The biogenesis of functionally mature mRNAs in mammalian cells is remarkably involved and inherently subject to inefficiencies and inaccuracies that result in the generation of abnormal translational reading frames. Mammalian mRNAs are transcribed initially as precursors, most of which contain multiple introns that must be removed by the process of pre-mRNA splicing. If transcription initiates incorrectly or an intron either fails to be removed or is removed using one or more abnormal splice sites, then product mRNA has the potential to harbor a premature termination codon (PTC) that could derive from an upstream reading frame, a retained intron, or a shift in the reading frame.
In order to cope with the generation of PTCs and their potential to result in deleterious proteins that function in new or dominant-negative ways, mammalian cells have evolved a pathway called nonsense-mediated mRNA decay (NMD) or mRNA surveillance (reviewed in references 20
, and 32
). This pathway surveys all translated mRNAs, whether they be normal or defective, in order to degrade those that prematurely terminate translation more than 50 to 55 nucleotides (nt) upstream of the final exon-exon junction (7
)—a feature of most PTCs but not most normal termination codons (34
). These and other data indicate that NMD is mechanistically linked to nuclear pre-mRNA splicing.
Depending on the particular mRNA and its conditions of expression, NMD can take place in association with nuclei or after export to the cytoplasm. One unresolved issue of NMD pertains to the precise cellular site of nucleus-associated NMD that, like cytoplasmic NMD, requires a process that is experimentally indistinguishable from cytoplasmic translation. In theory, nucleus-associated NMD could take place either during mRNA transport from the nucleus to the cytoplasm and depend on cytoplasmic ribosomes or within the nucleoplasm and depend on nuclear ribosomes (8
). The link between splicing and NMD exists for both nucleus-associated (7
) and cytoplasmic (41
) NMD. We have proposed that the link involves proteins that are deposited by the process of splicing at or near exon-exon junctions of product mRNA and remain bound to mRNA long enough to interact with translational factors. Recent studies using HeLa cell nuclear extracts and cross-linking in vitro have identified several proteins, including the nuclear matrix-associated splicing coactivator SRm160, that form a tight complex at or near exon-exon junctions as a direct consequence of splicing and remain associated with mRNA after its release from the spliceosome (26
). Thus, evidence now exists that pre-mRNA splicing can influence mRNp structure. Another unresolved issue of NMD pertains to how the translational apparatus interacts with splicing-marked mRNA in a way that elicits NMD.
NMD typifies not only mammalian cells but all cells that have been examined. trans
-acting factors known to be required for NMD are best understood for Saccharomyces cerevisiae
and Caenorhabditis elegans
, which are readily amenable to genetic analyses. Loss-of-function mutations affecting the S. cerevisiae
Upf1 protein (p) (also known as Nam7p, Sal1p, Ifs2p, or Mof4p), Upf2p (also known as Nmd2p, Isf1p, or Sua1p), Upf3p (also known as Sua6p), or any one of SMG-1 through SMG-7 of C. elegans
eliminate NMD without general effects on the decay of mRNAs lacking PTCs (4
). In yeast, polysome-associated mRNAs are substrates for NMD (50
). Consistent with this, all three Upf proteins associate with ribosomes (3
) and Upf1p binds release factors (RFs) 1 and 3 to enhance translation termination and elicit NMD (11
). Upf1p also forms a complex with Dcp2p (also known as Nmd1p [11
]), a protein required for the mRNA decapping step of NMD (13
). In fact, all three Upf proteins appear to function in translation termination and monitor translational fidelity since they interact (18
), deleting any single UPF gene results in a nonsense suppression phenotype (9
), and the mof4-1
allele of the UPF1 gene as well as an upf3
-Δ strain demonstrate increased programmed −1 frameshifting (10
). The C. elegans
orthologue to S. cerevisiae
Upf1p is the phosphoprotein SMG-2 (35
). SMG-4 appears to be the C. elegans
orthologue to S. cerevisiae
Upf3p and derives from alternatively spliced RNA (R. Aronoff, R. Baran, and J. Hodgkin, unpublished data). SMG-3 appears to be the C. elegans
orthologue to S. cerevisiae
Upf2p (S. Kuchma and P. Anderson, personal communication).
Until now, the only Upf-like or SMG-like factor identified for mammalian cells has been human (h) Upf1p (hUpf1p) (1
). hUpf1p is required for NMD in mammalian cells, as evidenced by the finding that a cysteine in place of an arginine at position 844 (R844C) within the RNA helicase domain has a dominant-negative effect on the pathway (42
). Despite sequence and, by extrapolation, functional similarities among S. cerevisiae
Upf1p, C. elegans
SMG-2, and hUpf1p indicating that NMD evolved before most eukaryotes (1
), there may be significant differences among the three organisms in the factors that elicit NMD. First, yeast Upf1p has never been reported to be phosphorylated, in contrast to both SMG-2 (35
) and hUpf1p (M. Pal, Y. Ishigaki, E. Nagy, and L. E. Maquat, unpublished data), although data demonstrating that epitope-tagged Upf1p can migrate as a doublet in acrylamide (4
) suggest that it may be posttranslationally modified. Second, expression of either yeast Upf1p in SMG-2
mutant worms or hUpf1p in upf1
mutant yeast fails to restore NMD (35
). Third, even a hybrid hUpf1p flanked by the extreme N and C termini of yeast Upf1p, which is capable of binding RFs 1 and 3 and functioning in nonsense suppression in yeast, fails to function in NMD in yeast (11
). Finally, four of the seven SMG factors are without known orthologues in either yeast or humans.
Here, we identify and describe human orthologues to S. cerevisae Upf2p and S. cerevisae Upf3p (C. elegans SMG-4). Using comparative genomics and rapid amplification of cDNA ends (RACE), the results of cDNA analyses indicate that there is a single human orthologue to S. cerevisae Upf2p, which we have called hUpf2p. In contrast, there are multiple human orthologues to S. cerevisae Upf3p (C. elegans SMG-4) that derive from two separate genes, one of which is X-linked and both of which produce alternatively spliced RNAs. The full-length versions are called hUpf3p-X and hUpf3p. Immunoprecipitations of epitope-tagged proteins transiently produced in HeLa cells demonstrate that hUpf1p, hUpf2p, hUpf3p-X, and hUpf3p copurify, providing evidence for a role in NMD. Indirect immunofluorescence assays of protein localization in HeLa cells reveal that hUpf1p is detected exclusively in the cytoplasm, hUpf2p is detected primarily in the cytoplasm, and hUpf3p-X is mostly nuclear. Results of protein shuttling in interspecies heterokaryons indicate that hUpf3p-X shuttles rapidly between nuclei and the cytoplasm. Apparent similarities and differences of these proteins in S. cerevisiae, C. elegans, and humans are discussed.