Abstract
RNA Polymerase II (RNAPII) uniquely possesses an extended carboxy terminal domain (CTD) on its largest subunit, Rpb1, comprising a repetitive Tyr1Ser2Pro3Thr4 Ser5Pro6Ser7 motif with potential phosphorylation sites. The phosphorylation of the CTD serves as a signal for the binding of various transcription regulators for mRNA biogenesis including the mRNA capping complex. In eukaryotes, the 5 prime capping of the nascent transcript is the first detectable mRNA processing event, and is crucial for the productive transcript elongation. The binding of capping enzyme, RNA guanylyltransferases to the transcribing RNAPII is known to be primarily facilitated by the CTD, phosphorylated at Ser5 (Ser5P). Here we report that the Saccharomyces cerevesiae RNA guanylyltransferase (Ceg1) has dual specificity and interacts not only with Ser5P but also with Ser7P of the CTD. The Ser7 of CTD is essential for the unconditional growth and efficient priming of the mRNA capping complex. The Arg159 and Arg185 of Ceg1 are the key residues that interact with the Ser5P, while the Lys175 with Ser7P of CTD. These interactions appear to be in a specific pattern of Ser5PSer7PSer5P in a tri-heptad CTD (YSPTSPPS YSPTSPSP YSPTSPPS) and provide molecular insights into the Ceg1-CTD interaction for mRNA transcription.
Highlights
Eukaryotic mRNAs are transcribed by RNA polymerase II (RNAPII) and the pre-mRNAs undergo several processing events such as 5 prime (5′) capping, splicing, polyadenylation etc. before becoming the mature mRNA
In a pull down assay, an interaction between Ceg[1] and the carboxy terminal domain (CTD) phosphorylated at Ser[5] and Ser[7] was observed (Fig. 1a)
An interaction between Ceg[1] and CTD was observed in cases where either all the three serines or at least two serines (Ser[5] and Ser7) were phosphorylated (Fig. 1a, second and third panel)
Summary
Eukaryotic mRNAs are transcribed by RNA polymerase II (RNAPII) and the pre-mRNAs undergo several processing events such as 5 prime (5′) capping, splicing, polyadenylation etc. before becoming the mature mRNA. The CTD was primarily known to be phosphorylated at Ser[2] (Ser2P) and Ser[5] (Ser5P) These two phosphorylations were considered essential for recruiting factors important for the activity of RNAPII during mRNA biogenesis[1,2]. Its function is obscure in budding yeast, it contributes to the expression of noncoding RNA and mRNA splicing in mammalian cells[1,8,9,10,11]. A distinct pattern of all the serine phosphorylations is observed in the protein-coding genes and the role for Ser7P in mRNA transcription is consistent with the observation that highly transcribed genes show high levels of this mark[2]. The actual phosphorylation pattern and sequence in CTD, which promote protein binding or dissociation or regulate their function remains unknown in vivo. We are dealing with a situation that hinders the in depth understanding of the properties and function of CTD in vivo
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