The Guanylyltransferase Domain of Mammalian mRNA Capping Enzyme Binds to the Phosphorylated Carboxyl-terminal Domain of RNA Polymerase II

C Kiong Ho,Verl Sriskanda,Susan Mccracken,David Bentley,Beate Schwer,Stewart Shuman

doi:10.1074/jbc.273.16.9577

Abstract

We have conducted a biochemical and genetic analysis of mouse mRNA capping enzyme (Mce1), a bifunctional 597-amino acid protein with RNA triphosphatase and RNA guanylyltransferase activities. The principal conclusions are as follows: (i) the mammalian capping enzyme consists of autonomous and nonoverlapping functional domains; (ii) the guanylyltransferase domain Mce1(211-597) is catalytically active in vitro and functional in vivo in yeast in lieu of the endogenous guanylyltransferase Ceg1; (iii) the guanylyltransferase domain per se binds to the phosphorylated RNA polymerase II carboxyl-terminal domain (CTD), whereas the triphosphatase domain, Mce1(1-210), does not bind to the CTD; and (iv) a mutation of the active site cysteine of the mouse triphosphatase elicits a strong growth-suppressive phenotype in yeast, conceivably by sequestering pre-mRNA ends in a nonproductive complex or by blocking access of the endogenous yeast triphosphatase to RNA polymerase II. These findings contribute to an emerging model of mRNA biogenesis wherein RNA processing enzymes are targeted to nascent polymerase II transcripts through contacts with the CTD. The phosphorylation-dependent interaction between guanylyltransferase and the CTD is conserved from yeast to mammals.

Highlights

We have conducted a biochemical and genetic analysis of mouse mRNA capping enzyme (Mce1), a bifunctional 597-amino acid protein with RNA triphosphatase and RNA guanylyltransferase activities
MRNA capping occurs by a series of three enzymatic reactions in which the 5Ј triphosphate terminus of the primary transcript is cleaved to a diphosphate by RNA triphosphatase, capped with GMP by RNA guanylyltransferase, and methylated at the N-7 position of guanine by RNA methyltransferase (1)
There must exist a mechanism to target cap formation in vivo to transcripts made by polymerase II (pol II), because the capping enzymes have no inherent specificity for modifying pre-mRNAs in vitro

Summary

Introduction

We have conducted a biochemical and genetic analysis of mouse mRNA capping enzyme (Mce1), a bifunctional 597-amino acid protein with RNA triphosphatase and RNA guanylyltransferase activities. The principal conclusions are as follows: (i) the mammalian capping enzyme consists of autonomous and nonoverlapping functional domains; (ii) the guanylyltransferase domain Mce1(211–597) is catalytically active in vitro and functional in vivo in yeast in lieu of the endogenous guanylyltransferase Ceg[1]; (iii) the guanylyltransferase domain per se binds to the phosphorylated RNA polymerase II carboxyl-terminal domain (CTD), whereas the triphosphatase domain, Mce1(1–210), does not bind to the CTD; and (iv) a mutation of the active site cysteine of the mouse triphosphatase elicits a strong growth-suppressive phenotype in yeast, conceivably by sequestering pre-mRNA ends in a nonproductive complex or by blocking access of the endogenous yeast triphosphatase to RNA polymerase II. The 573amino acid nematode protein consists of a carboxyl-terminal domain homologous to yeast Ceg[1] and an amino-terminal domain that has strong similarity to the superfamily of protein phosphatases that act via a covalent phosphocysteine intermediate

Methods

Results

Conclusion