The messenger RNA decapping and recapping pathway in Trypanosoma

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. 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...

Truncated, uncapped mRNA 5’ ends targeted by cytoplasmic recapping cluster at CAGE tags and some transcripts are alternatively spliced

“…The work presented here reinforces and extends upon these earlier observations [19,21]. Importantly, cytoplasmic capping also appears to have evolved independently in trypanosomes [35].…”

Conserved long-range base pairings are associated with pre-mRNA processing of human genes

“…Therefore, the same RNA structure-mediated mechanism of coupling between cleavage, polyadenylation, and splicing may also exist in 5'-UTRs, however the link between these processes and RNA recapping is currently missing. As the existence of decapping and recapping pathways has already been established [103,104], we expect that establishing connections between all steps of RNA processing will soon be a matter of intensive research.…”

mRNA 5′ ends targeted by cytoplasmic recapping cluster at CAGE tags and select transcripts are alternatively spliced

“…The newly added cap is then methylated by RNMT [18]. Importantly, alluding to its biological significance, cytoplasmic capping also appears to have evolved independently in trypanosomes [22]. Roughly 2000 mRNAs are targeted by cytoplasmic capping and those mRNAs are less frequently associated with translating polysomes when DN-cCE is expressed [19].…”

“…Collectively, these findings implied that novel 5 0 ends from truncated mRNAs should be observable among the nontranslating mRNAs in cells where cytoplasmic capping was unimpeded in addition to those where cytoplasmic capping had been blocked. Importantly, alluding to its biological significance, cytoplasmic capping also appears to have evolved independently in trypanosomes [22].…”