RNA polymerase II C-terminal domain phosphorylation patterns in Caenorhabditis elegans operons, polycistronic gene clusters with only one promoter

Abstract

The heptad repeat of the RNA polymerase II (RNAPII) C-terminal domain is phosphorylated at serine 5 near gene 5' ends and serine 2 near 3' ends in order to recruit pre-mRNA processing factors. Ser-5(P) is associated with gene 5' ends to recruit capping enzymes, whereas Ser-2(P) is associated with gene 3' ends to recruit cleavage and polyadenylation factors. In the gene clusters called operons in Caenorhabditis elegans, there is generally only a single promoter, but each gene in the operon forms a 3' end by the usual mechanism. Although downstream operon genes have 5' ends, they receive their caps by trans splicing rather than by capping enzymes. Thus, they are predicted to not need Ser-5 phosphorylation. Here we show by RNAPII chromatin immunoprecipitation (ChIP) that internal operon gene 5' ends do indeed lack Ser-5(P) peaks. In contrast, Ser-2(P) peaks occur at each mRNA 3' end, where the 3'-end formation machinery binds. These results provide additional support for the idea that the serine phosphorylation of the C-terminal domain (CTD) serves to bring RNA-processing enzymes to the transcription complex. Furthermore, these results provide a novel demonstration that genes in operons are cotranscribed from a single upstream promoter.

FIG. 1.

Ser-2 and Ser-5 phosphorylation across a C. elegans gene. The gene is depicted by filled boxes and angled lines representing exons and introns, respectively. Flanking intergenic regions are shown as lines. The arrow indicates the location of the promoter (2). (a and b) ChIP qPCR experiments using antibodies against Ser-5(P) (a) and Ser-2(P) (b) in the R10E4.2 gene, each normalized to the highest value. The results from each primer set are positioned immediately above the corresponding genomic location. Error bars represent standard errors of the means of data from two independent immunoprecipitation experiments. (c) ChIP qPCR signals of Ser-5(P) and Ser-2(P) as a ratio of total RNAPII in the R10E4.2 gene. Normalized ChIP signals of Ser-5(P) and Ser-2(P) (as in a and b) were divided by the normalized total RNAPII at each primer set. The graph shows best-fit lines.

FIG. 2.

In a two-gene operon (CEOPX012), the level of Ser-5(P) is high at the promoter and the level of Ser-2(P) is high at both 3′ ends. Shown are ChIP qPCR signals of Ser-5(P) (a) and Ser-2(P) (b) in a two-gene operon, each normalized to the highest value. The dashed lines separate genes. A gene in the opposite orientation, just 3′ of C44C1.2, is expressed at a very low level, and primers that query this region were not tested. The most 3′ primer pair is a negative control located in the center of a region lacking annotated genes; it is not adjacent to this operon. Error bars represent standard errors of the means of data from two independent immunoprecipitation experiments.

FIG. 3.

In a four-gene operon (CEOP3156), the level of Ser-5(P) is high at the promoter and the level of Ser-2(P) is high at each 3′ end. (a and b) ChIP qPCR signals of Ser-5(P) (a) and Ser-2(P) (b) in a four-gene operon, each normalized to the highest value. Error bars represent standard errors of the means of data from three independent immunoprecipitation experiments. (c) ChIP qPCR signals of Ser-2(P) as a ratio of total RNAPII in a four-gene operon. Normalization was done as described in the legend to Fig. 1c. The line connects adjacent points.

FIG. 4.

In an eight-gene operon (CEOP1484), the level of Ser-5(P) is high at the operon promoter and at an internal promoter and the level of Ser-2(P) is high at each 3′ end. Shown are data from ChIP qPCR experiments using antibodies against Ser-5(P) (a), Ser-2(P) (b), and total RNAPII (c) in an eight-gene operon, each normalized to the highest value. The location of a previously unknown internal promoter is marked by an asterisk. Error bars represent standard errors of the means of data from two independent immunoprecipitation experiments.