The identification of putative RNA polymerase II C-terminal domain associated proteins in red and green algae

Abstract

A tandemly repeated C-terminal domain (CTD) of the largest subunit of RNA polymerase II is functionally essential and strongly conserved in many organisms, including animal, yeast and plant models. Although present in simple, ancestral red algae, CTD tandem repeats have undergone extensive modifications and degeneration during the evolutionary transition to developmentally complex rhodophytes. In contrast, CTD repeats are conserved in both green algae and their more complex land plant relatives. Understanding the mechanistic differences that underlie these variant patterns of CTD evolution requires knowledge of CTD-associated proteins in these 2 lineages. To provide an initial baseline comparison, we bound potential phospho-CTD associated proteins (PCAPs) to artificially synthesized and phosphorylated CTD repeats from the unicellular red alga Cyanidioschyzon merolae and green alga Chlamydomonas reinhardtii. Our results indicate that red and green algae share a number of PCAPs, including kinases and proteins involved in mRNA export. There also are important taxon-specific differences, including mRNA splicing-related PCAPs recovered from Chlamydomonas but not Cyanidioschyzon, consistent with the relative intron densities in green and red algae. Our results also offer the first experimental indication that different proteins bind 2 distinct types of repeats in Cyanidioschyzon, suggesting a division of function between the proximal and distal CTD, similar to patterns identified in more developmentally complex model organisms.

Keywords: C-terminal domain; CDK, Cyclin-dependent kinase; CTD, Carboxyl-terminal domain; PCAPs, Phospho-CTD associated proteins; RNA polymerase II; green algae; phosphorylation; red algae.

Figure 1.

The CTD in green plants and red algae. The tree reflecting the relationships among green algae/plants and red algae was constructed based on the Tree of Life Web Project. Annotated CTDs for each genus are shown above the taxa included in the tree (CTD N-termini are at the top of each sequence). Sequences from multicellular red algae are shown in boxes; they have highly modified CTDs with no discernable repetitive structures that are present in unicellular (ancestral) forms. Green indicates regions with at least 2 continuous canonical (YSPxSPx) heptapeptides; yellow indicates the presence of isolated heptads, not in tandem with another canonical repeat; purple indicates the presence of the non-canonical motif “FSPTSPS;" red regions are without any canonical heptapeptides whatsoever. For more detail on these annotations, see our previous publication.

Figure 2.

PCAP purification process. The PCAP purification process is shown step by step as indicated by the direction of the arrows. The elution from each affinity column was subjected to SDS-PAGE followed by staining with Coomassie blue. The gels run on elutions are shown for each affinity column. M represents molecular weight (KDa) marker, 0.3 M and 0.5 M indicates elution with those concentrations of NaCl in BH buffer. The putative PCAPs from each elution highlighted in our results section are shown under the respective gels.