COUPLING BETWEEN TRANSCRIPTION AND mRNA PROCESSING

Mouse (n = 52) Budding / Fission yeast (n = 26 / 29)

Structure of RNA Pol II Patrick Cramer, David A. Bushnell, Roger D. Kornberg Science (2001) 249, 1863 CTD appears to be of sufficient size and flexibility to interact with multiple components of pre-mRNA processing machinery and to localize this machinery close to the pre-mRNA as it emerges from the exit groove of the pol II.

Before transcription initiation, the CTD is unphosphorylated and is associated with the mediator and components of the initiation machinery. ( Tyr Ser Pro Thr Ser Pro Ser ) n Initiation factor TFIIH (Cdk 7/cyclin H) phosphorylates Ser-5 of CTD shortly after initiation.

RNA Pol II is arrested by Negative Transcritption Elongation Factor (NELF/DSIF).

P-TEFb (Cdk 9/cyclin T) phosphorylates Ser-2 of CTD during elongation and neutalizes the repressive action by NELF.

Binding of Mammalian Capping Enzyme to CTD Phosphopeptides Mce1 (Mouse Capping Enzyme) binds avidly to CTD containing PO 4 on Ser2 or Ser5 of each heptad.

Stimulation of Guanylyltransferase Activity by CTD-PO4 Allosteric effector function of Ser5 phosphorylation is “ dominant ” over non-activating Ser2 phosphorylation.

Cap structure is added (as short as 22 nts). P-TEFb is recruited to the elongation complex, phosphorylate CTD and neutalizes the repressive action by NELF. Pol II CTD and transcription elongation factor Spt5 (subunit of DSIF) stimulates MCE1 GTase activity (Wen & Shatkin). Capping enzyme is recruited to the transcription complex via Pol II CTD. 40

HOW IS SPLICING POSSIBLE?

SUR2 gene: 40 kb exon intron 9.6 kb intron

2.4 Mb 260 kb intron Human Dystrophin gene

Like transcription factors, hnRNP proteins are modular proteins containing two types of domains: RNA-binding domain Protein-protein interaction domain The RNA-binding domains have much less specificity for binding to a specific RNA sequence than do the DNA- binding domains of activators and repressors.

Searching for proteins binding to RNA Pol II by two-hybrid screen identified several SR-like proteins known as SCAFs (SR-related CTD associated factors). SR (Ser-Arg) protein family: Characterized by one or two RBDs at their N-terminal and an Arg-Ser (RS) repeat domain at the C-terminal. Can bind the pre-mRNAs via their RBDs and interact with proteins via their RS domain simultaneously. It has been proposed that SR proteins help to bridge the two splice-sites.

Misteli T. and Spector D. (1999) Mol Cell 3: 697 Targeting of pre-mRNA Splicing Factor to Transcription Sites in Vivo

In higher eukaryotes, exons encode more than just amino acid sequence. Exons in long pre-mRNAs also contain binding sites for SR proteins called exonic splicing enhancers.

SR proteins bound to exonic splicing enhancers that span an exon interact with U1 snRNP bound to the 5’ splice site of the downstream intron and to U2AF and U2 snRNP bound to the 3’ end of the upstream intron. These protein-protein and RNA-protein interactions form a network of intermolecular interactions that span the length of an exon called a cross exon recognition complex. The cumulative effect of these multiple interactions specifies an exon, distinguishing it from intron sequence, a process called exon definition.

pre-mRNA

Coupling between Splicing Machinery and Transcription. In vitro splicing reactions are 1000-fold slower than in vivo. Transcription by a Pol II with only 5 copies of CTD repeats inhibits splicing. Pol IIO (phosphorylated form) has been shown to enhance in vitro splicing in the absence of transcription. Overexpression of CTD fusion proteins interferes with mRNA splicing in vivo. CTD peptides or antibodies against the CTD inhibit in vitro splicing.

Coupled transcription and splicing

U1snRNP U2snRNP Phosphorylated CTD Pol II (Adapted from T. D. Pollard and W. C. Earnshaw Cell Biology (2002)) snRNPs, SR proteins and splicing factors associate with phos- phorylated CTD { Cross-exon recognition complex

FUNCTIONAL LINK BETWEEN TRANSCRIPTION AND SPLICING??

Coupling between 3’ Cleavage and Transcription. Multi subunit protein complex:  CPSF (cleavage/polyadenylation specificity factor): Required for cleavage and polyadenylation. Binds to AAUAAA sequence just upstream of poly A site. 50

Coupling between 3’ Cleavage and Transcription. Multi subunit protein complex:  CPSF (cleavage/polyadenylation specificity factor): Required for cleavage and polyadenylation. Binds to AAUAAA sequence just upstream of poly A site.  CstF (cleavage stimulation factor): Required for cleavage only. Binds to downstream G/U rich sequence.

Coupling between 3’ Cleavage and Transcription. Multi subunit protein complex:  CPSF (cleavage/polyadenylation specificity factor): Required for cleavage and polyadenylation. Binds to AAUAAA sequence just upstream of poly A site.  CstF (cleavage stimulation factor): Required for cleavage only. Binds to downstream G/U rich sequence.  CF1 and CF2 (cleavage factor) : Required for pre-cleavage assembly and stability.

Coupling between 3’ Cleavage and Transcription. Multi subunit protein complex:  CPSF (cleavage/polyadenylation specificity factor): Required for cleavage and polyadenylation. Binds to AAUAAA sequence just upstream of poly A site.  CstF (cleavage stimulation factor): Required for cleavage only. Binds to downstream G/U rich sequence.  CF1 and CF2 (cleavage factor) : Required for pre-cleavage assembly and stability.  PAP (Poly A Polymerase): Catalyze Pol(A) synthesis. Also required for cleavage.

Coupling between 3’ Cleavage and Transcription. Ryan K etal. (2002) MCB 22: 1684

Coupling between 3’ Cleavage and Transcription. Pol II CTD stimulates in vitro cleavage reaction. RNA transcribed by truncated CTD was not efficiently polyadenylated in vivo.

Coupling between 3’ Cleavage and Transcription. Purified Pol II stimulates in vitro cleavage reaction. RNA transcribed by truncated CTD was not efficiently polyadenylated in vivo. CPSF is brought to the pre-initiation complex by the general transcription factor TFIID and dissociate from TFIID in elongating polymerase. CPSF and CstF both bind the unphosphorylated and phosphorylated form of Pol II.

Less Processing Less Transcription

Before transcription initiation, the CTD is unphosphorylated and is associated with the mediator and components of the initiation machinery. Partial phosphorylation of CTD during transcription initiation recruits the capping enzymes to cap the 5’ end of nascent transcript. CTD also activate the capping activity. Further phosphorylation of the CTD recruits components of the pre-mRNA splicing machinery. RNA Pol II reaches termination signals and factors required for clavage and 3’ polyadenylation of the transcript associated with the CTD and recognized sequences in the RNA.