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