BY511 - Lecture 14 - Transcription Elongation

hnRNA (pre-mRNA)

• Heterogeneous nuclear RNA is the primary transcript in eukaryotes before splicing.

RNA Processing

• This refers to the modifications of mRNA necessary before it exits the nucleus; exclusive to eukaryotic cells.

Processing of hnRNA to mRNA

• hnRNA undergoes processing involving adding a 5' cap, a 3' Poly(A) tail, and splicing to remove introns.
• The splicing mechanism entails breaking the exon-intron junctions and joining exons, resulting in mature mRNA.
• Mature mRNA is subsequently transported through nuclear pores for translation in the cytoplasm.

5' Cap

• A modified guanine nucleotide added to the 5' end of pre-mRNA.
• It protects mRNA from degradation and facilitates ribosome binding during translation.

3' Poly(A) Sequence

• A poly(A) tail at the 3' end safeguards mRNA from enzymatic degradation and aids in transcription termination, export from the nucleus, and translation.

snRNA (small nuclear RNA)

• Small RNA molecules (~200 nucleotides) that participate in splicing by removing introns.

Sequential Phosphorylation of RNA Pol II CTD Heptapeptide Tail

• Step I: Serine 5 is phosphorylated by TFIIH kinase, signaling for 5'-Capping factors.
• Step II: Serine 2 is phosphorylated by P-TEFb kinase, promoting recruitment of intron splicing factors.
• Step III: Dephosphorylation of Ser 5 by PPase leads to increased Ser 2 phosphorylation and recruitment of polyadenylation factors.

Serine 7

• Phosphorylation of Ser-7 occurs prior to transcription initiation by CDK7.
• Necessary for the recognition of RT-PCR by P-TEFb kinase for splicing factor recruitment.
• Mutations in Ser-7 can hinder snRNA gene expression.

RNA Polymerase II CTD Structure and Function

• The CTD consists of heptapeptide repeats, featuring seven amino acid sequences.
• Each heptad includes "Serine" at positions 2, 5, and 7, which are phosphorylated sequentially by specific kinases, recruiting factors for hnRNA processing.

Eukaryotic Transcription Elongation and Anticancer Drug Targeting

• P-TEFb is a kinase crucial for phosphorylating Ser 2 of the RNA polymerase II CTD, aiding in intron splicing factor recruitment.
• It plays a role in potential anticancer drug development.

Flavopiridol (P-TEFb Kinase Inhibitor) - Mechanism

• Inhibits P-TEFb by blocking ATP-binding, preventing phosphorylation of Ser 2 on RNA Pol II CTD.
• This disruption halts hnRNA processing, thereby blocking mRNA production and protein synthesis, leading to cancer cell apoptosis.

Alvocidib (CDK9 Inhibitor)

• A potent inhibitor of CDK9, which activates transcription elongation via phosphorylation of RNA polymerase II CTD.
• By inhibiting CDK9, alvocidib reduces transcription of key target genes, including MCL-1, impacting cancer cell survival.