The specific phosphorylation of the CTD regulates Pol II initiation and elongation in vivo through the orchestrated recruitment and displacement of positive and negative elongation factors (Majello and Napolitano, 2001). How these processes are regulated may partly be explained in that the CTD kinases discovered so far, exhibit preferences for the different amino acids present in the consensus repeat YSPTSPS. This suggests a mechanism by which the pattern of phosphorylation may be altered to create or destroy sites of protein- protein interaction. In addition, the presence of non-consensus repeats provides for further specificity, enabling factors to be recruited to specific regions of the CTD. Work by the group of Bentley suggests that this may indeed be the case, showing that consensus and non-consensus regions of the mammalian CTD possess different properties for the processing of pre-mRNA. The first 27 repeats of the CTD conform largely to the consensus sequence and support 5’ capping, whereas the non-consensus repeats 27-52 support 5’- capping, splicing and 3’ processing of RNA (Fong and Bentley, 2001).
The majority of CTD kinases target serine residues (for review see Kobor and Greenblatt, 2002). The change from phosphorylation at serine-5 of the CTD consensus repeat at promoter proximal regions, to serine-2 during elongation
and termination, has been shown to correlate with the change in composition, and activity of complexes present in these regions (Komarnitsky et al., 2000). The activity of the capping enzyme complex is dependent on serine-5 phosphorylation (Rodriguez et al., 2000; Schroeder et al., 2000), whereas the recruitment and activation of splicing factors requires serine-2 phosphorylation (Licatalosi et al., 2002). Based upon these observations, many laboratories have performed screens for proteins recognising different phosphorylated forms of CTD, the outcome of which being the identification of novel and known factors implicated in elongation, repression and pre-mRNA processing (reviewed in (Howe, 2002)). It is therefore possible that the phosphorylation of the last repeat by CKII, or the phosphorylation of CTD tyrosines by c-Abl and c-Arg regulates the recruitment to Pol II, of factors involved in pre-mRNA processing, or the DNA damage response.
Tyrosine-1 of the CTD is the only residue to be completely conserved between all repeats, and has been shown to be essential for viability in yeast (West and Corden, 1995). The phosphorylation of this residue may therefore be expected to affect some function of the CTD, and has been shown to overcome the requirement of Tat for the activation of the HIV promoter (Baskaran et al., 1999). However, in the experiments performed during the course of this work, no effect on the transcription of any specific genes has been seen using mutants no longer able to interact with the only currently described CTD tyrosine kinases. A role for tyrosine-1 phosphorylation in the regulation of pre-mRNA processing can not be ruled out: by recruiting specific splicing and 3’processing factors to the CTD, the preferential production of certain splice variants could be achieved in response to cellular stress. Alternatively, tyrosine-1 phosphorylation may control the presence of factors that target the degradation of the polymerase. We have shown here that mutants no longer able to interact with the only known CTD tyrosine kinases appear sensitive to degradation to the Pol IIB form. Elongation blocks caused by DNA lesions have been shown to target the destruction of RNA Pol II through the recruitment of the Rsp5 ubiquitin ligase to the CTD (Beaudenon et al., 1999; Huibregtse et al., 1997). Tyrosine phosphorylation of the CTD may thus play a role in regulating the stability of the Pol II large subunit, perhaps through the negative regulation of factors that target its digestion. However, this theory is not convincing, given that inhibitors of c-Abl and c-Arg tyrosine kinases fail to induce the Pol IIb form.
3.8
Outlook
The orchestration of transcriptional-, and co-transcriptional processes through phosphorylation of the Pol II CTD was undoubtedly an evolutionary development of major significance in higher eucaryotes. It can therefore be assumed, that pathways resulting in the modification of this domain have some influence on these processes. During this work I tried to identify genes that depend on signalling through the last repeat of Pol II LS for their regulation (activation of transcription). Despite much literature suggestive of the opposite, this does not appear to be the case. However, this does not exclude the possibility that signalling through the last repeat may regulate gene transcription in another way, for example, through pre-mRNA processing. Some preliminary tests were performed during the course of this work, but to little avail. Since the potential effects could be gene specific, the problem arises of knowing which genes are affected: using the chromatin IP (ChIP) technique in combination with a DNA array composed of many different promoter regions, it may be possible to identify promoters to which the c-Abl and CKII kinases are recruited.
The major finding of this work was the discovery that in vivo expression of a mutant lacking the last CTD repeat, resulted in the appearance of the Pol IIb form. This is the first time the Pol IIb form has been identified in vivo, and suggests that the last CTD repeat may regulate its formation. Since the Pol IIb form is probably inactive, degradation to this form may provide a novel mechanism for transcriptional control. Initial experiments to identify the protease responsible, or agents that induce its activation have proved unsuccessful. Further experiments are required to identify this protease, and other interacting partners of the last CTD repeat.