Identification of a Cytoplasmic Complex That Adds a Cap onto 5′-Monophosphate RNA

Abstract

Endonuclease decay of nonsense-containing ␤-globin mRNA in erythroid cells generates 5-truncated products that were reported previously to have a cap or caplike structure. We confirmed that this 5 modification is indistinguishable from the cap on full-length mRNA, and Western blotting, immunoprecipitation, and active-site labeling identified a population of capping enzymes in the cytoplasm of erythroid and nonerythroid cells. Cytoplasmic capping enzyme sediments in a 140-kDa complex that contains a kinase which, together with capping enzyme, converts 5-monophosphate RNA into 5-GpppX RNA. Capping enzyme shows diffuse and punctate staining throughout the cytoplasm, and its staining does not overlap with P bodies or stress granules. Expression of inactive capping enzyme in a form that is restricted to the cytoplasm reduced the ability of cells to recover from oxidative stress, thus supporting a role for capping in the cytoplasm and suggesting that some mRNAs may be stored in an uncapped state.The addition of the 5Ј cap is the first posttranscriptional step in pre-mRNA processing (8,25), and the cap plays a central role in subsequent steps of pre-mRNA processing, export, surveillance, translation, decay, and microRNA silencing through its binding by CBP80 (9) and eIF4E (22). The decay of most mammalian mRNAs begins with poly(A) shortening, after which the cap is removed and the body of the mRNA undergoes 3Ј-5Ј decay by the cytoplasmic exosome or 5Ј-3Ј decay by Xrn1 (7).While the action of a cytoplasmic poly(A) polymerase can restore a shortened poly(A) tail to one capable of supporting efficient translation (13), there is no evidence for the reversibility of decapping (30). In Saccharomyces cerevisiae, decapping is the rate-limiting step in mRNA decay, and this is followed by rapid 5Ј-3Ј degradation of the mRNA body by Xrn1 (7). Like in yeast, mammalian Dcp2 and Xrn1 are together recovered by immunoprecipitation and colocalize in P bodies (4), suggesting that unstable mRNAs decay similarly. However, until recently, no one had actually quantified the polarity of mammalian mRNA decay. Using a sensitive fluorescent resonance energy transfer-based assay to quantify the decay of each exon of a ␤-globin reporter mRNA, we found that the 5Ј and 3Ј ends decay simultaneously and showed that these processes are functionally linked (19). More surprisingly, we found that 5Ј decay is slow and relatively inefficient.mRNA containing a premature termination codon (PTC) is degraded by a process termed mRNA surveillance or nonsense-mediated mRNA decay. While it was previously thought that PTC-containing mRNAs are degraded while still associated with the nucleus (17), several recent studies point to P bodies as the major site of their decay (26, 31). An endonuclease activity in SMG6 also appears to be involved in this process, but Xrn1 must be knocked down to visualize downstream decay products (5, 10).Mutations in the ␤-globin gene comprise one of the largest cohorts of inherited disorders, and a PTC in exon 1 or 2 activates nonsense-...

Genome-wide analysis of promoter architecture in Drosophila melanogaster

“…Thus, these data support our conclusion that CAGE peaks in 39 UTRs are unlikely to represent novel sites of transcription initiation. We conclude that CAGE peaks in 39 UTRs are likely to be associated with transcript degradation products that might be recapped by a recently described cytoplasmic capping complex (Otsuka et al 2009). Thus, the CAGE peaks within 39 UTRs appear to represent 59 ends of cytoplasmic transcript fragments, and not independent promoters.…”

Potential role for snoRNAs in PKR activation during metabolic stress

“…1C as an example). Alternatively, a report identified a component of the cytosolic capping complex that adds a phosphate to monophosphorylated RNA, generating RNA with a 5′-diphosphate (34). Interestingly, RIG-I, a dsRNA-sensing protein, is activated by RNA with either a 5′-triphosphate or a 5′-diphosphate (35).…”

Multiple Nudix family proteins possess mRNA decapping activity

“…Demonstration of several Nudix proteins capable of potentially generating RNAs with a 5 ′ monophosphate or diphosphate raises an interesting question regarding the functional consequence of mRNAs with a 5 ′ diphosphate since these would not serve as a substrate for currently known 5 ′ -3 ′ exoribonucleases. However, the recent demonstration that a cytoplasmic 5 ′ -monophosphorylated mRNA can be recapped through a 5 ′ -diphosphorylated intermediate (Otsuka et al 2009;Mukherjee et al 2012) indicates that 5 ′ -diphosphorylated mRNAs may be more efficient substrates for recapping and entry of the mRNA into the translational pool. Whether decapping enzymes that can generate 5 ′ -end diphosphorylated RNAs are involved in facilitating the cytoplasmic recapping pathway remains to be determined.…”