Coupled Interactions in Eukaryotic Gene Expression Pathway: A Comprehensive Review

1. An extensive network of coupling among gene expression machines - Review Mani 03/03/04- 05/05/04

2. Eukaryotic Gene Expression • Transcription • Initiation, Elongation, Termination • 5’ cap, splicing, PolyA • Mature mRNA released from transcription site and exported to cytoplasm • RNA surveillance system

3. The Pathway • Each machine has its own unique reaction(s) to catalyze, and it also “interferes” with other machines in the pathway. • Not just between sequential steps!

4. The Pathway – cont’d A complex network of coupled interactions in gene expression

5. RNA pol II with RNA processing • Carboxy-terminal domain (CTD) and transcription elongation factors • CTD independently promotes each step of the pre-mRNA processing pathway • CTD is directly adjacent to the exit groove for the pre-mRNA • 650 A° CTD connected to Pol II through a 280 A° linker

6. RNA pol II with RNA processing – cont’d • Can interact with multiple components of the pre-mRNA processing machinery and localize the machinery close to the pre-mRNA as it exits

7. Pre-mRNA splicing with transcription elongation • Splicing machinery and elongation factors • Transcription elongation factor P-TEFb recruits another factor TAT-SF1 which in turn recruits splicing factors to the nascent pre-mRNA • This splicing machinery strongly stimulates transcription elongation • (Genes containing introns are more efficiently transcribed)

8. The “LONG intron” problem • human neurexin pre-mRNA is 480,000 nuc. long • Several hours pass b/w synthesis of 5’ and 3’ ends • Solution may lie in “tethering” splicing machinery to the CTD and elongation complex • Tethering splicing machinery to elongation complex reduces the kinetics of splicing machinery:pre-mRNA to zero-order

9. The “LONG intron” problem – cont’d • Also avoids the random non-specific RNA binding proteins acting on first-order kinetics • “Tether each newly synthesized exon and the adjacent splice sites to the CTD until the next exon emerges from the exit groove”

10. Transcription Factors and Splicing • Preinitiation complex and SR family splicing factors • SR family proteins bind to splicing enhancers (in exon sequences) • Transcription factors recruit SR proteins • Proximity of SR proteins may promote splicing

11. Transcription Factors and Splicing – cont’d • Transcription by different promoters can generate alternatively spliced mature mRNA • Two theories: • Particular SR proteins are recruited at different promoters, and are handed off to cognate splicing enhancers • Rate of transcription dictates the secondary structure of the pre-mRNA, and these could determine the availability of sequence elements

12. Capping/Polyadenylation and Splicing 5’ CAP binding proteins Terminal 3’ intron splicing factors CAP proximal 5’ splice site recognition Downstream polyadenylation complex

13. Termination and Initiation • May be mediated by a poly-adenylation factor • Human transcriptional coactivator PC4 interacts directly with polyadenylation factor CstF • PC4 may function as antiterminator by inhibiting CstF during elongation • They dissociate at polyadenylation site, so CstF can participate in polyadenylation and termination

14. mRNA Release and Transportation • Pre-mRNA with splicing mutations accumulate at site of transcription • Either abortive splice complexes are formed that don’t release, or splicing may be required for release • RNA export mutants accumulate at site of transcription as well – release and export could be coupled

15. This or That? • Anchored in an immobilized Gene Expression Factory and reels the template DNA in • Transcription machinery moves along DNA carrying the machines

16. Gene Expression Factory Model