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. T he earliest modification to the eukaryotic mRNA is the addition of a cap structure (m7GpppN; cap 0) at the 5′ end, to protect the mRNA from degradation and recruit factors that promote RNA splicing, export, and translation initiation (1). The cap is formed in the nucleus by three sequential enzymatic reactions: the 5′ triphosphate of the nascent mRNA is hydrolyzed to a diphosphate by RNA triphosphatase; the diphosphate end is capped with GMP by RNA guanylyltransferase; and the GpppRNA is methylated by (guanine N7) methyltransferase to form cap 0 (2). Nucleotides adjacent to the cap are typically methylated on the first and second nucleosides to form cap 1 and cap 2 structures, respectively. The most elaborate cap structure, called cap 4, is found in Trypanosoma brucei and other kinetoplastid parasites and consists of a standard cap 0 with 2′-O methylations on the first four ribose sugars (AmAmCmUm), and additional base methylations on the first adenine (m6,6A) and the fourth uracil (m3U) (3). Although additional methylations have been shown to enhance translation efficiency (4, 5), whether they affect the RNA decay process is unknown.All mRNA is subject to degradation by either a 5′-to-3′ or a 3′-to-5′ exonucleolytic pathway, generally initiated by shortening of the poly(A) tail (6). In the 5′-to-3′ pathway, the cap is removed as m7Gpp by the RNA decapping enzyme Dcp2, a member of the Nudix hydrolase superfamily, leaving a 5′-monophosphorylated RNA (pRNA) (7, 8). The exposed pRNA is progressively degraded by a 5′-to-3′ exonuclease (Xrn1/Rat1). Incompletely capped RNAs that lack the N7 methyl moiety, as well as defective mRNAs with premature termination codons, are decapped by a cel...

An ATP-dependent RNA ligase from Methanobacterium thermoautotrophicum (MthRnl) catalyzes intramolecular ligation of single-stranded RNA to form a closed circular RNA via covalent ligase-AMP and RNA-adenylylate intermediate. Here, we report the X-ray crystal structures of an MthRnl•ATP complex as well as the covalent MthRnl–AMP intermediate. We also performed structure-guided mutational analysis to survey the functions of 36 residues in three component steps of the ligation pathway including ligase-adenylylation (step 1), RNA adenylylation (step 2) and phosphodiester bond synthesis (step 3). Kinetic analysis underscored the importance of motif 1a loop structure in promoting phosphodiester bond synthesis. Alanine substitutions of Thr117 or Arg118 favor the reverse step 2 reaction to deadenylate the 5′-AMP from the RNA-adenylate, thereby inhibiting step 3 reaction. Tyr159, Phe281 and Glu285, which are conserved among archaeal ATP-dependent RNA ligases and are situated on the surface of the enzyme, are required for RNA binding. We propose an RNA binding interface of the MthRnl based on the mutational studies and two sulfate ions that co-crystallized at the active site cleft in the MthRnl–AMP complex.