Capped RNA Synthesis (E2050) | NEB

Family members of Kinetoplastidae include Trypanosoma and Leishmania species that synthesize cap 4, a hypermethylated form of the cap in spliced leader (SL) sequences (). Cap 4 in Kinetoplastidae is critical for pre-mRNA trans-splicing and translational efficiency. In T. brucei, the genes encoding AdoMet-dependent ribose 2′-O methyltransferases have been identified. The 2′-O methyltransferase TbMtr1 methylates the first transcribed nucleoside, TbMtr2 is responsible for methylation of the second nucleoside, while TbMtr3 has been implicated in methylation of nucleosides at positions 3 and 4. Each of these methyltransferases share sequence similarity to vaccinia VP39 and are presumed to function in an analogous manner.

Cap rna synthesis | tecontthemjakedutucafoncpetab

Synthesis and application of a chain‐terminating dinucleotide mRNA cap analog.

Capped RNA Synthesis (E2040) | NEB

Similar to fungi, several protozoan parasites such as Encephalitozoan cuniculi, Plasmodium falciparum, and Trypanosoma species encode capping machinery in three separate polypeptide chains. In these organisms, the RNA triphosphatase appears similar to the metal-dependent Cet1 triphosphtase. In Leishmania, capping enzymes are synthesized in two parts with the Cet1-like RNA triphosphatase activity encoded in a single polypeptide, whereas the RNA guanylyltransferase and guanine-N7 methyltransferase activities are encoded in a bifunctional polypeptide ().

RNA modifications (synthesis of 5 prime cap) - YouTube

While activities required for cap 0 synthesis are conserved, the way in which the enzymes are organized differs across eukaryal and viral evolution. In fungi, the three enzymatic activities are encoded by separate genes, one for the metal-dependent triphosphatase, one for the guanylyltransferase, and one for the guanine-N7 methyltransferase ()., In metazoa and plants, capping activities are encoded in two polypeptides, one for the guanine-N7 methyltransferase and one that contains a bifunctional enzyme with an N-terminal metal-independent RNA triphosphatase domain fused to a C-terminal RNA guanylyltransferase domain, ().

Figure 1 shows the time course of capped RNA synthesis from 1 µg control template.

Protein Synthesis Steps In Brief

Synthesis of mRNA in influenza virus is initiated by stealing cellular caps (), and the viral RNA-dependent RNA polymerase is responsible for this unique ‘cap snatching’ mechanism. The viral polymerase is a heterotrimer composed of three subunits: PA, PB1 and PB2. Although polymerase activity is localized to the PB1 subunit, the PB2 subunit recognizes and binds cellular 5′ capped transcripts in a manner that is stimulated by interactions between the 5′ end of the viral RNA template and PB1. A series of recent crystal structures revealed that the interface between PA1 and PB2 is important for viral polymerase function and identified the cap-binding site in PB2. Upon binding capped cellular transcripts, the PA N-terminal domain catalyzes endonucleolytic cleavage between nucleotides 10 and 13 to generate a short capped RNA oligoribonucleotide,–. The capped oligoribonucleotide products of this reaction are then used to prime synthesis of viral mRNAs, which are polyadenylated at their 3′ ends by the polymerase.

DNA / RNA Oligonucleotide Synthesizers | Biolytic

Several double-stranded RNA viruses synthesize the cap 1 structure. In some of the Reoviridae family members, each of the four capping activities for cap 1 synthesis are contained in a single multifunctional polypeptide chain and the 5′ end of transcripts are capped with cap 1 in four steps prior to viral RNA export to the host cell cytoplasm. Structural studies on the reovirus core showed that all essential enzymatic activities required for cap 1 synthesis reside in the λ2 polypeptide of the viral core including a guanylyltransferase, and two C-terminal λ2 methyltransferase domains with structural similarity to vaccinia virus VP39 (see below). Although the reovirus guanylyltransferase bears no resemblance to other cellular guanylyltransferases, it does contain a lysine residue that is known to form a lysyl-N-GMP adduct. Based on the respective positions of the active sites within the context of the virus core, these domains appeared poised to cap the RNA as it is extruded from the virus.Blue-tongue virus also encodes four enzymatic activities for cap 1 modification in a single polypeptide chain, VP4. Structural studies of VP4 showed a multidomain architecture that includes an N-terminal kinase-like domain predicted to be important for protein–protein interactions, central N7-methyltransferase and 2′-O methyltransferase domains, and a C-terminal domain that was proposed to contain both triphosphatase and guanylyltransferase activities despite its lack of structural similarity to any characterized RNA triphosphatase or guanylyltransferase enzymes ().

RNA capping and decapping; RNA methylation; cap‐binding proteins; ..

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.