Preservation of forelimb function by UPF1 gene therapy in a rat model of TDP-43-induced motor paralysis.

Nonsense-mediated mRNA decay (NMD) is an RNA surveillance mechanism that requires upframeshift protein 1 (UPF1). This study demonstrates that human UPF1 exerts protective effects in a rat paralysis model based on the amyotrophic lateral sclerosis (ALS)-associated protein, TDP-43 (transactive response DNA-binding protein 43 kDa). An adeno-associated virus vector (AAV9) was used to express TDP-43 throughout the spinal cord of rats, inducing reproducible limb paralysis, to recapitulate the paralysis in ALS. We selected UPF1 for therapeutic testing based on a genetic screen in yeast. The expression of human TDP-43 or human UPF1 in the spinal cord was titrated to less than twofold over the respective endogenous level. AAV9 human mycUPF1 clearly improved overall motor scores in rats also expressing TDP-43. The gene therapy effect of mycUPF1 was specific and reproducible compared with groups receiving either empty vector or green fluorescent protein vector controls. The gene therapy maintained forelimb motor function in rats that would otherwise become quadriplegic. This work helps validate UPF1 as a novel therapeutic for ALS and other TDP-43-related diseases and may implicate UPF1 and NMD involvement in the underlying disease mechanisms.

CBP80-promoted mRNP rearrangements during the pioneer round of translation, nonsense-mediated mRNA decay, and thereafter.

In mammalian cells, two different messenger ribonucleoproteins (mRNPs) serve as templates for protein synthesis. Newly synthesized mRNPs bound by the cap-binding protein heterodimer CBP80-CBP20 (CBC) initially undergo a pioneer round of translation. One purpose of this round of translation is to ensure the quality of gene expression, as exemplified by nonsense-mediated messenger RNA (mRNA) decay (NMD). NMD largely functions to eliminate mRNAs that prematurely terminate translation, although NMD also contributes to proper gene control, and it targets CBC-bound mRNPs. CBC-bound mRNPs are remodeled to eukaryotic translation initiation factor (eIF)4E-bound mRNPs in steps that (1) are a consequence of the pioneer round of translation and (2) occur independently of translation. Rather than supporting NMD, eIF4E-bound mRNPs provide for the bulk of cellular protein synthesis and are the primary targets of mRNA decay mechanisms that conditionally regulate gene expression. Here, we overview cellular processes by which CBC-bound mRNPs are remodeled to eIF4E-bound mRNPs. We also describe the molecular movements of certain factors during NMD in view of the influential role of CBP80.

Evidence that selenium deficiency results in the cytoplasmic decay of GPx1 mRNA dependent on pre-mRNA splicing proteins bound to the mRNA exon-exon junction.

Studies of transcripts for the selenoprotein glutathione peroxidase 1 (GPx1) have provided remarkable evidence for the coupling of pre-mRNA splicing in the nucleus and mRNA translation in the cytoplasm. Such evidence derives from the initial finding that GPx1 mRNA is a natural substrate of nonsense-mediated decay. Here, recent work on GPx1 RNA metabolism is reviewed and future directions of study are defined.

Evidence for a pioneer round of mRNA translation: mRNAs subject to nonsense-mediated decay in mammalian cells are bound by CBP80 and CBP20.

Nonsense-mediated decay (NMD) eliminates mRNAs that prematurely terminate translation. We used antibody to the nuclear cap binding protein CBP80 or its cytoplasmic counterpart eIF4E to immunopurify RNP containing nonsense-free or nonsense-containing transcripts. Data indicate that NMD takes place in association with CBP80. We defined other components of NMD-susceptible mRNP as CBP20, PABP2, eIF4G, and the NMD factors Upf2 and Upf3. Consistent with the dependence of NMD on translation, the NMD of CBP80-bound mRNA is blocked by cycloheximide or suppressor tRNA. These findings provide evidence that translation can take place in association with CBP80. They also indicate that CBP80-bound mRNA undergoes a "pioneer" round of translation, before CBP80-CBP20 are replaced by eIF4E, and Upf2 and Upf3 proteins dissociate from upstream of exon-exon junctions.

Mammalian heat shock p70 and histone H4 transcripts, which derive from naturally intronless genes, are immune to nonsense-mediated decay.

Nonsense-mediated decay (NMD), also called mRNA surveillance, is an evolutionarily conserved pathway that degrades mRNAs that prematurely terminate translation. To date, the pathway in mammalian cells has been shown to depend on the presence of a cis-acting destabilizing element that usually consists of an exon-exon junction generated by the process of pre-mRNA splicing. Whether or not mRNAs that derive from naturally intronless genes, that is, mRNAs not formed by the process of splicing, are also subject to NMD has yet to be investigated. The possibility of NMD is certainly reasonable considering that mRNAs of Saccharomyces cerevisiae are subject to NMD even though most derive from naturally intronless genes. In fact, mRNAs of S. cerevisiae generally harbor a loosely defined splicing-independent destabilizing element that has been proposed to function in NMD analogously to the spliced exon-exon junction of mammalian mRNAs. Here, we demonstrate that nonsense codons introduced into naturally intronless genes encoding mouse heat shock protein 70 or human histone H4 fail to elicit NMD. Failure is most likely because each mRNA lacks a cis-acting destabilizing element, because insertion of a spliceable intron a sufficient distance downstream of a nonsense codon within either gene is sufficient to elicit NMD.

Cloning of a novel phosphatidylinositol kinase-related kinase: characterization of the human SMG-1 RNA surveillance protein.

We have cloned and characterized a new member of the phosphatidylinositol kinase (PIK)-related kinase family. This gene, which we term human SMG-1 (hSMG-1), is orthologous to Caenorhabditis elegans SMG-1, a protein that functions in nonsense-mediated mRNA decay (NMD). cDNA sequencing revealed that hSMG-1 encodes a protein of 3031 amino acids containing a conserved kinase domain, a C-terminal domain unique to the PIK-related kinases and an FKBP12-rapamycin binding-like domain similar to that found in the PIK-related kinase mTOR. Immunopurified FLAG-tagged hSMG-1 exhibits protein kinase activity as measured by autophosphorylation and phosphorylation of the generic PIK-related kinase substrate PHAS-1. hSMG-1 kinase activity is inhibited by high nanomolar concentrations of wortmannin (IC(50) = 105 nm) but is not inhibited by a FKBP12-rapamycin complex. Mutation of conserved residues within the kinase domain of hSMG-1 abolishes both autophosphorylation and substrate phosphorylation, demonstrating that hSMG-1 exhibits intrinsic protein kinase activity. hSMG-1 phosphorylates purified hUpf1 protein, a phosphoprotein that plays a critical role in NMD, at sites that are also phosphorylated in whole cells. Based on these data, we conclude that hSMG-1 is the human orthologue to C. elegans SMG-1. Our data indicate that hSMG-1 may function in NMD by directly phosphorylating hUpf1 protein at physiologically relevant sites.

Nonsense-mediated decay of mRNA for the selenoprotein phospholipid hydroperoxide glutathione peroxidase is detectable in cultured cells but masked or inhibited in rat tissues.

Previous studies of mRNA for classical glutathione peroxidase 1 (GPx1) demonstrated that hepatocytes of rats fed a selenium-deficient diet have less cytoplasmic GPx1 mRNA than hepatocytes of rats fed a selenium-adequate diet. This is because GPx1 mRNA is degraded by the surveillance pathway called nonsense-mediated mRNA decay (NMD) when the selenocysteine codon is recognized as nonsense. Here, we examine the mechanism by which the abundance of phospholipid hydroperoxide glutathione peroxidase (PHGPx) mRNA, another selenocysteine-encoding mRNA, fails to decrease in the hepatocytes and testicular cells of rats fed a selenium-deficient diet. We demonstrate with cultured NIH3T3 fibroblasts or H35 hepatocytes transiently transfected with PHGPx gene variants under selenium-supplemented or selenium-deficient conditions that PHGPx mRNA is, in fact, a substrate for NMD when the selenocysteine codon is recognized as nonsense. We also demonstrate that the endogenous PHGPx mRNA of untransfected H35 cells is subject to NMD. The failure of previous reports to detect the NMD of PHGPx mRNA in cultured cells is likely attributable to the expression of PHGPx cDNA rather than the PHGPx gene. We conclude that 1) the sequence of the PHGPx gene is adequate to support the NMD of product mRNA, and 2) there is a mechanism in liver and testis but not cultured fibroblasts and hepatocytes that precludes or masks the NMD of PHGPx mRNA.

Evidence that phosphorylation of human Upfl protein varies with intracellular location and is mediated by a wortmannin-sensitive and rapamycin-sensitive PI 3-kinase-related kinase signaling pathway.

Human Upf1 protein (p), a group 1 RNA helicase, has recently been shown to function in nonsense-mediated mRNA decay (NMD) in mammalian cells. Here, we demonstrate that the estimated 3 x 10(6) copies of hUpf1 p per exponentially growing HeLa cell are essentially equally distributed among polysomal, subpolysomal, and ribosome-free fractions. We also demonstrate that hUpf1p binds RNA and is a phosphoprotein harboring phosphoserine and phosphothreonine. hUpf1p is phosphorylated to the highest extent when polysome-associated and to the lowest extent when ribosome free. We find that serum-induced phosphorylation of hUpf1p is inhibited by wortmannin at a concentration that selectively inhibits PI 3-kinase related kinases and, to a lesser extent, by rapamycin. These and other data suggest that phosphorylation is mediated by a wortmannin-sensitive and rapamycin-sensitive PI 3-kinase-related kinase signaling pathway. Comparisons are made of hUpf1p to Upf1p and SMG-2, which are the orthologs to hUpf1p in Saccharomyces cerevisiae and Caenorhabditis elegans, respectively.

Identification and characterization of human orthologues to Saccharomyces cerevisiae Upf2 protein and Upf3 protein (Caenorhabditis elegans SMG-4).

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 spliceosome deposits multiple proteins 20-24 nucleotides upstream of mRNA exon-exon junctions.

Eukaryotic mRNAs exist in vivo as ribonucleoprotein particles (mRNPs). The protein components of mRNPs have important functions in mRNA metabolism, including effects on subcellular localization, translational efficiency and mRNA half-life. There is accumulating evidence that pre-mRNA splicing can alter mRNP structure and thereby affect downstream mRNA metabolism. Here, we report that the spliceosome stably deposits several proteins on mRNAs, probably as a single complex of approximately 335 kDa. This complex protects 8 nucleotides of mRNA from complete RNase digestion at a conserved position 20-24 nucleotides upstream of exon-exon junctions. Splicing-dependent RNase protection of this region was observed in both HeLa cell nuclear extracts and Xenopus laevis oocyte nuclei. Immunoprecipitations revealed that five components of the complex are the splicing-associated factors SRm160, DEK and RNPS1, the mRNA-associated shuttling protein Y14 and the mRNA export factor REF. Possible functions for this complex in nucleocytoplasmic transport of spliced mRNA, as well as the nonsense-mediated mRNA decay pathway, are discussed.

Nonsense-mediated decay of glutathione peroxidase 1 mRNA in the cytoplasm depends on intron position.

mRNA for glutathione peroxidase 1 (GPx1) is subject to cytoplasmic nonsense-mediated decay (NMD) when the UGA selenocysteine (Sec) codon is recognized as nonsense. Here, we demonstrate by moving the sole intron of the GPx1 gene that either the Sec codon or a TAA codon in its place elicits NMD when located >/=59 bp but not

Pre-mRNA splicing alters mRNP composition: evidence for stable association of proteins at exon-exon junctions.

We provide direct evidence that pre-mRNA splicing alters mRNP protein composition. Using a novel in vitro cross-linking approach, we detected several proteins that associate with mRNA exon-exon junctions only as a consequence of splicing. Immunoprecipitation experiments suggested that these proteins are part of a tight complex around the junction. Two were identified as SRm160, a nuclear matrix-associated splicing coactivator, and hPrp8p, a core component of U5 snRNP and spliceosomes. Glycerol gradient fractionation showed that a subset of these proteins remain associated with mRNA after its release from the spliceosome. These results demonstrate that the spliceosome can leave behind signature proteins at exon-exon junctions. Such proteins could influence downstream metabolic events in vivo such as mRNA transport, translation, and nonsense-mediated decay.

Identical germ-line mutations in the triosephosphate isomerase alleles of two brothers are associated with distinct clinical phenotypes.

We describe here a new stop mutation at triosephosphate isomerase (TPI) position 145 in a Hungarian family for which the first mutation (240 Phe-->Leu) was published earlier. The entire genomic TPI locus (exons, introns and promoter) was sequenced and found to be identical in the two compound-heterozygote brothers. Both brothers have the same well-compensated level of non-spherocytic hemolytic anemia and very high levels of the TPI substrate dihydroxyacetonephosphate (DHAP), but only one brother manifests neurologic disorders. Differences in nonsense-mediated mRNA decay may be at the basis of the differences in phenotype expression although it cannot be excluded the interaction with a modifier gene. Based on our earlier results, the development of neurodegeneration may be decisively modulated by the cellular environment of the mutant proteins initiating the process of focal apoptosis of neurons in glycolytic, peroxisomal and prion-induced neurological diseases.

A mutated human homologue to yeast Upf1 protein has a dominant-negative effect on the decay of nonsense-containing mRNAs in mammalian cells.

All eukaryotic cells analyzed have developed mechanisms to eliminate the production of mRNAs that prematurely terminate translation. The mechanisms are thought to exist to protect cells from the deleterious effects of in-frame nonsense codons that are generated by routine inefficiencies and inaccuracies in RNA metabolism such as pre-mRNA splicing. Depending on the particular mRNA and how it is produced, nonsense codons can mediate a reduction in mRNA abundance either (i) before its release from an association with nuclei into the cytoplasm, presumably but not certainly while the mRNA is being exported to the cytoplasm and translated by cytoplasmic ribosomes, or (ii) in the cytoplasm. Here, we provide evidence for a factor that functions to eliminate the production of nonsense-containing RNAs in mammalian cells. The factor, variously referred to as Rent1 (regulator of nonsense transcripts) or HUPF1 (human Upf1 protein), was identified by isolating cDNA for a human homologue to Saccharomyces cerevisiae Upf1p, which is a group I RNA helicase that functions in the nonsense-mediated decay of mRNA in yeast. Using monkey COS cells and human HeLa cells, we demonstrate that expression of human Upf1 protein harboring an arginine-to-cysteine mutation at residue 844 within the RNA helicase domain acts in a dominant-negative fashion to abrogate the decay of nonsense-containing mRNA that takes place (i) in association with nuclei or (ii) in the cytoplasm. These findings provide evidence that nonsense-mediated mRNA decay is related mechanistically in yeast and in mammalian cells, regardless of the cellular site of decay.

At least one intron is required for the nonsense-mediated decay of triosephosphate isomerase mRNA: a possible link between nuclear splicing and cytoplasmic translation.

Mammalian cells have established mechanisms to reduce the abundance of mRNAs that harbor a nonsense codon and prematurely terminate translation. In the case of the human triosephosphate isomerase (TPI gene), nonsense codons located less than 50 to 55 bp upstream of intron 6, the 3'-most intron, fail to mediate mRNA decay. With the aim of understanding the feature(s) of TPI intron 6 that confer function in positioning the boundary between nonsense codons that do and do not mediate decay, the effects of deleting or duplicating introns have been assessed. The results demonstrate that TPI intron 6 functions to position the boundary because it is the 3'-most intron. Since decay takes place after pre-mRNA splicing, it is conceivable that removal of the 3'-most intron from pre-mRNA "marks" the 3'-most exon-exon junction of product mRNA so that only nonsense codons located more than 50 to 55 nucleotides upstream of the "mark" mediate mRNA decay. Decay may be elicited by the failure of translating ribosomes to translate sufficiently close to the mark or, more likely, the scanning or looping out of some component(s) of the translation termination complex to the mark. In support of scanning, a nonsense codon does not elicit decay if some of the introns that normally reside downstream of the nonsense codon are deleted so the nonsense codon is located (i) too far away from a downstream intron, suggesting that all exon-exon junctions may be marked, and (ii) too far away from a downstream failsafe sequence that appears to function on behalf of intron 6, i.e., when intron 6 fails to leave a mark. Notably, the proposed scanning complex may have a greater unwinding capability than the complex that scans for a translation initiation codon since a hairpin structure strong enough to block translation initiation when inserted into the 5' untranslated region does not block nonsense-mediated decay when inserted into exon 6 between a nonsense codon residing in exon 6 and intron 6.