In C. elegans embryos, mRNA transcription initiates at the 3-4 cell stage in the somatic cells. In the germline precursor, the transcriptional repressor PIE-1 prevents gene expression, possibly by interfering with phosphorylation of the C-terminal domain (CTD) of RNA Polymerase II. The Pol II CTD is composed of a heptapeptide repeat (YSPTSPS), which is differentially phosphorylated during the transcription cycle; unphosphorylated Pol II is recruited to a promoter and as the transcription pre-initiation complex forms, CDK-7/CyclinH phosphorylates the CTD serine 5. During the transition to mRNA elongation, CTD phosphorylation shifts to serine 2 due to the activity of CDK-9/CyclinT. While the CTD may be modified by a variety of factors, the mechanisms regulating the transcription cycle have not been well described in vivo. During early embryogenesis the somatic and germline precursors have distinct patterns of Pser5 and Pser2 levels and localization. Pser2 is detected in somatic cells after zygotic transcription begins, but is absent in the transcriptionally silent germline precursor. Pser5 levels parallel Pser2 in the somatic nuclei. The germline precursors have little or no nucleoplasmic Pser5, however they do contain two distinct foci, similar to those in the transcriptionally silent germline cells of Drosophila embryos (Seydoux and Dunn (1997) Devt 124:2191). We have examined regulation of Pol II during early embryogenesis and studied the Pser5 foci in more detail. Appearance of these foci depends on parts of the transcription pre-initiation complex, such as TFIIB and the mediator complex component RGR-1. However, the PSer5 foci appear in all somatic cells when transcription is inhibited through interference with TFIID components, which function at the last step in transcription initiation. Thus, these structures may represent stalled transcriptional loci or storage/recycling areas that accumulate factors such as Pol II from aborted transcription events. To better understand the Pser5 foci, we have examined other nuclear structures. Consistent with a role in stalled or aborted transcription, the Pser5 foci did not co-localize with antibodies to nuclear speckles, nor did they co-localize with antibodies specific to nucleoli. Cajal bodies have been described as sites for recycling components of the transcription machinery, however these structures are not well described in C. elegans . To approach this, we have used RNAi to inhibit expression of
smn-1 , which is found in Cajal bodies or related structures known as Gems. While overall transcription levels are diminished in the
smn-1(RNAi) embryos, the Pser5 foci are not affected. Thus, the Pser5 foci may represent a discrete functional compartment of a structure such as a Cajal body, or a distinct type of nuclear structure. We have also examined the regulation of CTD phosphorylation in embryonic and germline nuclei. Mitotic and pachytene regions of the germline have high levels of Pser5 and Pser2, reflecting the robust transcription in these nuclei. However, the most proximal oocytes, which have arrested in diakinesis prior, have no detectable Pser5 or Pser2. Suprisingly, we have found that disruption of the ubiquitination pathway through interference with ubiquitin activating enzyme,
uba-1 , or a ubiquitin conjugating enzyme,
ubc-2(
let-70) , causes Pser5, but not Pser2, to appear in the proximal oocytes and in 1-2 cell embryos. This increase in Pser5 in
uba-1(RNAi) oocytes and embryos may represent not inappropriate transcription because Pser2 levels are not increased and because the early embryonic reporter PES-10::GFP appears at the appropriate time. We have also investigated to role of several ubiquitin conjugating factors that modify Pol II in yeast. Interference with expression of these genes in C. elegans did not affect Pser5 levels, suggesting that the ubiquitination pathway may not be directly modifying Pol II. In addition, levels of unphosphorylated Pol II are normal in the
uba-1 or
ubc-2/let-70(RNAi) oocytes and embryos. These results suggest that ubiquitination is important for regulating early steps of the transcription cycle and that loss of ubiquitination causes persistent or inappropriate serine 5 phosphorylation.