Nonsense-mediated mRNA decay (NMD), also called mRNA surveillance, is an important pathway used by all organisms that have been tested to degrade mRNAs that prematurely terminate translation and, as a consequence, eliminate the production of aberrant proteins that could be potentially harmful. In mammalian cells, NMD appears to involve splicing-dependent alterations to mRNA as well as ribosome-associated components of the translational apparatus. To date, human (h) Upf1 protein (p) (hUpf1p), a group 1 RNA helicase named after its Saccharomyces cerevisiae orthologue that functions in both translation termination and NMD, has been the only factor shown to be required for NMD in mammalian cells. Here, we describe human orthologues to S. cerevisiae Upf2p and S. cerevisiae Upf3p (Caenorhabditis elegans SMG-4) based on limited amino acid similarities. The existence of these orthologues provides evidence for a higher degree of evolutionary conservation of NMD than previously appreciated. Interestingly, human orthologues to S. cerevisiae Upf3p (C. elegans SMG-4) derive from two genes, one of which is X-linked and both of which generate multiple isoforms due to alternative pre-mRNA splicing. We demonstrate using immunoprecipitations of epitope-tagged proteins transiently produced in HeLa cells that hUpf2p interacts with hUpf1p, hUpf3p-X, and hUpf3p, and we define the domains required for the interactions. Furthermore, we find by using indirect immunofluorescence that hUpf1p is detected only in the cytoplasm, hUpf2p is detected primarily in the cytoplasm, and hUpf3p-X localizes primarily to nuclei. The finding that hUpf3p-X is a shuttling protein provides additional indication that NMD has both nuclear and cytoplasmic components.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 premRNA 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, 28, 30, 31, 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 (7,8,41,43,44,48,49)-a feature of most PTCs but not most normal termination codons (34). These and other data i...

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Premature translation termination codon (PTC)-mediated effects on nuclear RNA processing have been shown to be associated with a number of human genetic diseases; however, how these PTCs mediate such effects in the nucleus is unclear. A PTC at nucleotide (nt) 2018 that lies adjacent to the 5 element of a bipartite exon splicing enhancer within the NS2-specific exon of minute virus of mice P4 promoter-generated pre-mRNA caused a decrease in the accumulated levels of P4-generated R2 mRNA relative to P4-generated R1 mRNA, although the total accumulated levels of P4 product remained the same. This effect was seen in nuclear RNA and was independent of RNA stability. The 5 and 3 elements of the bipartite NS2-specific exon enhancer are redundant in function, and when the 2018 PTC was combined with a deletion of the 3 enhancer element, the exon was skipped in the majority of the viral P4-generated product. Such exon skipping in response to a PTC, but not a missense mutation at nt 2018, could be suppressed by frame shift mutations in either exon of NS2 which reopened the NS2 open reading frame, as well as by improvement of the upstream intron 3 splice site. These results suggest that a PTC can interfere with the function of an exon splicing enhancer in an open reading frame-dependent manner and that the PTC is recognized in the nucleus.Premature termination codons (PTCs) decrease the accumulated levels of most known mRNAs in which they reside (reviewed in reference 28). In the yeast Saccharomyces cerevisiae, the UPF genes are involved in the degradation of PTCcontaining RNAs in the cytoplasm (review in reference 37). Similarly, the SMG proteins of Caenorhabditis elegans are required for the rapid decay of PTC-containing unc-54 myosin heavy-chain mRNAs (reviewed in reference 41).PTC-mediated effects on nuclear RNA processing have been shown to be associated with a number of human genetic diseases (reviewed in references 28 and 29); however, how PTCs mediate such effects in the nucleus is still unclear. In mammalian cells, PTCs have been shown to decrease the levels of nucleus-associated mRNAs by a posttranscriptional mechanism, often attributed to mRNA decay (3,6,9,28,45). Experiments done with PTCs in the human TPI gene suggest that nonsense-mediated decay (NMD) occurs on a fully spliced nucleus-associated mRNA molecule, possibly during mRNA export to the cytoplasm (4, 5, 9, 28).There is also increasing evidence that PTCs may affect mammalian RNA processing events other than decay. For example, several instances of PTC-associated exon skipping have been described (15,19,28,33). Perhaps the best-studied example is skipping of the 66-nucleotide (nt) exon 51 of fibrillin FBN1 RNA, which was detected when nonsense but not missense mutations were present within this exon, which was independent of protein synthesis, and for which normal splicing was restored when the nonsense codon was shifted out of frame with the initiation codon (14, 23). Retention of introns upstream of PTC-containing exons has also been reported for P4-ge...

The alternatively spliced 290-nucleotide NS2-specific exon of the parvovirus minute virus of mice (MVM), which is flanked by a large intron upstream and a small intron downstream, constitutively appears both in the R1 mRNA as part of a large 5'-terminal exon (where it is translated in open reading frame 3 [ORF3]), and in the R2 mRNA as an internal exon (where it is translated in ORF2). We have identified a novel bipartite exon enhancer element, composed of CA-rich and purine-rich elements within the 5' and 3' regions of the exon, respectively, that is required to include NS2-specific exon sequences in mature spliced mRNA in vivo. These two compositionally different enhancer elements are somewhat redundant in function: either element alone can at least partially support exon inclusion. They are also interchangeable: either element can function at either position. Either a strong 3' splice site upstream (i.e., the exon 5' terminus) or a strong 5' splice site downstream (i.e., the exon 3' terminus) is sufficient to prevent skipping of the NS2-specific exon, and a functional upstream 3' splice site is required for inclusion of the NS2-specific exon as an internal exon into the mature, doubly spliced R2 mRNA. The bipartite enhancer functionally strengthens these termini: the requirement for both the CA-rich and purine-rich elements can be overcome by improvements to the polypyrimidine tract of the upstream intron 3' splice site, and the purine-rich element also supports exon inclusion mediated through the downstream 5' splice sites. In summary, a suboptimal large-intron polypyrimidine tract, sequences within the downstream small intron, and a novel bipartite exonic enhancer operate together to yield the balanced levels of R1 and R2 observed in vivo. We suggest that the unusual bipartite exonic enhancer functions to mediate proper levels of inclusion of the NS2-specific exon in both singly spliced R1 and doubly spliced R2.

We have previously shown that efficient excision of the upstream large intron from P4-generated pre-mRNA of the autonomous parvovirus minute virus of mice depends upon at least the initial presence of sequences within the downstream small intron (Q.

How premature translation termination codons (PTCs) mediate effects on nuclear RNA processing is unclear. Here we show that a PTC at nucleotide (nt) 385 in the NS1/2 shared exon of P4-generated pre-mRNAs of the autonomous parvovirus minute virus of mice caused a decrease in the accumulated levels of doubly spliced R2 relative to singly spliced R1, although the total accumulated levels of R1 plus R2 remained the same. The effect of this PTC was evident within nuclear RNA, was mediated by a PTC and not a missense transversion mutation at this position, and could be suppressed by improvement of the large intron splice sites and by mutation of the AUG that initiated translation of R1 and R2. In contrast to the PTC at nt 385, the reading frame-dependent effect of the PTC at nt 2018 depended neither on the initiating AUG nor the normal termination codon for NS2; however, it could be suppressed by a single nucleotide deletion mutation in the upstream NS1/2 common exon that shifted the 2018 PTC out of the NS2 open reading frame. This suggested that there was recognition and communication of reading frame between exons on a pre-mRNA in the nucleus prior to or concomitant with splicing. Premature termination codons (PTCs)1 have been shown to result in decreased levels of PTC-containing mRNAs in many organisms, and there is an increasing appreciation of the effect of PTCs on RNA levels in mammalian cells. In some of these cases, PTCs have been shown to affect nuclear-associated mRNA abundance by a process termed nonsense-mediated decay, which has been suggested to degrade fully spliced mRNAs in the nucleus, possibly during mRNA export (reviewed in Refs.1 and 2).PTCs have also been implicated in altering nuclear RNA processing events other than decay in mammalian cells, which results in both intron retention and exon skipping, suggesting that PTCs may influence splice site selection (1). In two cases, exon skipping of the 66-nucleotide exon 51 of fibrillin FBN1 RNA (3) and exon skipping and intron retention for the P4-generated pre-mRNAs that generate the nonstructural proteins of the autonomous parvovirus minute virus of mice (MVM) (4, 5), the effects of PTCs on RNA processing have been shown to be reading frame-dependent.MVM is an autonomous parvovirus that is organized into two overlapping transcription units that produce three major classes of RNA (6 -8) (see Fig. 1). Transcripts R1 and R2 are generated form a promoter (P4) at map unit 4 and encode the viral nonstructural proteins NS1 and NS2, respectively, whereas the R3 transcripts are generated from a promoter at map unit 38 (P38) and encode the viral capsid proteins (6, 9). Both NS1 and NS2 play essential roles in viral replication and cytotoxicity (10), and so maintenance of their relative steady state levels, which is controlled at least in part by alternative splicing, is critical to the viral life cycle (Refs. 11-13; reviewed in Ref. 14). All MVM mRNAs generated during infection or following transfection are very stable (15), and no viral proteins are known to p...

ObjectivesTo match responsive neurostimulator (RNS) and polysomnographic data to determine if RNS detections and stimulations correlate with measurements of sleep disordered breathing and continuous glucose measurements (CGM).Materials and methodsIn a patient with an RNS with detection/stimulation leads implanted bi-temporally detection-stimulation counts were matched by time with coinciding polysomnogram and CGM data.ResultsTemporal dispersion of RNS DSC were independent of measures of sleep apnea, hypopnea or glucose.ConclusionHippocampal nighttime responsive neurostimulation therapies did not appear to worsen measures of normal or abnormal sleep.