Killing the messenger: new insights into nonsense-mediated mRNA decay (original) (raw)

The emerging picture by which these PTC-containing transcripts are recognized is proving increasingly complex and interesting. Although textbooks often depict precursor mRNA molecules and mRNA molecules as lonely travelers in a complex nucleus, this image could hardly be farther from the truth. From the moment of transcriptional initiation, the mRNAs in the nucleus are in the company of numerous proteins. Soon after transcription is completed, the 5′ end of the nascent mRNA is modified (“capped”), and a protein complex associates with the newly capped end of the molecule. In the cytoplasm, translational initiation and decapping reactions compete for the site, to determine whether protein synthesis or mRNA degradation carries the day. Mature mRNA species in the nucleus are also protected from decapping, although the mechanism for this protection is likely to be distinct from that which occurs in the cytoplasm. If, as is generally assumed, translational initiation occurs solely in the cytoplasm, or even if a single round of pioneer translation occurs within this compartment, it is unlikely that initiation complexes would be sufficiently abundant to protect the 5′ end of mRNAs from degradation.

The next set of events for most transcripts involves splicing, the removal of intervening sequences (introns) from between the coding domains (exons). Intron removal is orderly but not processive; introns are removed from a given pre-mRNA species in a characteristic but not invariant order, which, in large genes, does not correspond to simple progression from 5′ to 3′ (although, in general the 5′-end introns are processed prior to those at the 3′ end). Distinct small nuclear ribonuclear proteins recognize the branch site, the 3′ end of the intron, and the 5′ end of the intron. The resulting multisubunit RNA-protein complex, the spliceosome, facilitates cleavage at the 5′ end of the intron and formation of a lariat at a branch point close to the 3′ end of the intron, after which it cleaves the 3′ end of the intron and ligates the two exon ends.

During splicing, a protein complex is deposited about 20–24 nucleotides upstream of the splice site. Proteins so far identified in this splice junction complex include Y14 (an RNA-binding protein) (10), Aly/Ref (an RNA-binding and export factor) (11), RNPS1 (an RNA-binding protein previously implicated in splicing) (12), SRm160 (a protein that associates with the splicing complex but that does not bind RNA) (13, 14), DEK (a 45-kDa phosphoprotein that binds SRm160 and is part of the spliceosome complex) (15), and magoh (which binds to Y14 and TAP, a protein involved in mRNA export to the cytoplasm) (16). To date, none of these proteins seems to be essential for NMD in yeast.

Both Y14 and Aly/Ref bind only spliced mRNA species; they do not bind to unspliced, intron-containing mRNA or to intronless mRNA species. Aly/Ref is associated initially with the spliceosome, but following intron removal, it translocates on the mRNA to the site of splicing complex formation, upstream of the former intron-exon boundary. Y14 does not appear to be part of the spliceosome and thus probably depends on Aly/Ref for positioning on the spliced mRNA. The other components of the postsplicing marker complex bind one or more of these proteins and are transported out of the nucleus with the mature mRNA.

According to recently emerging models (14, 17), the formation of this marker complex represents a key step in NMD, because it links the intranuclear process of splicing to translation, a predominantly (if not exclusively: see below) cytoplasmic event. The protein product of the hUPF3 gene binds to Y14 protein in the nucleus on the spliced mRNA. The other two UPF gene products, hUPF2p and hUPF1p, are thought to reside at the periphery of the nucleus and in the cytoplasm, respectively (18). The hUPF2p appears to provide a bridge between hUPF3p and hUPF1p in the complex. The hUPF1p protein appears to be one of the key structural and functional elements to nonsense-containing mRNA degradation, providing links between the exon-exon boundary marks, the translation termination complex, and the mRNA cap complex (19).

In the cytoplasm, these proteins are stripped from the mRNA with the first passage of a ribosome during translation. If, however, translation terminates at a PTC, hUPF1p present at the proximal exon-exon boundary can interact with proteins in the translational termination complex and can then interact with DCP2p, thereby activating the decapping protein DCP1p. Once the cap is removed, the mRNA is rapidly degraded by the action of constitutively active intracellular 5′→3′ exonucleases. Because these complexes do not bind 3′ to the constitutive termination codon in mammalian mRNAs, those mRNA species that lack a PTC are protected from NMD.

To date, only a small number of mRNAs have been examined for binding of complexes, and it seems likely that NMD pathways for various mRNA species will prove idiosyncratic. For instance, special arrangements may be needed for mRNA quality control in the small number of genes that contain introns within their 3′ untranslated regions. Moreover, given the sheer number of splicing events that occur in the nucleus, it seems too much to ask that every exon-exon boundary be marked with a splicing complex. There is, as yet, no evidence for an upper limit to the distance between the splicing complex and the upstream PTC, so the system could probably operate efficiently as long as some exon-exon boundaries near the 3′ end of the gene receive a mark. Finally, selection for efficient NMD may have varied between different gene products, and there may be some for which a degree of variation at the C-terminal end of the protein is permissible or even advantageous. The extent to which cells tolerate the accumulation of a given PTC-bearing mRNA varies greatly and cannot be predicted in advance, a point to which I return later.