuAuuuuuGuGuuuuGuuuuuGuuuAuuAuuuuAUGuGuuuuuAuA
uUUGuuGGAuuuAUUuGCC***GCCAuAuuAC****AGuuAuuuAuuuuuuGuAAuAuG
AuuuuGCAGuuGAuAAuGG**AuuuuuuGuuGuuuuuGuuGuuuGuuuAGuuuuGuAuu
uGAuuuuuGAuAGuuAuuAuAuuGuuGuuGAAAuuuG**GuuUGuuA**UUGGAGUUAU
AGAAUAAGAUCAAAUAAGUUAAUAAUA
AAAAAUAAGUAUUUUGAUAUUAUUAAAGUAAAAGAGGAAUUUUGGGCGGAAGAGAAGGA
GACAGGAGAGGAAAUGAAGGAGAAAGGUUUUGAGAGGGGGGUUUUUUGAGGGGAGGAAA
AAGAAUUUUGAAUUUGAACUAUUUGUUUAAGUUAUGGGAGAGAAGCAAGGAGGAGAAAA
GUAGGGGAAUUUUGAGGAGuuuuuuuuAUUC**GuuuuuuGuuGuGuuuuuuAGAGuGu
uuuuCuuuGuuGuGuCGuuGuuuGuCGACGuuuuuGCGuuuGUUUUGuAAuuuAuuAuC
AuCCCAuUUUUUAuuGuuGAuGuuuuuuGAuuuuuuuUAuuuuAuuuuuGuuuuuuuuu
uuuAGGGuuuuuuGuuAuuGAuuuAuuuuAuuuAuuuuuGuGuuuuGuuuuuGuuuAuu
AuuuuAUGuGuuuuuAuAuUUGuuGGAuuuAUUuGCC***GCCAuAuuAC****AGuuA
uuuAuuuuuuGuAAuAuGAuuuuGCAGuuGAuAAuGG**AuuuuuuGuuGuuuuuGuuG
uuuGuuuAGuuuuGuAuuuGAuuuuuGAuAGuuAuuAuAuuGuuGuuGAAAuuuG**Gu
uUGuuA**UUGGAGUUAUAGAAUAAGAUCAAAUAAGUUAAUAAUA
AAAAAUAAGUAUUUUGAUAUUAUUAAAGUAAAAGAGGAAUUUUGGGCGGAAGAGAAGGA
GACAGGAGAGGAAAUGAAGGAGAAAGGUUUUGAGAGGGGGGUUUUUUGAGGGGAGGAAA
AAGAAUUUUGAAUUUGAACUAUUUGUUUAAGUUAUGGGAGAGAuAuuGCAuuuuuAuuu
uuGuuuuGuuuuuuAuGuGAuuuuuuuuuGuuuAAuAAuuuGuUAGuuGGuGAuA****
GuuuuAuGGAuGuuuuuuuuAUUC**GuuuuuuGuuGuGuuuuuuAGAGuGuuuuuCuu
uGuuGuGuCGuuGuuuGuCGACGuuuuuGCGuuuGUUUUGuAAuuuAuuAuCAuCCCAu
UUUUUAuuGuuGAuGuuuuuuGAuuuuuuuUAuuuuAuuuuuGuuuuuuuuuuuuAGGG
uuuuuuGuuAuuGAuuuAuuuuAuuuAuuuuuGuGuuuuGuuuuuGuuuAuuAuuuuAU
GuGuuuuuAuAuUUGuuGGAuuuAUUuGCC***GCCAuAuuAC****AGuuAuuuAuuu
uuuGuAAuAuGAuuuuGCAGuuGAuAAuGG**AuuuuuuGuuGuuuuuGuuGuuuGuuu
AGuuuuGuAuuuGAuuuuuGAuAGuuAuuAuAuuGuuGuuGAAAuuuG**GuuUGuuA*
*UUGGAGUUAUAGAAUAAGAUCAAAUAAGUUAAUAAUA
AAAAAUAAGUAUUUUGAUAUUAUUAAAGUAAAAGAGGAAUUUUGGGCGGAAGAGAAGGA
GACAGGAGAGGAAAUGAAGGAGAAAGGUUUUGAGAGGGGGGUUUUUUGAGGGGAGGAAA
AAGAAUUUUGAAUUUGAACuUAUUUG***AAuuuG*UAuuUGuuGuuuuGuAuuGuuuu
uuuAuuGuAuAuuGCAuuuuuAuuuuuGuuuuGuuuuuuAuGuGAuuuuuuuuuGuuuA
AuAAuuuGuUAGuuGGuGAuA****GuuuuAuGGAuGuuuuuuuuAUUC**GuuuuuuG
uuGuGuuuuuuAGAGuGuuuuuCuuuGuuGuGuCGuuGuuuGuCGACGuuuuuGCGuuu
GUUUUGuAAuuuAuuAuCAuCCCAuUUUUUAuuGuuGAuGuuuuuuGAuuuuuuuUAuu
uuAuuuuuGuuuuuuuuuuuuAGGGuuuuuuGuuAuuGAuuuAuuuuAuuuAuuuuuGu
GuuuuGuuuuuGuuuAuuAuuuuAUGuGuuuuuAuAuUUGuuGGAuuuAUUuGCC***G
CCAuAuuAC****AGuuAuuuAuuuuuuGuAAuAuGAuuuuGCAGuuGAuAAuGG**Au
uuuuuGuuGuuuuuGuuGuuuGuuuAGuuuuGuAuuuGAuuuuuGAuAGuuAuuAuAuu
GuuGuuGAAAuuuG**GuuUGuuA**UUGGAGUUAUAGAAUAAGAUCAAAUAAGUUAAU
AAUA
AAAAAUAAGUAUUUUGAUAUUAUUAAAGUAAAAGAGGAAUUUUGGGCGGAAGAGAAGGA
GACAGGAGAGGAAAUGAAGGAGAAAGGUUUUGAGAGGGGGGUUUUUUGAGuuGuGGuuu
AuGuuGuuuAAuuuAuAuAGuuuAAUUUUGuAuuA*UUGuAuuACuUAUUUG***AAuu
uG*UAuuUGuuGuuuuGuAuuGuuuuuuuAuuGuAuAuuGCAuuuuuAuuuuuGuuuuG
uuuuuuAuGuGAuuuuuuuuuGuuuAAuAAuuuGuUAGuuGGuGAuA****GuuuuAuG
GAuGuuuuuuuuAUUC**GuuuuuuGuuGuGuuuuuuAGAGuGuuuuuCuuuGuuGuGu
CGuuGuuuGuCGACGuuuuuGCGuuuGUUUUGuAAuuuAuuAuCAuCCCAuUUUUUAuu
GuuGAuGuuuuuuGAuuuuuuuUAuuuuAuuuuuGuuuuuuuuuuuuAGGGuuuuuuGu
uAuuGAuuuAuuuuAuuuAuuuuuGuGuuuuGuuuuuGuuuAuuAuuuuAUGuGuuuuu
AuAuUUGuuGGAuuuAUUuGCC***GCCAuAuuAC****AGuuAuuuAuuuuuuGuAAu
AuGAuuuuGCAGuuGAuAAuGG**AuuuuuuGuuGuuuuuGuuGuuuGuuuAGuuuuGu
AuuuGAuuuuuGAuAGuuAuuAuAuuGuuGuuGAAAuuuG**GuuUGuuA**UUGGAGU
UAUAGAAUAAGAUCAAAUAAGUUAAUAAUA
AAAAAUAAGUAUUUUGAUAUUAUUAAAGUAAAAGAGGAAUUUUGGGCGGAAGAGAAGGAGA
CAGGAGAGGAuuuuAAuUGuuuAAuGuuGAuuuuuGAuuuuuuAuuAuuuuGuuuG*UUUG
AuuuGuAuuuGuuuGuuGGuuuGuG***UUUGuuuuuAuuGuuGuGGuuuAuGuuGuuuAA
uuuAuAuAGuuuAAUUUUGuAuuA*UUGuAuuACuUAUUUG***AAuuuG*UAuuUGuuGu
uuuGuAuuGuuuuuuuAuuGuAuAuuGCAuuuuuAuuuuuGuuuuGuuuuuuAuGuGAuuu
uuuuuuGuuuAAuAAuuuGuUAGuuGGuGAuA****GuuuuAuGGAuGuuuuuuuuAUUC*
*GuuuuuuGuuGuGuuuuuuAGAGuGuuuuuCuuuGuuGuGuCGuuGuuuGuCGACGuuuu
uGCGuuuGUUUUGuAAuuuAuuAuCAuCCCAuUUUUUAuuGuuGAuGuuuuuuGAuuuuuu
uUAuuuuAuuuuuGuuuuuuuuuuuuAGGGuuuuuuGuuAuuGAuuuAuuuuAuuuAuuuu
uGuGuuuuGuuuuuGuuuAuuAuuuuAUGuGuuuuuAuAuUUGuuGGAuuuAUUuGCC***
GCCAuAuuAC****AGuuAuuuAuuuuuuGuAAuAuGAuuuuGCAGuuGAuAAuGG**Auu
uuuuGuuGuuuuuGuuGuuuGuuuAGuuuuGuAuuuGAuuuuuGAuAGuuAuuAuAuuGuu
GuuGAAAuuuG**GuuUGuuA**UUGGAGUUAUAGAAUAAGAUCAAAUAAGUUAAUAAUA
AAAAAUAAGUAUUUUGAUAUUAUUAAAGUAAAAGuuuuuAuuuuuuuuuuGuGAuuuAU
UUUGGuuGCGuuuGuuAuuAuGuAuGuAuuAuuGuGuAuGAuCuAGGuuAuGuuuuAuu
GuGuAuuuuAAuUGuuuAAuGuuGAuuuuuGAuuuuuuAuuAuuuuGuuuG*UUUGAuu
uGuAuuuGuuuGuuGGuuuGuG***UUUGuuuuuAuuGuuGuGGuuuAuGuuGuuuAAu
uuAuAuAGuuuAAUUUUGuAuuA*UUGuAuuACuUAUUUG***AAuuuG*UAuuUGuuG
uuuuGuAuuGuuuuuuuAuuGuAuAuuGCAuuuuuAuuuuuGuuuuGuuuuuuAuGuGA
uuuuuuuuuGuuuAAuAAuuuGuUAGuuGGuGAuA****GuuuuAuGGAuGuuuuuuuu
AUUC**GuuuuuuGuuGuGuuuuuuAGAGuGuuuuuCuuuGuuGuGuCGuuGuuuGuCG
ACGuuuuuGCGuuuGUUUUGuAAuuuAuuAuCAuCCCAuUUUUUAuuGuuGAuGuuuuu
uGAuuuuuuuUAuuuuAuuuuuGuuuuuuuuuuuuAGGGuuuuuuGuuAuuGAuuuAuu
uuAuuuAuuuuuGuGuuuuGuuuuuGuuuAuuAuuuuAUGuGuuuuuAuAuUUGuuGGA
uuuAUUuGCC***GCCAuAuuAC****AGuuAuuuAuuuuuuGuAAuAuGAuuuuGCAG
uuGAuAAuGG**AuuuuuuGuuGuuuuuGuuGuuuGuuuAGuuuuGuAuuuGAuuuuuG
AuAGuuAuuAuAuuGuuGuuGAAAuuuG**GuuUGuuA**UUGGAGUUAUAGAAUAAGA
UCAAAUAAGUUAAUAAUA
AAAAAUAAGUAUUUUGAUAUUAUUAAAGUAAAuAuGuuuuuAuuuuuuuuuuGuGAuuu
AUUUUGGuuGCGuuuGuuAuuAuGuAuGuAuuAuuGuGuAuGAuCuAGGuuAuGuuuuA
uuGuGuAuuuuAAuUGuuuAAuGuuGAuuuuuGAuuuuuuAuuAuuuuGuuuG*UUUGA
uuuGuAuuuGuuuGuuGGuuuGuG***UUUGuuuuuAuuGuuGuGGuuuAuGuuGuuuA
AuuuAuAuAGuuuAAUUUUGuAuuA*UUGuAuuACuUAUUUG***AAuuuG*UAuuUGu
uGuuuuGuAuuGuuuuuuuAuuGuAuAuuGCAuuuuuAuuuuuGuuuuGuuuuuuAuGu
GAuuuuuuuuuGuuuAAuAAuuuGuUAGuuGGuGAuA****GuuuuAuGGAuGuuuuuu
uuAUUC**GuuuuuuGuuGuGuuuuuuAGAGuGuuuuuCuuuGuuGuGuCGuuGuuuGu
CGACGuuuuuGCGuuuGUUUUGuAAuuuAuuAuCAuCCCAuUUUUUAuuGuuGAuGuuu
uuuGAuuuuuuuUAuuuuAuuuuuGuuuuuuuuuuuuAGGGuuuuuuGuuAuuGAuuuA
uuuuAuuuAuuuuuGuGuuuuGuuuuuGuuuAuuAuuuuAUGuGuuuuuAuAuUUGuuG
GAuuuAUUuGCC***GCCAuAuuAC****AGuuAuuuAuuuuuuGuAAuAuGAuuuuGC
AGuuGAuAAuGG**AuuuuuuGuuGuuuuuGuuGuuuGuuuAGuuuuGuAuuuGAuuuu
uGAuAGuuAuuAuAuuGuuGuuGAAAuuuG**GuuUGuuA**UUGGAGUUAUAGAAUAA
GAUCAAAUAAGUUAAUAAUA
editing in progress...edited
M F L F F F C D
L F W L R L L L C M Y Y C V W S R L C F
I V Y F N C L M L I F D F L L F C L F
D L Y L F V G L C L F L L L W F M L
F N L Y S L I L Y Y C I T Y L N L Y
L L F C I V F L L Y I A F L F L F C F
L C D F F L F N N L L V G D S F M D
V F F I R F L L C F L E C F S L L C R
C L S T F L R L F C N L L S S H F L L
L M F F D F F Y F I F V F F F W C F L
L L I Y F I Y F C V L F L F I I L C V F
I F V G F I C R H I T V I Y F L ter
M F L F F F C D
L F W L R L L L C M Y Y C V W S R L C F
I V Y F N C L M L I F D F L L F C L F
D L Y L F V G L C L F L L L W F M L
F N L Y S L I L Y Y C I T Y L N L Y
L L F C I V F L L Y I A F L F L F C F
L C D F F L F N N L L V G D S F M D
V F F I R F L L C F L E C F S L L C R
C L S T F L R L F C N L L S S H F L L
L M F F D F F Y F I F V F F F W C F L
L L I Y F I Y F C V L F L F I I L C V F
I F V G F I C R H I T V I Y F L ter
Bhat et al. (1990)
edited T. brucei ATPase subunit 6 RNA
||||||||||||||
...AAAGAGCAGGAAAGGUUAGGGGGAGGAGAGAAGAAAGGGAAAGUUGUGAUUUUGGAGUUAUAG... |·|||||||||| UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC...
...AAAGAGCAGGAAAGGUUAGGGGGAGGAGAGAAGAAAGGGAAAGUUGUGAUUGGAGUUAUAG... ||·|||||||||| UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC...
...AAAGAGCAGGAAAGGUUAGGGGGAGGAGAGAAGAAAGGGAAAGUUGUGuuAUUGGAGUUAUAG... ·|||||·|||||||||| UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC...
...AGAGCAGGAAAGGUUAGGGGGAGGAGAGAAGAAAGGGAAAGUUGuuUGuuAUUGGAGUUAUAG... ·||·|||||·|||||||||| UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC...
...AAAGAGCAGGAAAGGUUAGGGGGAGGAGAGAAGAAAGGGAAAGGuuUGuuAUUGGAGUUAUAG... |·||·|||||·|||||||||| UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC...
...GAGCAGGAAAGGUUAGGGGGAGGAGAGAAGAAAGGGAAAuuuGGuuUGuuAUUGGAGUUAUAG... ·||||·||·||·|||||·|||||||||| UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC...
...GCAGGAAAGGUUAGGGGGAGGAGAGAAGAAAGGuuGAAAuuuGGuuUGuuAUUGGAGUUAUAG... ||··||||·||·||·|||||·|||||||||| UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC...
...AGGAAAGGUUAGGGGGAGGAGAGAAGAAAGuuGuuGAAAuuuGGuuUGuuAUUGGAGUUAUAG... ··|||··||||·||·||·|||||·|||||||||| UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC...
...GAAAGGUUAGGGGGAGGAGAGAAGAAAuuGuuGuuGAAAuuuGGuuUGuuAUUGGAGUUAUAG... ||···|||··||||·||·||·|||||·|||||||||| UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC...
...AAAGGUUAGGGGGAGGAGAGAAGAAuAuuGuuGuuGAAAuuuGGuuUGuuAUUGGAGUUAUAG... ||||···|||··||||·||·||·|||||·|||||||||| UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC...
...AGGUUAGGGGGAGGAGAGAAGAuuAuAuuGuuGuuGAAAuuuGGuuUGuuAUUGGAGUUAUAG... ||·||||···|||··||||·||·||·|||||·|||||||||| UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC...
...GUUAGGGGGAGGAGAGAAGuuAuuAuAuuGuuGuuGAAAuuuGGuuUGuuAUUGGAGUUAUAG... |·||||·||||···|||··||||·||·||·|||||·|||||||||| UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC...
5’...UUAGGGGGAGGAGAGAuAGuuAuuAuAuuGuuGuuGAAAuuuGGuuUGuuAUUGGAGUUAUAG...3’ ····|··|·|·|||·||||·||||···|||··||||·||·||·|||||·|||||||||| 3’ UUUUUUUUUUUUAUUAAUAGUAUAGUGACAGUUUUAGACUAAGCAAUAGCCUCAAUAUC...5’
AnchorInformationU tail
directed by guide RNAs (gRNAs) -- 60-70 nt RNAs with post-transcriptionally added oligo(U) tails
Edited region specified by a single gRNA = block
Editing starts at the 3’ end of the pre-edited mRNA. Editing directed by the first gRNA creates the mRNA sequence which will be recognized by the next gRNA. This creates an overall 3’ to 5’ direction for editing.
3’3’Block 1Block 2
UUUUU
Block 35’5’
|||||||
RNA editing in kinetoplastid mitochondria
gRNA
mRNA
RNA editing in kinetoplastid mitochondria
ND8
ND9
ND7
COII
MURF2
CR4CR5
RPS12
A6CYB
COIII
CR322kb
MAXICIRCLE(~50 copies)
gRNA 1
gRNA 2
gRNA 3
~1kb
MINICIRCLE(>1000 different molecules)
12 pre-edited RNAs
1000s of gRNAs
U
U
U
Insertion editingDeletion editingUUU5'-
3'-
... ... - 3'
- 5'
5'-
3'-
... ... - 3'- 5'
p
5'-
3'-
... ... - 3'
- 5'
5'-
3'-
... ... - 3'
- 5'
5'-
3'-
... ... - 3'- 5'
p
UU5'-
3'-
... ... - 3'
- 5'
UTPUMP
UGAU
GCAAUA
UG AU
UA
GC
AA
5'-
3'-
... ... - 3'- 5'
pUG AU
UA
GC
AA
UG AU
UAAA
A UG
ACU
UG
AC
A
U
5'-
3'-
... ... - 3'
- 5'
p UG
AC
A
U
AUG
ACU G C
CleavageEndoribonuclease
U addition TUTase
LigationRNA ligase
U removalExo Uase
RNA editing mechanism
Proteins of the T. brucei 20S Editosome
A1 81 interactionA2 63 interactionA3 42 interaction*A4 24 interactionA5 19 interaction*A6 18 interaction*
B1 90 nuclease*B2 67 nuclease*B3 61 nuclease*
B4 46 interaction*B5 44 interaction
B6 49 interaction*B7 47 interaction*B8 41 interaction*
C1 100 ExoUaseC2 99 ExoUase
L1 52 RNA ligaseL2 48 RNA ligase
T2 57 TUTase
H1 61 Helicase Helicase
OB-fold
OB-fold?
Z Z OB-fold
OB-fold?
ZZ OB-fold
Z Z? OB-fold
Ligase tau K
Ligase tau K
5’3’exo Endo/Exo/Phos
5’3’exo Endo/Exo/Phos
RNase III dsRBMU1-like
RNase III dsRBMU1-like
RNase III dsRBMU1-like
U1-like
U1-like
U1-like
RNase III? PumU1-like
RNase III? PumU1-like
PAP PAP-assoc.cat.
name potential role motifs
Colored box indicates role demonstrated
A6 (18)
C2(99)U-
A2 (63)
L1
T2
A1 (81)
L2
INSERTION DELETION
20S editosome interactions in T. brucei
by yeast two hybrid, Co-IP, TAP-Tag, catalytic enhancement, knockdown
20-30% of disease-causing mutations in humans involve pre-mRNA splicing
Cystic fibrosis – C T mutation creates cryptic splice site which creates a short extra ‘exon’ that contains a stop codon
Myotonic dystrophy – mis-regulation of alternative splicing
Errors in RNA editing also produce disease
Wilm’s tumor is due to mis-editing
Fabry disease is due to mis-editing
RNA processing and disease