A rule-based kinetic model of RNA polymerase II C-terminal domain phosphorylation

Abstract

The complexity of many RNA processing pathways is such that a conventional systems modelling approach is inadequate to represent all the molecular species involved. We demonstrate that rule-based modelling permits a detailed model of a complex RNA signalling pathway to be defined. Phosphorylation of the RNA polymerase II (RNAPII) C-terminal domain (CTD; a flexible tail-like extension of the largest subunit) couples pre-messenger RNA capping, splicing and 3' end maturation to transcriptional elongation and termination, and plays a central role in integrating these processes. The phosphorylation states of the serine residues of many heptapeptide repeats of the CTD alter along the coding region of genes as a function of distance from the promoter. From a mechanistic perspective, both the changes in phosphorylation and the location at which they take place on the genes are a function of the time spent by RNAPII in elongation as this interval provides the opportunity for the kinases and phosphatases to interact with the CTD. On this basis, we synthesize the available data to create a kinetic model of the action of the known kinases and phosphatases to resolve the phosphorylation pathways and their kinetics.

Keywords: Kappa; rule-based modelling; transcription.

Figure 1.

Phosphorylation of serines 2, 5 and 7 (Ser2, Ser5 and Ser7) on the CTD of RNAPII. (a) Ser5 and Ser7 are phosphorylated at the promoter by TFIIH/Kin28. Ser2 phosphorylation increases towards the 3′ end of the gene and is a requirement for 3′ end maturation. Serine phosphorylation has been linked to cotranscriptional splicing (the excision of an intron, in red, is illustrated). (b) The actions of the kinases Kin28, Ctk1, Bur1 and Srb10, and the phosphatases Rtr1, Fcp1 and Ssu72 on an individual heptapeptide are indicated by a change of state of one of Ser2, Ser5, Ser7 to P from U, or vice versa. The specific actions of these proteins at the promoter, during elongation, at the 3′ UTR and on the free RNAPII (recycling) must be distinguished. Higher reaction rates are indicated by broader arrows. Two reactions supported by biochemical evidence but not by in vivo data are indicated by dashed arrows.

Figure 2.

Ser2, Ser5 and Ser7 phosphorylation in the coding regions of single-intron genes. (a) Model predictions of the ratios of Ser2∼P, Ser5∼P and Ser7∼P to RNAPII. (b–d) Scatter plots of the ratios of Ser2∼P, Ser5∼P and Ser7∼P to RNAPII genome-wide for genes with introns between 300 and 600 nucleotides in length derived from the data in [11]. (See electronic supplementary material, figure S1 for intron and exon length distributions.) Data sampled from the promoter region (100 nucleotides upstream from the transcription start site (TSS)) are plotted in green, exon 1 (blue), the intron (red) and exon 2 (grey). In (b–d), the black lines indicate the model simulations of (a) thresholded and scaled to best approximate the measured phosphorylation.

Figure 3.

Sensitivity of (a) RNAPII, (b) Ser2∼P and (c) Ser5∼P to the binding rate p0 and unbinding rate p3. The colour scale white to red indicates the effect on the model prediction (lowest to highest sum of square differences) for each combination of p0 and p3 surveyed, taking p0 = 0.1 and p3 = 100 as the reference.

Figure 4.

Paused RNAPII, Ser2 and Ser5 phosphorylation in the coding regions of single-intron genes. (a) Phosphorylation and RNAPII density along the Ribo1 gene at 240 s after induction (data from [12]). Phosphorylations at the promoter (Prom), exon1 (E1), 3′ splice site (3SS), 5′ end of exon2 (5′-E2) and 3′ end of exon2 (3′-E2) of Ribo1 are indicated. Note that Ser2∼P, Ser5∼P and the density of RNAPII are each normalized to their respective values on induction, and hence are not comparable to each other on an absolute scale. (b) Model simulation of Ribo1 with a pause introduced 600 nucleotides downstream of the TSS. (a) Real and (b) simulated pauses in RNAPII lead to an accumulation of RNAPII that reverses the typical reduction in the absolute level of Ser5∼P and raises the level of Ser2∼P.