RNA Processing I (3)

Where is the CTD of RNA Pol II?

On its large subunit

What sequence encodes that domain?

YSPTSPS: 52 repeats of this heptapeptide so it’s essential

Structure of the CTD

Extends out of RNA Pol II and has unique conformations or is entirely unstructured

Which phosphorylated AAs of YSPTSPS affect transcription?

The Serines at positions 2 and 5

What enzyme phosphorylates Ser5?

TF2H

When does RNA Pol II stall?

After 25-60 nt/ after the phosphorylation of Ser5

1st reason for stalling

Capping of the 5’ end of mRNA

Capping process

7-methylguanylate CAP added through a 5’-5’ triphosphate linkage= hard for exonucleases to cut

Advantages of CAP

1. Protection

2. Facilitated export

3. Recognition by translation factors

2nd reason for stalling

Methylation of 2’ hydroxyl of the first and (second: in vertebrates) nt

What enzyme phosphorylates Ser2?

CDK9

Other phosphorylated elements of stalled complex

DSIF and NELF

Effect of phosphorylation of DSIF

Clamp holds DNA down

Effect of phosphorylation of NELF

No stalling!

What proteins does P-Ser2 recruit?

• Splicing factors

• Polyadenylation factors

• Export factors

Events of mRNA processing

1. 5’ capping

2. Cleavage at poly(A) site

3. Polyadenylation (3’ poly A tail)

4. RNA splicing

CO-TRANSCRIPTIONAL

Splicing

Pre mRNA to mRNA

Process of splicing

Removal of introns bc they are not needed for translation: only regulatory elements

Hybridization experiments

Allow to visualize discrepancy between mRNA size and gene size

Loop outs indicated…

that the probe did not interact with the DNA introns

Conserved border sequences of introns (3)

1. Splice donor (GU)

2. Branch point (A)

3. Splice acceptor (AG)

Spliceosome

5 snRNPs

1 snRNP or small nuclear ribonucleoprotein particle

1 snRNA (U1,2,4,5,6) + 6-10 proteins

Splice donor snRNA

U1

Branch point snRNA

U2: the adenosine is not complementarily paired and bulges out!

Problem with U1

Limited homology with splice site (mutations)

Correction of problem

Introduction of a compensatory mutation in U1= proof that RNA:RNA paring is critical for spliceosome function

1st trans-esterification

2’ hydroxyl group of branch point attacks 5’ phosphate of the G residue of intron = lariat formation

2nd trans-esterification

3’ oxygen of exon attacks 5’ phosphate group of the exon = release of the lariat

How can a lariat be observed?

By using radio-labelled probes in vitro

How does the spliceosome assemble?

Sequentially: U1 and U2 first followed by U4, 5 and 6

The spliceosome becomes active when…

U1 and U4 snRNAs leave

Role of the debranching enzyme

Cuts and linearizes the intron

Do self-splicing introns also result in the formation of a lariat?

Yes in Group II introns but not in Group I introns

1% of human introns have…

A/CU…AC splice sites instead if GU…AG = need less snRNPs

Trans-splicing

Formation of mRNAs from 2 different pre-mRNAs