Abstract
Positive Transcription Elongation Factor b (P-TEFb) is a kinase consisting of Cdk9 and Cyclin T that releases RNA Polymerase II (Pol II) into active elongation. It can assemble into a larger Super Elongation Complex (SEC) consisting of additional elongation factors. Here, we use a miRNA-based approach to knock down the maternal contribution of P-TEFb and SEC components in early Drosophila embryos. P-TEFb or SEC depletion results in loss of cells from the embryo posterior and in cellularization defects. Interestingly, the expression of many patterning genes containing promoter-proximal paused Pol II is relatively normal in P-TEFb embryos. Instead, P-TEFb and SEC are required for expression of some non-paused, rapidly transcribed genes in pre-cellular embryos, including the cellularization gene Serendipity-α. We also demonstrate that another P-TEFb regulated gene, terminus, has an essential function in embryo development. Similar morphological and gene expression phenotypes were observed upon knock down of Mediator subunits, providing in vivo evidence that P-TEFb, the SEC and Mediator collaborate in transcription control. Surprisingly, P-TEFb depletion does not affect the ratio of Pol II at the promoter versus the 3’ end, despite affecting global Pol II Ser2 phosphorylation levels. Instead, Pol II occupancy is reduced at P-TEFb down-regulated genes. We conclude that a subset of non-paused, pre-cellular genes are among the most susceptible to reduced P-TEFb, SEC and Mediator levels in Drosophila embryos.
Highlights
RNA Polymerase II (Pol II) is responsible for the transcription of protein-coding genes in eukaryotes, and differs from all other RNA Polymerases in that its largest subunit contains a Cterminal domain (CTD) consisting of a heptapeptide repeat with the consensus YSPTSPS [reviewed in 1]
Positive Transcription Elongation Factor b (P-TEFb) and Drosophila Development proximal pausing involves the kinase P-TEFb, which phosphorylates negative elongation factors, allowing Pol II to enter into productive elongation
We find that several genes with paused Pol II can be expressed relatively normally in P-TEFb depleted embryos, whereas expression of some non-paused genes is substantially reduced
Summary
RNA Polymerase II (Pol II) is responsible for the transcription of protein-coding genes in eukaryotes, and differs from all other RNA Polymerases in that its largest subunit contains a Cterminal domain (CTD) consisting of a heptapeptide repeat with the consensus YSPTSPS [reviewed in 1]. Phosphorylation of the CTD is linked to various steps in the transcription cycle. Hypophosphorylated Pol II is recruited to the transcription start site (TSS) and forms a pre-initiation complex. Phosphorylation of Ser in the heptapeptide repeat is coupled to initiation of transcription. The transition into transcription elongation involves CTD Ser phosphorylation. CTD phosphorylation links transcription with RNA processing, since the CTD forms a platform for the assembly and action of enzymes involved in capping (Ser phosphorylated CTD), splicing and polyadenylation (Ser phosphorylated CTD) [reviewed in 2]
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