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The messenger RNA decapping and recapping pathway in Trypanosoma

Anna V. Ignatochkinaa, Yuko Takagib, Yancheng Liuc, Kyosuke Nagataa, and C. Kiong Ho
12/11/2018
PNAS
 Significance

The 5′ end of eukaryotic mRNA is capped and methylated to protect mRNA from degradation and enhance protein synthesis. However, the cap can be removed from mRNA by decapping. We identified a recapping enzyme with 5′-monophosphate RNA kinase activity from trypanosome and provide evidence that decapped transcripts can be recapped to regenerate translatable mRNA. The kinase activity is dependent on mRNA leader sequence and is stimulated by hypermethylation found in the trypanosome mRNA. We also identify a trypanosome decapping enzyme that removes cap structure from the mRNA, but is less active on hypermethylated capped mRNA. These results suggest that hypermethylated cap structure can influence certain transcripts to be preferentially decapped or recapped during the parasite life cycle.
 

Abstract

The 5′ terminus of trypanosome mRNA is protected by a hypermethylated cap 4 derived from spliced leader (SL) RNA. Trypanosoma brucei nuclear capping enzyme with cap guanylyltransferase and methyltransferase activities (TbCgm1) modifies the 5′-diphosphate RNA (ppRNA) end to generate an m7G SL RNA cap. Here we show that T. brucei cytoplasmic capping enzyme (TbCe1) is a bifunctional 5′-RNA kinase and guanylyltransferase that transfers a γ-phosphate from ATP to pRNA to form ppRNA, which is then capped by transfer of GMP from GTP to the RNA β-phosphate. A Walker A-box motif in the N-terminal domain is essential for the RNA kinase activity and is targeted preferentially to a SL RNA sequence with a 5′-terminal methylated nucleoside. Silencing of TbCe1 leads to accumulation of uncapped mRNAs, consistent with selective capping of mRNA that has undergone trans-splicing and decapping. We identify T. brucei mRNA decapping enzyme (TbDcp2) that cleaves m7GDP from capped RNA to generate pRNA, a substrate for TbCe1. TbDcp2 can also remove GDP from unmethylated capped RNA but is less active at a mature cap 4 end and thus may function in RNA cap quality surveillance. Our results establish the enzymology and relevant protein catalysts of a cytoplasmic recapping pathway that has broad implications for the functional reactivation of processed mRNA ends.