Conservation of the mammalian RNA polymerase II largest-subunit C-terminal domain

Summary We have isolated and sequenced a portion of the gene encoding the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II from three mammals. These mammalian sequences include one rodent and two primate CTDs. Comparisons of the new sequences to mouse and Chinese hamster show a high degree of conservation among the mammalian CTDs. Due to synonymous codon usage, the nucleotide differences between hamster, rat, ape, and human result in no amino acid changes. The amino acid sequence for the mouse CTD appears to have one different amino acid when compared to the other four sequences. Therefore, except for the one variation in mouse, all of the known mammalian CTDs have identical amino acid sequences. This is in marked contrast to the situation among more divergent species. The present study suggests that there is a strong evolutionary pressure to maintain the primary structure of the mammalian CTD. Offprint requests to: J.L. Corden     

Requirements of the RNA polymerase II C-terminal domain for reconstituting pre-mRNA 3' cleavage

RNA polymerase II (RNAP II) has previously been shown to be required for the pre-mRNA polyadenylation cleavage reaction in vitro. This activity was found to reside solely in the C-terminal domain (CTD) of the enzyme's largest subunit. Using a deletion analysis of glutathione S-transferase-CTD fusion proteins, we searched among the CTD's 52 imperfectly repetitive heptapeptides for the minimal subset that possesses this property. We found that heptads in the vicinity of 30 to 37 contribute modestly more than other sections, but that no specific subsection of the CTD is necessary or sufficient for cleavage. To investigate further the heptad requirements for cleavage, we constructed a series of all-consensus CTDs having 13, 26, 39, and 52 YSPTSPS repeats. We found that the nonconsensus CTD heptads are together responsible for only 20% of the wild-type cleavage activity. Analysis of the all-consensus CTD series revealed that the remaining 80% of the CTD-dependent cleavage activity directly correlates with CTD length, with significant activity requiring approximately 26 or more repeats. These results are consistent with a scaffolding role for the RNAP II CTD in the pre-mRNA cleavage reaction.     

Arabidopsis RNA polymerase II C-terminal domain phosphatase-like 1 targets mitogen-activated protein kinase cascades to suppress plant immunity

Mitogen-activated protein kinase (MAPK) cascades play pivotal roles in plant defense against phytopathogens downstream of immune receptor complexes. The amplitude and duration of MAPK activation must be strictly controlled, but the underlying mechanism remains unclear. Here, we identified Arabidopsis CPL1 (C-terminal domain phosphatase-like 1) as a negative regulator of microbe-associated molecular pattern (MAMP)-triggered immunity via a forward-genetic screen. Disruption of CPL1 significantly enhanced plant resistance to Pseudomonas pathogens induced by the bacterial peptide flg22. Furthermore, flg22-induced MPK3/MPK4/MPK6 phosphorylation was dramatically elevated in cpl1 mutants but severely impaired in CPL1 overexpression lines, suggesting that CPL1 might interfere with flg22-induced MAPK activation. Indeed, CPL1 directly interacted with MPK3 and MPK6, as well as the upstream MKK4 and MKK5. A firefly luciferase-based complementation assay indicated that the interaction between MKK4/MKK5 and MPK3/MPK6 was significantly reduced in the presence of CPL1. These results suggest that CPL1 plays a novel regulatory role in suppressing MAMP-induced MAPK cascade activation and MAMP-triggered immunity to bacterial pathogens.     

The diverse roles of RNA polymerase II C-terminal domain phosphatase SCP1

RNA polymerase II carboxyl-terminal domain (pol II CTD) phosphatases are a newly emerging family of phosphatases that are members of DXDX (T/V). The subfamily includes Small CTD phosphatases, like SCP1, SCP2, SCP3, TIMM50, HSPC129 and UBLCP. Extensive study of SCP1 has elicited the diversified roles of the small C terminal domain phosphatase. The SCP1 plays a vital role in various biological activities, like neuronal gene silencing and preferential Ser5 dephosphorylation, acts as a cardiac hypertrophy inducer with the help of its intronic miRNAs, and has shown a key role in cell cycle regulation. This short review offers an explanation of the mechanism of action of small CTD phosphatases, in different biological activities and metabolic processes. [BMB Reports 2014; 47(4): 192-196]     

Heterogeneity in mammalian RNA 3′ end formation

Precisely directed cleavage and polyadenylation of mRNA is a fundamental part of eukaryotic gene expression. Yet, 3′ end heterogeneity has been documented for thousands of mammalian genes, and usage of one cleavage and polyadenylation signal over another has been shown to impact gene expression in many cases. Building upon the rich biochemical and genetic understanding of the 3′ end formation, recent genomic studies have begun to suggest that widespread changes in mRNA cleavage and polyadenylation may be a part of large, dynamic gene regulatory programs. In this review, we begin with a modest overview of the studies that defined the mechanisms of mammalian 3′ end formation, and then discuss how recent genomic studies intersect with these more traditional approaches, showing that both will be crucial for expanding our understanding of this facet of gene regulation.