nonsense - mediated mrna decay ( nmd ) is a specific rna - degradation process by which eukaryotic cells can eliminate mrnas that contain premature translation - termination codons ( ptcs ) . such mrnas encode truncated polypeptides that could , when translated , exert dominant - negative effects ; it is therefore advantageous to cells to minimize translation of these mrnas . most of the mutations that introduce a ptc into the human -globin mrna cause a form of -thalassemia that is recessive ; heterozygous carriers of these mutations are thought to be protected by degradation of the mutant mrnas via nmd . a mutation that escapes nmd similarly , certain ptcs in the human ror2 mrna , which encodes an orphan receptor tyrosine kinase , fail to trigger nmd and cause a dominantly inherited form of brachydactyly type b . finally , in b and t lymphocytes , the somatic mutation and recombination events of the immune system frequently lead to the production of ptc - containing mrnas . the nmd pathway is thought to prevent the expression of truncated immunoglobulin and t - cell - receptor polypeptides that would arise from such aberrant mrnas . three proteins have been shown to be directly and specifically involved in nmd in saccharomyces cerevisiae : upf1p / nam7p , upf2p / nmd2p and upf3p ( reviewed in ) . functional homologs of upf1p , upf2p and upf3p have been described in humans and in other organisms , and the three upf proteins can interact with each other . human upf1p ( hupf1p ) is cytoplasmic and associated with polysomes ; most hupf2p localizes to the nuclear periphery ; and the two human upf3p homologs ( encoded by two genes , hupf3a and hupf3b ) are shuttling proteins that are found predominantly in the nucleus . the interaction of upf1p with the translation - termination factors erf1 and erf3   links nmd biochemically to the termination of translation . a central question about the mechanism of nmd is how the cell distinguishes a premature from a normal stop codon ( figure 1 ) . remarkably , the position of introns seems to play a critical role in metazoans : a ptc is functionally defined as an in - frame stop codon that is located at least 50 nucleotides upstream of the last exon - exon junction in a spliced mrna ( see figure 1 ; reviewed in ) . consequently , naturally intronless ptc - containing mrnas are not usually destroyed by nmd , and transfection of ptc - containing cdnas of human  globin yields ' nmd immune ' mrnas . furthermore , insertion of an intron more than 50 nucleotides downstream of a normal stop codon triggers nmd of an mrna that in fact has an intact open reading frame . as might be expected , it is unusual to find an intron in the 3 ' untranslated region of an mrna . when such introns do occur , they are almost always positioned less than 50 nucleotides downstream of the stop codon . these observations collectively support a model in which splicing deposits a ' mark ' at or near exon - exon junctions and thus tags the position of excised introns for later ' inspection ' by the translation machinery . in yeast , in which only few genes have introns , a downstream sequence element seems to serve as such a positional mark . when the mrna first passes through the ribosome , nmd is thought to be triggered when the mark is encountered downstream from the position of translation termination   ( figure 1 ) . recent work has identified a multi - subunit complex that could serve as a mark of intron removal ( figure 2 ) . this exon - junction complex ( ejc ) consists of at least five proteins : the splicing - associated factors srm160 , dek and rnps1 , the mrna export factor ref / aly and the mrna - shuttling protein y14 . it is deposited approximately 20 nucleotides upstream of exon - exon junctions   and at least part of it is exported together with the spliced rna to the cytoplasm ( see below ) . using a shortened -globin mrna with a first exon of either 38 or 17 nucleotides ( ' /38 ' or ' /17 ' , respectively ) as a reporter , le hir et al .   showed that the /38 mrna but not the /17 bound the ejc after splicing . deposition of the ejc onto the mrna was associated with a substantial increase in rna export from the nucleus . this increase can be explained by the ability of the ejc to interact with the export factors ref / aly and tap / p15 , which in turn interact with the nuclear pore complex ( reviewed in ) . as the ejc travels from the nucleus to the cytoplasm , its composition changes ( figure 2 ) : only antibodies against srm160 ( weakly ) and y14 ( strongly ) co - immunoprecipitated spliced /38 mrna from the cytoplasm , suggesting that dek , rnps1 and ref / aly dissociate from the ejc before or soon after export . although it can not yet be determined conclusively whether the ejc has a role in nmd , there are some strong clues that it might . as mentioned above , the y14 and srm160 proteins escort the spliced mrna into the cytoplasm . furthermore , recent reports implicate the ejc component rnps1 in nmd   and show an association between the ejc and the nmd factors hupf3 and hupf2 . used a tethering approach to examine the role of the different components of the ejc in nmd . using as a reporter gene a human -globin mrna with binding sites for the bacteriophage ms2 coat protein in its 3 ' untranslated region ( utr ) , they examined whether ejc proteins fused to the ms2 coat protein could trigger nmd when the ms2 binding sites were located at an appropriate distance ( over 50 nucleotides ) downstream of the stop codon . like any of the hupf proteins , when tethered in this manner rnps1 , and to a lesser extent also y14 , induced nmd of the reporter -globin mrna in transfected hela cells ( with rnps1 , 22 - 34% of the rna remained compared with control rna without binding sites ; with y14 , 56 - 72% remained ) . importantly , this effect could be partially inhibited by a dominant - negative mutant of hupf1 . rnps1 is known to shuttle between the nucleus and the cytoplasm of hek293 human embryonic kidney cells , and thus could contribute to nmd in both subcellular locations . when the ejc components were tagged individually , only rnps1 could co - immunoprecipitate hupf1 , hupf2 and hupf3 from lysates of transfected hek293 cells ; this result suggests that rnps1 is the component of the ejc that interacts with the nmd factors . on the other hand , kim et al .   found using a tag - specific antibody that tagged hupf3a or hupf3b could co - immunoprecipitate y14 . although these experiments suggest a direct interaction between y14 and hupf3a / b , the possibility that proteins ( such as rnps1 ) present in the in vitro translation reactions could mediate the interaction can not be excluded . in support of the hypothesis that hupf3 is a bona fide component of the ejc at post - splicing stages of mrna maturation , kim et al . showed that flag - tagged hupf3b stably associated with spliced mrna at a position similar to that of the ejc . taken together , the experiments of lykke - andersen et al .   and kim et al provide biochemical support for the idea that the ejc subunits rnps1 and/or y14 are involved in nmd by providing a bridge between the splicing ' mark ' and the upf proteins ( figure 2 ) , which in turn are linked to translation termination . for nmd to protect the cell from aberrant proteins , it is crucial to destroy ptc - containing mrnas as soon as possible and before multiple truncated polypeptides have been translated . could this destruction happen as early as when the mrna is still in the nucleus ? although the possibility of ' nuclear translation ' has recently received renewed attention , cytoplasmic translation appears to be required for nmd ; the first round of translation , that is , the first time that the mrna passes through the ribosome , is considered to be particularly important . what distinguishes the initial round from subsequent rounds of translation ? conceivably , nmd could take place while the mrna is being exported from the nucleus . during this phase , nuclear factors bound to the mrna could be removed and potentially replaced by cytosolic proteins . is this initial round of translation initiated by the canonical translation initiation factors or does it involve any special translation initiation factors ? in the nucleus , the mgpppn cap structure of mrnas is bound by a heterodimeric cap - binding complex ( cbc , consisting of cbp20 and cbp80 ) , which can affect splicing and 3 ' end processing . it appears that mrnas are exported with their 5 ' ends first , exposing the cbc to the cytoplasm early in the transport process . the initiation factor eif4e binds the cap structure in the cytoplasm , displacing the cbc , and mediates translation by recruiting eif4 g , other translation factors and ultimately the ribosome . this raises the question of whether the cbc and the cytoplasmic eif4e exchange before , during or after the initial round of translation ( figure 2 ) . notably , both yeast and mammalian cbcs can , like eif4e , interact with eif4 g ( via their cbp80 subunits ) , and the yeast cbc has been shown to be able to support cell - free translation , albeit quite inefficiently . recently , ishigaki et al .   proposed that a " pioneer round of translation " may be initiated while the mrna is still associated with the cbc , and that this could act to survey the mrna for the presence of ptcs . two versions of a human -globin mrna served as a reporter system for the experiments of ishigaki et al . : a -globin mrna with a ptc ( ' -globin ptc ' ) and one without ( ' -globin normal ' ) . following immunoprecipitation with antibodies against cbp80 , the level of co - immunoprecipitated -globin ptc rna was reduced to 20% of the level of -globin normal rna . this reduction quantitatively reflects nmd of the -globin ptc rna in the total cellular rna fraction , and corresponds to the level of reduction in the -globin ptc rna ( 15% ) that was precipitatable with antibodies to eif4e . thus , the authors suggest that surveillance and nmd of -globin ptc take place while the mrna is associated with cbp80 and before the remaining intact rna is handed over to eif4e . .   also showed that hupf2 , hupf3 ( but not hupf1 ) and the nuclear poly(a)-binding protein ( pabp2 ) co - immunoprecipitated with anti - cbp80 antibodies in an rna - dependent manner , whereas complexes precipitated with the anti - eif4e antibody did not contain any of these proteins . given that hupf2 and hupf3 are involved in nmd , these results can be interpreted to suggest that nmd occurs while the mrna is still bound by cbp80 , but not detectably when the rna is bound to eif4e . do the majority of mrnas undergo the initial round of translation without the support of eif4e , which was thought to be a very important translation initiation factor ? this idea offers a very interesting and unexpected twist to our perception of the translation and nmd pathway , and will surely stimulate many interesting future experiments . before accepting the notion of a ' pioneer round ' of mrna translation under the guidance of cbp80 as a general aspect of mrna export , however , some additional considerations may be warranted . at least in vitro , moreover , a yeast strain with the cbp80 gene deleted does not display any overt translational defects nor any lack of nmd ; cbp80 is thus not essential either for nmd or for translation in yeast . the function of the cbc in mammalian cells may differ from its functions in yeast , however , and/or it may act as a more efficient translation factor in vivo than in vitro . the finding that the amount of -globin ptc mrna associated with cbp80 was reduced to 20% of the level of -globin normal mrna   certainly suggests that translation and nmd can occur before cbc has been exchanged for eif4e . only about 15%-30% of the cbc and eif4e proteins and their associated rnas from the extract was accessible to immunoprecipitation , however , and we therefore do not know about the mrnas associated with the other 70 - 85% of these proteins . comparisons of mrna levels were made between ptc - containing and wild - type rnas each co - immunoprecipitated with either cbp80 or eif4e , but we do not know the percentage of the -globin ptc and -globin normal mrnas present in the cells that was analyzed . thus , we do not yet know to what extent the analyzed mrna - protein complexes ( mrnps ) are representative of the majority of mrnas being exported from the nucleus and being surveyed for nmd . undoubtedly , future experiments will address this question as well as some of the intriguing implications of this work . from a broader perspective , the recent progress highlights the dynamic nature of mrnp remodeling during mrna processing , export , translation and degradation . we are on the way to a better understanding of how the different levels of gene expression are integrated and coordinated , how they affect and influence each other , and what the players and mechanisms are at the biochemical level . ( a ) in the nucleus , a normal intron - containing transcript is subjected to multiple modifications , such as splicing , addition of a mgpppn cap and polyadenylation , to yield a mature mrna . the exon - exon junction complex ( ejc ) is associated with the mrna just upstream of each splice site . ( b ) if an mrna carries a premature termination codon ( ptc ) , however , it can be degraded by the nmd system . note that the physiological stop codon has no ejc positioned downstream of it , whereas nmd - sensitive ptcs are usually followed by at least one ejc ( curved arrows in ( a , b ) ) . the ejc is therefore proposed to play an important role in mrna surveillance . a model of the changes in ptc - containing mrna - protein complexes ( mrnps ) during nuclear export . a ptc - containing transcript , depicted as in figure 1 , concomitantly , the cap - binding complex ( cbc , consisting of cbp80 and cbp20 ) , the exon junction complex ( ejc , which consists of the dek , ref / aly , rnps1 , srm160 and y14 proteins ) , the nmd protein hupf3 and the export factor tap / p15 are all added , to give a fully processed mrnp . before or soon after export from the nucleus through the nuclear pore complex ( npc ) , dek , rnps1 and ref / aly dissociate from the ejc and hupf2 binds . it is not clear whether the first round of translation can be mediated by cbc ( left ) or whether cbc is first exchanged for eif4e . after this first round , the ptc - containing mrna undergoes nonsense - mediated decay .
when pre - mrnas are spliced , a multi - component complex is deposited onto them , close to the sites of intron removal . 
 new findings suggest that these exon - exon junction complexes and the complexes that bind mrna caps are key effectors of the fate of spliced mrnas and may regulate whether mrnas containing premature stop codons are degraded .