B3130.25w.Lecture.15

Topics for Lecture 15

• Review of Lecture 14

• Coordination of Gene Expression

  • Phosphorylation of the CTD

  • The CTD and pre-mRNA Processing

  • The CTD and Histone Modifications

Review: 3'-end formation and termination

• Key components and processes include:

  • Transcription Start Site (TSS)

  • RNA Polymerase II (pol II)

  • Stop codon and Cleavage site

  • Endonucleolytic cleavage of nascent RNA

  • Polyadenylation

  • Role of XRN2

RNase Protection Assays for RNA Processing

• Purpose:

  • Examining splicing efficiency

  • Examining cleavage efficiency

Review: Alternative Polyadenylation

• Types of Polyadenylation:

  • Tandem UTR with Proximal and Distal Polyadenylation Signals (PAS)

  • Different mRNA isoforms produced based on PAS choices

  • Alternative last exon (ALE) results in different mRNA isoforms

Overview of Coordinating Gene Expression

• Historical view:

  • Pre-mRNA processing was seen as strictly post-transcriptional (after transcription completion).

• Current understanding:

  • Processing is primarily co-transcriptional.

• The CTD of RNAP II connects transcription with processing steps.

CTD Phosphorylation

• Sequence: Tyr-Ser-Pro-Thr-Ser-Pro-Ser

• Phosphorylation detected on all non-Pro residues.

• Identification of phosphorylated forms: Tyr1-P, Ser2-P, Thr4-P, Ser5-P, Ser7-P

• Distribution of phosphorylations:

  • Ser5-P: localizes at the 5′ end

  • Ser2-P: localizes at the 3′ end

CTD Kinases

• TFIIH phosphorylates Ser5

  • Mechanism: Phosphorylation near the 5′ end

  • Enzymes involved: CDK7 (mammals) and Kin28 (yeast)

• Cdk9 phosphorylates Ser2 in mammals

  • Promotes elongation by overcoming transcriptional pausing.

• Phosphorylation of Ser2 dependent on prior Ser5 phosphorylation.

CTD Kinases in Yeast

• Yeast do not exhibit pausing, so Ser2 phosphorylated downstream.

• Kinases:

  • Bur1 phosphorylates Ser2 near promoters

  • Ctk1 dominates Ser2 phosphorylation downstream.

CTD Phosphorylation: Summary

• Unphosphorylated RNAP II associates with promoters.

• RNAP II clears the promoter after Ser5 phosphorylation.

• Recruitment of P-TEFb dependent on Ser5-P CTD, which leads to Ser2 phosphorylation.

The CTD and Pre-mRNA Processing

• The CTD's function was initially mysterious but is crucial for RNAP II transcript processing.

  • Only RNAP II transcripts experience capping, splicing, and polyadenylation.

α-Amanitin Resistant RNAP II and Processing

• Experiment to observe effects of truncated CTD on processing.

• Shows RNAP II's CTD needed for efficient capping, splicing, and cleavage.

5′ Capping

• Occurs co-transcriptionally when the transcript is ~30 nt long.

• Capping enzymes bind to the Ser5-P CTD, essential for efficient capping.

• RNAP II with a truncated CTD shows significantly reduced capping efficiency.

Splicing is Co-Transcriptional

• Evidence shows splicing happens as transcription proceeds.

• CTD coordinates recruitment of splicing factors to nascent RNA during elongation.

CTD’s Role in Splicing

• α-Amanitin-resistant RNAP model demonstrates the necessity of the CTD for splicing in vivo.

Molecular Insights: Interaction with Spliceosome

• CTD phosphorylated on Ser5 interacts with spliceosome during splicing.

The CTD and 3′-End Processing

• CTD crucial in cleavage/polyadenylation processes.

• Recruitment of cleavage/polyadenylation factors relies on Ser2 phosphorylation.

Impacts of CTD Truncation on Cleavage

• Truncated CTD results in impaired 3'-end cleavage in vivo as shown through RNase protection assays.

Summary: Coupling Transcription with Processing

• Processing factors interact with the CTD, influenced by phosphorylation.

Histone Modifications: The “Histone Code”

• Histone modifications include acetylation, methylation, and phosphorylation.

  • Particularly important modifications:

    • Methylation of H3K4 associated with active gene promoters

    • Methylation of H3K36 increases towards the 3' end of active genes.

The CTD and Histone Methyltransferases (HMTs)

• CTD phosphorylation influences the placement of histone modifications.

• Set1: Responsible for H3K4 methylation; recruited by Ser5-P CTD.

• Set2: Responsible for H3K36 methylation; recruited during elongation by Ser2-P CTD.