Enzymatically stable 5′ mRNA cap analogs: Synthesis and binding studies with human DcpS decapping enzyme

Abstract

Four novel 5′ mRNA cap analogs have been synthesized with one of the pyrophosphate bridge oxygen atoms of the triphosphate linkage replaced with a methylene group. The analogs were prepared via reaction of nucleoside phosphor/phosphon-1-imidazolidates with nucleoside phosphate/phosphonate in the presence of ZnCl₂. Three of the new cap analogs are completely resistant to degradation by human DcpS, the enzyme responsible for hydrolysis of free cap resulting from 3′ to 5′ cellular mRNA decay. One of the new analogs has very high affinity for binding to human DcpS. Two of these analogs are Anti Reverse Cap Analogs which ensures that they are incorporated into mRNA chains exclusively in the correct orientation. These new cap analogs should be useful in a variety of biochemical studies, in the analysis of the cellular function of decapping enzymes, and as a basis for further development of modified cap analogs as potential anti-cancer and anti-parasite drugs.

Introduction

Eukaryotic messenger RNAs are modified at their 5′-ends by addition of a 7-methylguanosine attached by a 5′–5′ triphosphate bridge to the first nucleotide of the mRNA chain (Fig. 1). The cap structure plays a pivotal role in mRNA metabolism. It is required for mRNA processing, transport, translation, and is an important determinant in mRNA turnover.¹

mRNA turnover plays an important role in the control of gene expression contributing to the control of mRNA levels in the cell.² There are two general pathways of eukaryotic mRNA degradation, both initiated by the shortening of mRNA 3′-polyadenyl tail. In 5′ to 3′ mRNA decay, the cap structure is cleaved by a specific pyrophosphatase Dcp1/Dcp2. This generates 7-methylGDP and an RNA with a 5′ exposed phosphate which is a substrate for 5′ to 3′ exonucleolytic decay by nuclease Xrn1. In the 3′ to 5′ pathway of mRNA decay, shortening of the poly(A)-tail of the RNA exposes the 3′ end of the RNA to a complex of 3′ to 5′ exonucleases, known as the exosome, which progressively degrade the mRNA. The products of this 3′ to 5′ RNA degradation are the monophosphates of the RNA and the mRNA cap. The free mRNA cap is hydrolyzed by a scavenger decapping enzyme known as DcpS. Current data suggest that the 5′ to 3′ pathway of mRNA degradation may be dominant in yeast, whereas at least in vitro the 3′ to 5′ pathway may be the major pathway in vertebrate cells.2, 3, 4

Sequence and mutagenesis analyses indicate that DcpS enzymes are members of the HIT family of pyrophosphatases containing a conserved histidine triad in the active site.⁴ DcpS decapping enzymes catalyze the hydrolysis of capped dinucleotides (m⁷GpppN) and short oligonucleotides (up to ∼8 nucleotides) resulting from exosome mediated 3′–5′ mRNA decay. DcpS is unable to hydrolyze the cap structure linked to a long mRNA chain. This property protects functional mRNAs from degradation by DcpS. Crystallographic studies of DcpS in complex with m⁷GpppG and m⁷GpppA suggest that negative allosteric interactions between cap analogs and the enzyme are likely responsible for the inability of the enzyme to cleave the cap on a long mRNA chain.⁵ DcpS cleaves the cap 5′–5′ pyrophosphate bond to release m⁷GMP and ppN-. This contrasts with the RNA decapping enzyme Dcp2/Dcp1 which cleaves the cap on a long mRNA chain producing m⁷GDP and pN-. DcpS cleavage proceeds through nucleophilic attack of His-277 on the γ-phosphate. Cap analogs resulting from 3′ to 5′ RNA degradation are predicted to be toxic to cells as they are inhibitors of a variety of cap-interacting proteins and could be misincorporated into nucleic acids. Many aspects of the activity and regulation of DcpS remain unknown. Furthermore, the contribution of 5′ to 3′ versus 3′ to 5′ decay in higher eukaryotes also remains unclear. Our group is interested in developing tools that will help provide a better understanding of the mechanism and role of both DcpS and Dcp2/Dcp1 decapping. One approach to the analysis of these decapping enzymes interaction with the cap is through the use of modified cap analogs that are resistant to hydrolysis of the triphosphate bridge. These resistant cap analogs can be used for biophysical studies of DcpS or Dcp2/Dcp1, as inhibitors in vitro and may enable the synthesis of mRNAs with enhanced stability. They also enabled us to initiate experiments aimed at understanding the relationships between mRNA translation and decapping in higher eukaryotes.

There are a number of approaches that might be applicable to make oligophosphates immune to hydrolysis by cellular enzymes including the use of phosphorothioates.6, 7, 8 Another commonly used strategy is to replace a pyrophosphate bridge oxygen by a methylene group. The methylene group precludes any enzymatic cleavage due to the extreme stability of P–C bonds.9, 10, 11, 12, 13, 14, 15, 16 In a brief preliminary report, we have used the latter approach with the goal of producing analogs that retain high affinity for the DcpS and Dcp2/Dcp1 enzymes.¹⁷ Here, we present a detailed description of their synthesis and susceptibility toward human DcpS enzyme.