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Post-transcriptional gene control
Subjects, covered in the lecture
• Processing of eukaryotic pre-mRNA-capping
-polyadenylation
-splicing
-editing
• Nuclear transport
Processing of eukaryotic pre-mRNA: the classical texbook picture
Alternative picture: co-transcriptional pre-mRNA processing
• This picture is more realistic than the previous one, particularly for long pre-mRNAs
Heterogenous ribonucleoprotein patricles (hnRNP) proteins
• In nucleus nascent RNA transcripts are associated with abundant set of proteins
• hnRNPs prevent formation of secondary structures within pre-mRNAs
• hnRNP proteins are multidomain with one or more RNA binding domains and at least one domain for interaction with other proteins
• some hnRNPs contribute to pre-mRNA recognition by RNA processing enzymes
• The two most common RNA binding domains are RNA recognition motifs (RRMs) and RGG box (five Arg-Gly-Gly repeats interspersed with aromatic residues)
3D structures of RNA recognition motif (RRM ) domains
Capping
p-p-p-N-p-N-p-N-p….
p-p-N-p-N-p-N-p…
G-p-p-p-N-p-N-p-N-p…
CH3
G-p-p-p-N-p-N-p-N-p…
CH3 CH3
GMP mCE (another subunit)
Capping enzyme (mCE)
methyltransferasesS-adenosyl methionine
The capping enzyme
• A bifunctional enzyme with both 5’-triphosphotase and guanyltransferase activities
• In yeast the capping enzyme is a heterodimer
• In metazoans the capping enzyme is monomeric with two catalytic domains
• The capping enzyme specific only for RNAs, transcribed by RNA Pol II (why?)
Capping mechanism in mammals
DNA
Growing RNA
Capping enzyme is allosterically controlled by CTD domains of RNA Pol II and another stimulatory factor hSpt5
Polyadenylation
• Poly(A) signal recognition
• Cleavage at Poly(A) site
• Slow polyadenylation
• Rapid polyadenylation
• G/U: G/U or U rich region
• CPSF: cleavage and polyadenylation specificity factor
• CStF: cleavage stimulatory factor
• CFI: cleavage factor I
• CFII: cleavage factor II
PAP: Poly(A) polymerase
CPSF
PAP
PABPII- poly(A) binding protein II
PABP II functions:
1. rapid polyadenylation
2. polyadenylation termination
pp
Pol II
ctd
mRNA
PolyA – binding factors
Link between polyadenylation and transcription
Pol II gets recycled
mRNA gets cleaved and polyadenylated
degradation
cap
polyA
cap
splicing,nuclear transport
pp
aataaa
FCP1 Phosphatase removes phospates from CTDs
cap
Splicing
The size distribution of exons and introns in human, Drosophila and C. elegans genomes
Consensus sequences around the splice site
YYYY
Molecular mechanism of splicing
Small nuclear RNAs U1-U6 participate in splicing
• snRNAs U1, U2, U4, U5 and U6 form complexes with 6-10 proteins each, forming small nuclear ribonucleoprotein particles (snRNPs)
• Sm- binding sites for snRNP proteins
The secondary structure of snRNAs
Additional factors of exon recognition
ESE - exon splicing enhancer sequences
SR – ESE binding proteins
U2AF65/35 – subunits of U2AF factor, binding to pyrimidine-rich regions and 3’ splice site
Binding of U1 and U2 snRNPs
Binding of U4, U5 and U6 snRNPs
The essential steps in splicing
Rearrangement of base-pair interactions between snRNAs, release of U1 and U4 snRNPs
The catalytic core, formed by U2 and U6 snRNPs catalyzes the first transesterification reaction
Further rearrangements between U2, U6 and U5 lead to second transesterification reaction
The spliced lariat is linearized by debranching enzyme and further degraded in exosomes
Not all intrones are completely degraded. Some end up as functional RNAs, different from mRNA
pp
Pol IIctd
mRNA
SCAFs: SR- like CTD – associated factors
cap
SRssnRNPs
Intron
Co-transciptional splicing
Self-splicing introns
• Under certain nonphysiological conditions in vitro, some introns can get spliced without aid of any proteins or other RNAs
• Group I self-splicing introns occur in rRNA genes of protozoans
• Group II self-splicing introns occur in chloroplasts and mitochondria of plants and fungi
Group I introns utilize guanosine cofactor, which is not part of RNA chain
Comparison of secondary structures of group II self-splicing introns and snRNAs
Spliceosome
• Spliceosome contains snRNAs, snRNPs and many other proteins, totally about 300 subunits.
• This makes it the most complicted macromolecular machine known to date.
• But why is spliceosome so extremely complicated if it only catalyzes such a straightforward reaction as an intron deletion? Even more, it seems that some introns are capable to excise themselves without aid of any protein, so why have all those 300 subunits?
• No one knows for sure, but there might be at least 4 reasons:
• 1. Defective mRNAs cause a lot of problems for cells, so some subunits might assure correct splicing and error correction
• 2. Splicing is coupled to nuclear transport, this requires accessory proteins
• 3. Splicing is coupled to transcription and this might require more additional accessory proteins
• 4. Many genes can be spliced in several alternative ways, which also might require additional factors
One gene – several proteins
• Cleavage at alternative poly(A) sites
• Alternative promoters
• Alternative splicing of different exons
• RNA editing
Alternative splicing, promoters & poly-A cleavage
RNA editing
• Enzymatic altering of pre-mRNA sequence
• Common in mitochondria of protozoans and plants and chloroplasts, where more than 50% of bases can be altered
• Much rarer in higher eukaryotes
Editing of human apoB pre-mRNA
The two types of editing1) Substitution editing• Chemical altering of individual nucleotides• Examples: Deamination of C to U or A to I
(inosine, read as G by ribosome)
2) Insertion/deletion editing•Deletion/insertion of nucleotides (mostly uridines) •For this process, special guide RNAs (gRNAs) are required
Guide RNAs (gRNAs) are required for editing
Organization of pre-rRNA genes in eukaryotes
Electron micrograph of tandem pre-rRNA genes
Small nucleolar RNAs
• ~150 different nucleolus restricted RNA species• snoRNAs are associated with proteins, forming small
nucleolar ribonucleoprotein particles (snoRNPs)• The main three classes of snoRNPs are envolved in
following processes:
a) removing introns from pre-rRNA
b) methylation of 2’ OH groups at specific sites
c) converting of uridine to pseudouridine
What is this pseudouridine good for?
• Pseudouridine is found in RNAs that have a tertiary structure that is important for their function, like rRNAs, tRNAs, snRNAs and snoRNAs
• The main role of and other modifications appears to be the maintenance of three-dimensional structural integrity in RNAs
Uridine ( U ) Pseudouridine ()
Where do snoRNAs come from?
• Some are produced from their own promoters by RNA pol II or III
• The majority of snoRNAs come from introns of genes, which encode proteins involved in ribosome synthesis or translation
• Some snoRNAs come from intrones of genes, which encode nonfuctional mRNAs
Assembly of ribosomes
Processing of pre-tRNAs
RNase P cleavage site
Splicing of pre-tRNAs is different from pre-mRNAs and pre-rRNAs
• The splicing of pre-tRNAs is catalyzed by protein only
• A pre-tRNA intron is excised in one step, not by two transesterification reactions
• Hydrolysis of GTP and ATP is required to join the two RNA halves
Macromolecular transport across the nuclear envelope
The central channel• Small metabolites, ions and globular
proteins up to ~60 kDa can diffuse freely through the channel
• Large proteins and ribonucleoprotein complexes (including mRNAs) are selectively transported with the assistance of transporter proteins
Two different kinds of nuclear location sequences basic hydrophobic
importin importin importin
nuclear import
Proteins which are transported into nucleus contain nuclear location sequences
Artifical fusion of a nuclear localization signal to a
cytoplasmatic protein causes its import to nucleus
Mechanism for nuclear “import”
Mechanism for nuclear “export”
Mechanism for mRNA transport to cytoplasm
Example of regulation at nuclear transport level: HIV mRNAs
After mRNA reaches the cytoplasm...
• mRNA exporter, mRNP proteins, nuclear cap-binding complex and nuclear poly-A binding proteins dissociate from mRNA and gets back to nucleus
• 5’ cap binds to translation factor eIF4E• Cytoplasmic poly-A binding protein (PABPI)
binds to poly-A tail• Translation factor eIF4G binds to both eIF4E and
PABPI, thus linking together 5’ and 3’ ends of mRNA