RNA PROCESSING DNA MATURE RNA PROTEIN PRECURSOR RNAs pre-rRNA pre-tRNA pre-mRNA Cleavage Nucleotide...

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





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







AGUUGUGA**UUGGAGUUAUAGAAUAAGAUCAAAUAAGUUAAUAAUA







AGUUGuuUGuuA**UUGGAGUUAUAGAAUAAGAUCAAAUAAGUUAAUAAUA






UUGCCAAACUUUUAGAAGAAAGAGCAGGAAAGGUUAGGGGGAGGAGAGAAGAAAGuuGu

uGAAAuuuG**GuuUGuuA**UUGGAGUUAUAGAAUAAGAUCAAAUAAGUUAAUAAUA






UUGCCAAACUUUUAGAAGAAAGAGCAGGAAAGGUUAGGGGGuuuAGuuuuGuAuuuGAu

uuuuGAuAGuuAuuAuAuuGuuGuuGAAAuuuG**GuuUGuuA**UUGGAGUUAUAGAA

UAAGAUCAAAUAAGUUAAUAAUA






UUGCCAAACUUUUAGAAGAAAGAGCAGGAAAGG**AuuuuuuGuuGuuuuuGuuGuuuG

uuuAGuuuuGuAuuuGAuuuuuGAuAGuuAuuAuAuuGuuGuuGAAAuuuG**GuuUGu

uA**UUGGAGUUAUAGAAUAAGAUCAAAUAAGUUAAUAAUA





ACACCCAUUUUUAGGAGGAUAAGAGGGGAGAAAAGGGGAAAUGGAAuUUGuuGGAuuuA

UUuGCC***GCCAuAuuAC****AGuuAuuuAuuuuuuGuAAuAuGAuuuuGCAGuuGA

uAAuGG**AuuuuuuGuuGuuuuuGuuGuuuGuuuAGuuuuGuAuuuGAuuuuuGAuAG

uuAuuAuAuuGuuGuuGAAAuuuG**GuuUGuuA**UUGGAGUUAUAGAAUAAGAUCAA

AUAAGUUAAUAAUA





ACACCCAUUUUUAGGAGGAUAAGAGGGGAGAAAAGGGGuuuAuuAuuuuAUGuGuuuuu

AuAuUUGuuGGAuuuAUUuGCC***GCCAuAuuAC****AGuuAuuuAuuuuuuGuAAu

AuGAuuuuGCAGuuGAuAAuGG**AuuuuuuGuuGuuuuuGuuGuuuGuuuAGuuuuGu

AuuuGAuuuuuGAuAGuuAuuAuAuuGuuGuuGAAAuuuG**GuuUGuuA**UUGGAGU

UAUAGAAUAAGAUCAAAUAAGUUAAUAAUA





ACACCCAUUUUUAGGAGGAUAAGAGGGuuuuuuGuuAuuGAuuuAuuuuAuuuAuuuuu

GuGuuuuGuuuuuGuuuAuuAuuuuAUGuGuuuuuAuAuUUGuuGGAuuuAUUuGCC**

*GCCAuAuuAC****AGuuAuuuAuuuuuuGuAAuAuGAuuuuGCAGuuGAuAAuGG**

AuuuuuuGuuGuuuuuGuuGuuuGuuuAGuuuuGuAuuuGAuuuuuGAuAGuuAuuAuA

uuGuuGuuGAAAuuuG**GuuUGuuA**UUGGAGUUAUAGAAUAAGAUCAAAUAAGUUA

AUAAUA





ACACCCAUUUUUAGGAGGAUAuuuuAuuuuuGuuuuuuuuuuuuAGGGuuuuuuGuuAu

uGAuuuAuuuuAuuuAuuuuuGuGuuuuGuuuuuGuuuAuuAuuuuAUGuGuuuuuAuA

uUUGuuGGAuuuAUUuGCC***GCCAuAuuAC****AGuuAuuuAuuuuuuGuAAuAuG

AuuuuGCAGuuGAuAAuGG**AuuuuuuGuuGuuuuuGuuGuuuGuuuAGuuuuGuAuu

uGAuuuuuGAuAGuuAuuAuAuuGuuGuuGAAAuuuG**GuuUGuuA**UUGGAGUUAU

AGAAUAAGAUCAAAUAAGUUAAUAAUA




GUAGGGGAAUUUUGAGGAGuuuuuuuuAUUC**GuuuuuuGuuGuGuuuuuuAGAGuGu

uuuuCuuuGuuGuGuCGuuGuuuGuCGACGuuuuuGCGuuuGUUUUGuAAuuuAuuAuC

AuCCCAuUUUUUAuuGuuGAuGuuuuuuGAuuuuuuuUAuuuuAuuuuuGuuuuuuuuu

uuuAGGGuuuuuuGuuAuuGAuuuAuuuuAuuuAuuuuuGuGuuuuGuuuuuGuuuAuu

AuuuuAUGuGuuuuuAuAuUUGuuGGAuuuAUUuGCC***GCCAuAuuAC****AGuuA

uuuAuuuuuuGuAAuAuGAuuuuGCAGuuGAuAAuGG**AuuuuuuGuuGuuuuuGuuG

uuuGuuuAGuuuuGuAuuuGAuuuuuGAuAGuuAuuAuAuuGuuGuuGAAAuuuG**Gu

uUGuuA**UUGGAGUUAUAGAAUAAGAUCAAAUAAGUUAAUAAUA



AAGAAUUUUGAAUUUGAACUAUUUGUUUAAGUUAUGGGAGAGAuAuuGCAuuuuuAuuu

uuGuuuuGuuuuuuAuGuGAuuuuuuuuuGuuuAAuAAuuuGuUAGuuGGuGAuA****

GuuuuAuGGAuGuuuuuuuuAUUC**GuuuuuuGuuGuGuuuuuuAGAGuGuuuuuCuu

uGuuGuGuCGuuGuuuGuCGACGuuuuuGCGuuuGUUUUGuAAuuuAuuAuCAuCCCAu

UUUUUAuuGuuGAuGuuuuuuGAuuuuuuuUAuuuuAuuuuuGuuuuuuuuuuuuAGGG

uuuuuuGuuAuuGAuuuAuuuuAuuuAuuuuuGuGuuuuGuuuuuGuuuAuuAuuuuAU

GuGuuuuuAuAuUUGuuGGAuuuAUUuGCC***GCCAuAuuAC****AGuuAuuuAuuu

uuuGuAAuAuGAuuuuGCAGuuGAuAAuGG**AuuuuuuGuuGuuuuuGuuGuuuGuuu

AGuuuuGuAuuuGAuuuuuGAuAGuuAuuAuAuuGuuGuuGAAAuuuG**GuuUGuuA*

*UUGGAGUUAUAGAAUAAGAUCAAAUAAGUUAAUAAUA



AAGAAUUUUGAAUUUGAACuUAUUUG***AAuuuG*UAuuUGuuGuuuuGuAuuGuuuu

uuuAuuGuAuAuuGCAuuuuuAuuuuuGuuuuGuuuuuuAuGuGAuuuuuuuuuGuuuA

AuAAuuuGuUAGuuGGuGAuA****GuuuuAuGGAuGuuuuuuuuAUUC**GuuuuuuG

uuGuGuuuuuuAGAGuGuuuuuCuuuGuuGuGuCGuuGuuuGuCGACGuuuuuGCGuuu

GUUUUGuAAuuuAuuAuCAuCCCAuUUUUUAuuGuuGAuGuuuuuuGAuuuuuuuUAuu

uuAuuuuuGuuuuuuuuuuuuAGGGuuuuuuGuuAuuGAuuuAuuuuAuuuAuuuuuGu

GuuuuGuuuuuGuuuAuuAuuuuAUGuGuuuuuAuAuUUGuuGGAuuuAUUuGCC***G

CCAuAuuAC****AGuuAuuuAuuuuuuGuAAuAuGAuuuuGCAGuuGAuAAuGG**Au

uuuuuGuuGuuuuuGuuGuuuGuuuAGuuuuGuAuuuGAuuuuuGAuAGuuAuuAuAuu

GuuGuuGAAAuuuG**GuuUGuuA**UUGGAGUUAUAGAAUAAGAUCAAAUAAGUUAAU

AAUA


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



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

RNA PROCESSING DNA MATURE RNA PROTEIN PRECURSOR RNAs pre-rRNA pre-tRNA pre-mRNA Cleavage Nucleotide...

Documents

Transcript of RNA PROCESSING DNA MATURE RNA PROTEIN PRECURSOR RNAs pre-rRNA pre-tRNA pre-mRNA Cleavage Nucleotide...