An unappreciated role for RNA surveillance
© Hillman et al.; licensee BioMed Central Ltd. 2004
Received: 3 November 2003
Accepted: 2 January 2004
Published: 2 February 2004
Nonsense-mediated mRNA decay (NMD) is a eukaryotic mRNA surveillance mechanism that detects and degrades mRNAs with premature termination codons (PTC+ mRNAs). In mammals, a termination codon is recognized as premature if it lies more than about 50 nucleotides upstream of the final intron position. More than a third of reliably inferred alternative splicing events in humans have been shown to result in PTC+ mRNA isoforms. As the mechanistic details of NMD have only recently been elucidated, we hypothesized that many PTC+ isoforms may have been cloned, characterized and deposited in the public databases, even though they would be targeted for degradation in vivo.
We analyzed the human alternative protein isoforms described in the SWISS-PROT database and found that 144 (5.8% of 2,483) isoform sequences amenable to analysis, from 107 (7.9% of 1,363) SWISS-PROT entries, derive from PTC+ mRNA.
For several of the PTC+ isoforms we identified, existing experimental evidence can be reinterpreted and is consistent with the action of NMD to degrade the transcripts. Several genes with mRNA isoforms that we identified as PTC+ - calpain-10, the CDC-like kinases (CLKs) and LARD - show how previous experimental results may be understood in light of NMD.
Alternative pre-mRNA splicing endows genes with the potential to produce a menagerie of protein products. After pre-mRNA is transcribed, a complex system of regulation determines which one of several possible versions of mature mRNA will be produced (reviewed in ). Alternative splicing is particularly important in human gene expression, as it affects half or more of human genes [2, 3]. The diversity-generating capacity of alternative splicing can be staggering: one notable example, the dscam gene of Drosophila melanogaster, is hypothetically capable of producing 38,016 unique alternative isoforms . However, functional roles for most alternative isoforms remain undiscovered.
During pre-mRNA processing, the spliceosome removes intron sequences. As this occurs, a set of proteins called the exon-junction complex is deposited 20-24 nucleotides upstream of the sites of intron removal [8–11]. The components of this complex serve the dual roles of facilitating export of the mature mRNA to the cytoplasm and remembering the gene structure . According to the current model, as a ribosome traverses the mRNA in its first pioneering round of translation, it displaces all exon-junction complexes in its path [13–16]. For normal mRNAs, whose termination codons are on or near the final exon, the ribosome will have displaced all exon-junction complexes. By contrast, if any exon-junction complexes remain when the ribosome reaches the stop codon, a series of interactions ensues that leads to the decapping and degradation of the mRNA. This model explains the basis of the '50 nucleotide rule' for mammalian NMD: if a termination codon is more than about 50 nucleotides upstream of the final exon, it is a PTC and the mRNA that harbors it will be degraded . The mechanisms for NMD differ among yeast , flies , and mammals - and may be different still in other eukaryotes.
Degradation of PTC+ mRNAs is generally thought to occur as a quality-surveillance system -preempting translation of potentially dominant-negative, carboxy-terminal truncated proteins . PTC+ transcripts are aberrantly produced in several ways. The somatic recombination that underlies immune-system diversity frequently generates recombined genes whose transcripts contain a PTC . Inefficient or faulty splicing will often generate a frameshift in the resulting mRNA, inducing a PTC to come into frame. Also, the high processivity of RNA polymerase yields a relatively high error rate, 1 in 10,000 bases [22, 23], commonly introducing premature stops. DNA mutations are a source of potentially heritable PTCs. It is estimated that 30% of inherited disorders in humans are caused by a PTC . The numerous diseases whose pathogenesis has been linked to NMD-inducing PTC mutations include aniridia due to the PAX6 gene , Duchenne muscular dystrophy due to the dystrophin gene , and Marfan syndrome due to the FBN1 gene .
In addition to its quality-control role in degrading aberrantly produced PTC+ mRNAs, NMD has also been shown experimentally to act on a handful of wild-type PTC+ mRNAs [28–35]. In Caenorhabditis elegans, for example, expression of the ribosomal proteins L3, L7a, L10a and L12 and the SR proteins SRp20 and SRp30b are regulated posttranscriptionally via the coupling of alternative splicing and NMD [31, 32]. In each case, productive isoforms were shown to be produced in vivo, as well as unproductive isoforms with a PTC. Regulated splicing to generate the unproductive isoforms is used as a means of downregulating protein expression, as these mRNA isoforms are degraded by NMD rather than translated to make protein. This system, which we have termed regulated unproductive splicing and translation (RUST), is also used in humans [28–30]. For example, the SR protein SC35 has been shown to autoregulate its own expression using RUST . When levels of SC35 protein are elevated, SC35 binds its own pre-mRNA, inducing the production of PTC+ SC35 mRNA. The PTC+ SC35 mRNA is destabilized by NMD, resulting in lower levels of SC35 protein. A similar autoregulatory RUST system was also recently discovered to control production of polypyrimidine tract binding protein (PTB) .
In a previous study, we found that 35% of human mRNA alternative isoforms reliably inferred from expressed sequence tags (ESTs) are PTC+, rendering them apparent targets of NMD (see  and a conference report at . Therefore, many wild-type alternative mRNA isoforms may not be translated into functional protein, but instead are targeted for degradation by NMD. The vast majority of PTC+ isoforms identified in that study represent previously unrecognized potential targets of NMD. However, EST databases contain expressed sequence for many isoforms that are otherwise uncharacterized. Therefore, it was not obvious how many of the isoforms identified in that study as PTC+ were functionally relevant or even previously known. It was also not obvious to what extent those PTC+ isoforms represented instances of RUST regulation or simply errors or deregulation in pre-mRNA processing. Regardless, it is clear that NMD has a vital role in regulating mammalian gene expression, as inhibition of NMD is embryonic lethal for mouse .
To understand the biological significance of PTC+ isoforms and the prevalence of NMD on wild-type transcripts, it is necessary to expand beyond existing isolated RUST examples, while retaining a focus on functionally characterized genes. For this reason, we analyzed the human alternative isoforms described in the SWISS-PROT database. Common routes for gene isoform sequences to be determined and entered into databases include the cloning of intronless mini-genes and the sequencing of unexpected PCR bands. By either method, gene structure cannot be directly observed, and therefore PTCs may be overlooked. Further computational and experimental analyses will also often be oblivious to these features. Because the cloning and characterization of many isoforms predates our current understanding of NMD action, we hypothesized that unrecognized potential targets of NMD may be present even in curated databases like SWISS-PROT. We found that many of these alternative protein isoforms derive from PTC+ mRNAs. This is particularly surprising as SWISS-PROT is a heavily curated database of expressed protein sequences. According to the current NMD model, these PTC+ mRNAs should be degraded, and therefore the protein isoforms should not be expressed at high abundance. To resolve this apparent conflict, we examined existing experimental evidence and found that, in several cases, results described in the scientific literature are readily explained by NMD action.
Results and discussion
SWISS-PROT protein isoforms from PTC+mRNAs
NMD-candidate SWISS-PROT isoforms
SH3BP2; 3BP2; RES4-23
ANTXR1; ATR; TEM8
CFLAR; CLARP; MRIT; CASH
CMKLR1; DEZ; CHEMR23
CUGBP1; BRUNOL2; CUGBP; NAB50
DFFB; DFF2; DFF40; CAD
DNAJB2; HSJ1; HSPF3
EPHA3; ETK1; ETK; HEK
ESR2; NR3A2; ESTRB
USP9Y; USP10; DFFRY
B DELTA S2
GFRA1; GDNFRA; TRNR1; RETL1
IL17RB; IL17BR; EVI27
IL1RL1; ST2; T1; DER4
CHS1; LYST; CHS
MAPK11; PRKM11; SAPK2
ABCC3; CMOAT2; MRP3; MLP2
PPEF1; PPEF; PPP7C
PSEN1; PSNL1; AD3; PS1
ADARB1; RED1; DRADA2
SFRS5; SRP40; HRS
SHOX2; SHOT; OG12X
SON; NREBP; DBP5; C21ORF50; KIAA1019
TNFRSF10B; DR5; TRAILR2; TRICK2; KILLER; ZTNFR9
TNFRSF6; APT1; FAS; FAS1
TNFRSF25; TNFRSF12; WSL1; WSL; APO3; DR3; DDR3
UBCE7IP3; C20ORF18; XAP4
(XE7X; XE7; DXYS155E); (XE7Y; XE7; DXYS155E)
Next we examined existing reports for experimental evidence that would refute or support action of NMD on these PTC+ isoforms. We found that published descriptions of these PTC+ isoforms sometimes do describe the isoforms as containing premature termination codons. However, these articles almost universally lack any mention of NMD, even as they often describe data that is suggestive of NMD action. Among the many well-characterized proteins found in our study to have at least one PTC+ splice variant, three examples demonstrate how previously published experimental results may be interpreted in the light of NMD degradation of alternative mRNA isoforms.
CDC-like kinases CLK1, CLK2 and CLK3
Having observed conservation amongst the human paralogs, we examined the gene structures of the mouse orthologs of each CLK (Figure 5b) to determine if the splicing pattern was shared across species. We identified mouse orthologs through existing RefSeq database annotation. EST evidence of alternative splicing showed that all three mouse CLK orthologs showed the same pattern of alternative splicing, skipping exon 4 to induce a PTC, as seen in the human CLKs. The significance of this evolutionary conservation is underscored by the recent finding that low-abundance alternative exons are 'mostly not conserved' between human and mouse . For the CLK genes, the alternative exons and the introns flanking them are among the most similar regions of these genes (Figure 5b).
We next searched for evidence of more distant conservation of CLK alternative splicing. We identified the single CLK homolog in the sea squirt by using a hidden Markov model of CLKs to search the Ciona intestinalis genome (see Materials and methods). EST evidence clearly indicated that the same alternative splicing pattern seen in human and mouse is also conserved in C. intestinalis. We were not able to observe a set of similar splicing patterns in D. melanogaster (data not shown).
CLK1 has been shown to indirectly affect its own splicing : the presence of high levels of CLK1 protein favors generation of the truncated PTC+ splice variant. However, instead of coding for an inactive, truncated protein isoform, we propose that this PTC+ mRNA isoform may be simply degraded by NMD. Autoregulation of this type would be analogous with that seen for the splicing factors SC35  and PTB . Both SC35 and PTB proteins promote the alternative splicing of PTC+ isoforms of their own mRNAs, which are then degraded by NMD.
Death-domain-containing receptors such as LARD (also known as TNFRSF12, DR3 and Apo3; SWISS-PROT entry Q99831) are known to regulate the balance between lymphocyte proliferation and apoptosis . The term death domain refers to a conserved intracellular region present in receptors such as Fas and tumor necrosis factor receptor 1 (TNFR-1) that is capable of inducing apoptosis when the receptor has bound its ligand (in these cases, Fas ligand and tumor necrosis factor α (TNFα) respectively). The regulation of functional death receptor expression is important in maintaining the balance between lymphocyte proliferation and apoptosis in vivo.
We found that the five truncated LARD mRNA isoforms shown expressed in unstimulated lymphocytes all have PTCs (isoforms 2, 3, 4, 5 and 6). (Note that SWISS-PROT uses a different numbering scheme in which isoforms 2-6 are known as 12, 3, 5, 6 and 7, respectively.) Only the full-length apoptosis-promoting isoform 1, expressed in activated lymphocytes, is free of a PTC. Although there is presently no evidence of transcript degradation, this precise correlation between PTC-containing isoform expression and lymphocyte activation suggests that the role of alternative splicing in regulating lymphocyte apoptosis may be mediated by NMD.
We found that 144 of the human alternative isoforms described in SWISS-PROT derive from mRNAs that contain PTCs. These mRNAs are apparent targets for NMD, and we expect that most are degraded by this system. In many cases, existing experimental evidence is consistent with this expectation. Because our analysis was restricted to only human entries and many SWISS-PROT records could not be reliably analyzed, it is likely that there remain more unidentified putative NMD targets. We are beginning a collaborative project with SWISS-PROT to identify and suitably annotate these entries. The relevance of this effort is highlighted by the many instances in which existing experimental data can be explained in the light of NMD action.
Materials and methods
SWISS-PROT isoform extraction and assembly
We analyzed each of the 1,641 SWISS-PROT v.41 human entries containing a VARSPLIC line in its feature table . Information contained in each VARSPLIC line was used to assemble protein isoform sequences for 4,556 isoforms from 1,636 unique SWISS-PROT entries. Five entries could not be analyzed due to ambiguous VARSPLIC annotation.
Identification of corresponding cDNA/mRNA sequences
Although SWISS-PROT contains cross-references to cDNA/mRNA sequences for major protein isoforms, cross-references do not exist for many alternative isoforms. To find the cDNA/mRNA sequence corresponding to each SWISS-PROT protein isoform, we used BLAST version 2.2.4  to align each protein isoform sequence to translated cDNA/mRNA sequences from all GenBank  and RefSeq cDNA/mRNA sequences in these databases as of 22 March 2003 . In these alignments, we required ≥ 99% identity over the full length of the SWISS-PROT isoform. In cases of multiple matches, we selected 100% identical matches over 99% identical matches and RefSeq matches over GenBank matches. For SWISS-PROT isoforms matching multiple entries from the same database at the same percent identity, the match associated with the longest cDNA/mRNA sequence was chosen. These rules associated 2,871 alternatively spliced human SWISS-PROT protein isoforms from 1,496 SWISS-PROT entries with a corresponding cDNA/mRNA sequence from either RefSeq or Genbank.
Retrieving coding sequences and genomic loci
We used LocusLink  to map each cDNA/mRNA sequence to the correct human genomic contig sequence from the National Center for Biotechnology Information (NCBI) human genome build 30 . The coding sequence (CDS) feature of each GenBank or RefSeq record was used to identify the location of the termination codon. Of the 2,871 alternatively spliced human SWISS-PROT protein isoforms we associated with corresponding cDNA/mRNA sequences, 2,742 had GenBank or RefSeq records that were not polycistronic, allowing us to unambiguously determine the termination codon location for these records. These 2,742 alternative isoforms represented 1,463 unique SWISS-PROT entries.
Assessing NMD candidacy
The SPIDEY mRNA-to-genomic DNA alignment program  was used to determine the location of introns in each cDNA/mRNA alternative isoform sequence. SPIDEY takes as input a cDNA/mRNA sequence and the corresponding genomic sequence, and it generates an alignment that establishes the gene structure. Of the 2,742 alternatively spliced human SWISS-PROT protein isoforms for which both a cDNA/mRNA sequence and stop codon location could be identified, 2,483 resulted in high-confidence SPIDEY alignments, leading us to discard 259 from our analysis. These 2,483 isoform sequences represented 1,363 unique SWISS-PROT entries. We compared the intron positions to the position of the termination codon for each remaining cDNA/mRNA alternative isoform sequence. If the termination codon was found to be more than 50 nucleotides upstream of the final intron, we deemed the transcript to be PTC+ and a candidate target for NMD according to the model of mammalian PTC recognition . One hundred and seventy-seven alternatively spliced human isoforms from 130 SWISS-PROT entries were identified as possible PTC+ splice variants using these criteria. These predictions required further screening, however, to confirm the veracity of the SPIDEY alignments on which they were based.
We manually reviewed all 177 putatively PTC+ alignments and discarded 33 because of demonstrable errors in the SPIDEY alignments. These errors included a variety of malformed intron predictions and poly(A) tails mistakenly annotated as 3' exons. Isoforms that remained following the application of these manual filters were deemed high-confidence PTC+ mRNAs. This was the case for 5.8% of the isoforms (144 of 2,483) from 7.9% of the unique SWISS-PROT entries studied (107 of 1,363). The SPIDEY alignment for each of these is included in Additional data file 1; the sequence identifiers for each step are included in Additional data file 2.
Human CLK1, CLK2 and CLK3 (SWISS-PROT IDs P49759, P49760, and P49761) were among those SWISS-PROT entries we selected for further examination. They were mapped to RefSeq and GenBank entries, as shown in Figure 2. LocusLink was used to associate each CLK gene sequence to its corresponding genomic contig. For each gene, SPIDEY version 1.35 was run twice, using the vertebrate splice-site setting, to align it with its contig sequence and determine its gene structure. This first SPIDEY alignment was used to define the extent of each gene's locus: the region containing all the coding sequence, introns, and 1,000 nucleotides of flanking sequence on each side. The second SPIDEY alignment was made using just this locus. Custom scripts (available from the authors on request), GFF2PS , and manual editing were used to generate the graphical representations of the gene structures shown in Figure 4. Intron and exon sequences were then extracted using the SPIDEY results to delineate exon and intron boundaries. Corresponding exons and introns were globally aligned using ALIGN version 2.0u  with default parameters.
Mouse CLKs were identified using RefSeq annotation (NM_009905 - which skipped exon 4 and had a PTC, NM_007712, and NM_007713). Genomic loci sequences were generated and gene structures determined for each mouse CLK gene using SPIDEY, as above. The loci sequences were then used to search the mouse ESTs from dbEST (1 May 2003)  using WU-BLAST 2.0MP (23 May 2003)  with default parameters. Hits with E-values of 10-30 or better were aligned to the locus sequence using SPIDEY. These alignments were examined for evidence of PTC-inducing alternative splicing. The GI numbers for ESTs that exhibited the alternative splicing pattern shown in Figure 3 for each of the mouse CLKs are: CLK1 (full-length): 25118521, 21852543, 12560958, and others; CLK2 (exon 4 skipping): 22822098; CLK3 (exon 4 skipping): 26079129.
The C. intestinalis CLK homolog was identified from the database of predicted peptides [62, 63] by searching (HMMSEARCH V2.2G)  with a HMMER model of known CLKs. This model was generated using HMMBUILD (default parameters) and calibrated using HMMCALIBRATE from a CLUSTAL W V1.83  alignment of the following CLK sequences: NP_004062, NP_003984, NP_003983, NP_031738, BAB33079, NP_031740, NP_065717, AAH43963, NP_599167, NP_031739, NP_477275, EAA12103, NP_741928, BAB67874, and NP_850695. The most significant hit (E-value: 4.4e-243) from C. intestinalis was ci0100143784. Visual inspection of other, less significant hits revealed that they align with only the kinase domain of the CLK model and none contains the LAMMER motif characteristic of CLKs. A maximum-likelihood tree was generated using PROTML V2.3B3  using ci0100143784 and the three full-length human CLKs. This tree revealed that the C. intestinalis CLK is orthologous to human CLK2. The corresponding cDNA transcript sequence, ci0100143784, was retrieved from the database of predicted transcripts .
As above, the locus for this gene was extracted from the genomic contig sequence, Scaffold18, and used to search the database of C. intestinalis ESTs. The following ESTs showed the full-length pattern with no PTC: 24144377, 24820603, 24627564, 24627468, 24866887, and 2482449. The following ESTs showed the alternatively spliced pattern that generates a PTC: 24888181, 24606693, 24823992, and 24893089.
The C. intestinalis CLK gene was found to have only 11 exons, whereas human and mouse CLK2 have 13. To determine which exons were homologous, we generated a CLUSTALW multiple-sequence alignment of the known CLK protein sequences listed above and C. intestinalis CLK and used this alignment to identify corresponding regions of DNA sequence. This unambiguously indicated the exon-to-exon alignment shown in Figure 4.
Additional data files
We thank Rajiv Bhatnagar, Liana Lareau, Don Rio, Marco Blanchette, Jasper Rine, Karsten Weis and Aaron Garnett for helpful discussions. This work was supported by a Searle Scholarship (01-L-116), NIH grants K22 HG00056 and T32 HG00047, and the Guidant Bioengineering Summer Research Program.
- Black DL: Mechanisms of alternative pre-messenger RNA splicing. Annu Rev Biochem. 2003, 72: 291-336. 10.1146/annurev.biochem.72.121801.161720.PubMedView ArticleGoogle Scholar
- Modrek B, Lee C: A genomic view of alternative splicing. Nat Genet. 2002, 30: 13-19. 10.1038/ng0102-13.PubMedView ArticleGoogle Scholar
- Kan Z, States D, Gish W: Selecting for functional alternative splices in ESTs. Genome Res. 2002, 12: 1837-1845. 10.1101/gr.764102.PubMedPubMed CentralView ArticleGoogle Scholar
- Celotto AM, Graveley BR: Alternative splicing of the Drosophila Dscam pre-mRNA is both temporally and spatially regulated. Genetics. 2001, 159: 599-608.PubMedPubMed CentralGoogle Scholar
- Leeds P, Peltz SW, Jacobson A, Culbertson MR: The product of the yeast UPF1 gene is required for rapid turnover of mRNAs containing a premature translational termination codon. Genes Dev. 1991, 5: 2303-2314.PubMedView ArticleGoogle Scholar
- Kinniburgh AJ, Maquat LE, Schedl T, Rachmilewitz E, Ross J: mRNA-deficient beta o-thalassemia results from a single nucleotide deletion. Nucleic Acids Res. 1982, 10: 5421-5427.PubMedPubMed CentralView ArticleGoogle Scholar
- Frischmeyer PA, Dietz HC: Nonsense-mediated mRNA decay in health and disease. Hum Mol Genet. 1999, 8: 1893-1900. 10.1093/hmg/8.10.1893.PubMedView ArticleGoogle Scholar
- Le Hir H, Izaurralde E, Maquat LE, Moore MJ: The spliceosome deposits multiple proteins 20-24 nucleotides upstream of mRNA exon-exon junctions. EMBO J. 2000, 19: 6860-6869. 10.1093/emboj/19.24.6860.PubMedPubMed CentralView ArticleGoogle Scholar
- Le Hir H, Moore MJ, Maquat LE: Pre-mRNA splicing alters mRNP composition: evidence for stable association of proteins at exon-exon junctions. Genes Dev. 2000, 14: 1098-1108.PubMedPubMed CentralGoogle Scholar
- Lykke-Andersen J, Shu MD, Steitz JA: Communication of the position of exon-exon junctions to the mRNA surveillance machinery by the protein RNPS1. Science. 2001, 293: 1836-1839. 10.1126/science.1062786.PubMedView ArticleGoogle Scholar
- Reichert VL, Le Hir H, Jurica MS, Moore MJ: 5' exon interactions within the human spliceosome establish a framework for exon junction complex structure and assembly. Genes Dev. 2002, 16: 2778-2791. 10.1101/gad.1030602.PubMedPubMed CentralView ArticleGoogle Scholar
- Le Hir H, Gatfield D, Izaurralde E, Moore MJ: The exon-exon junction complex provides a binding platform for factors involved in mRNA export and nonsense-mediated mRNA decay. EMBO J. 2001, 20: 4987-4997. 10.1093/emboj/20.17.4987.PubMedPubMed CentralView ArticleGoogle Scholar
- Dostie J, Dreyfuss G: Translation is required to remove Y14 from mRNAs in the cytoplasm. Curr Biol. 2002, 12: 1060-1067. 10.1016/S0960-9822(02)00902-8.PubMedView ArticleGoogle Scholar
- Maquat LE: Nonsense-mediated mRNA decay. Curr Biol. 2002, 12: R196-R197. 10.1016/S0960-9822(02)00747-9.PubMedView ArticleGoogle Scholar
- Buhler M, Wilkinson MF, Muhlemann O: Intranuclear degradation of nonsense codon-containing mRNA. EMBO Rep. 2002, 3: 646-651. 10.1093/embo-reports/kvf129.PubMedPubMed CentralView ArticleGoogle Scholar
- Ishigaki Y, Li XJ, Serin G, Maquat LE: Evidence for a pioneer round of mRNA translation: mRNAs subject to nonsense-mediated decay in mammalian cells are bound by CBP80 and CBP20. Cell. 2001, 106: 607-617. 10.1016/S0092-8674(01)00475-5.PubMedView ArticleGoogle Scholar
- Nagy E, Maquat LE: A rule for termination-codon position within intron-containing genes: when nonsense affects RNA abundance. Trends Biochem Sci. 1998, 23: 198-199. 10.1016/S0968-0004(98)01208-0.PubMedView ArticleGoogle Scholar
- Gonzalez CI, Ruiz-Echevarria MJ, Vasudevan S, Henry MF, Peltz SW: The yeast hnRNP-like protein Hrp1/Nab4 marks a transcript for nonsense-mediated mRNA decay. Mol Cell. 2000, 5: 489-499. 10.1016/S1097-2765(00)80443-8.PubMedView ArticleGoogle Scholar
- Gatfield D, Unterholzner L, Ciccarelli FD, Bork P, Izaurralde E: Nonsense-mediated mRNA decay in Drosophila: at the intersection of the yeast and mammalian pathways. EMBO J. 2003, 22: 3960-3970. 10.1093/emboj/cdg371.PubMedPubMed CentralView ArticleGoogle Scholar
- Cali BM, Anderson P: mRNA surveillance mitigates genetic dominance in Caenorhabditis elegans. Mol Gen Genet. 1998, 260: 176-184. 10.1007/s004380050883.PubMedView ArticleGoogle Scholar
- Li S, Wilkinson MF: Nonsense surveillance in lymphocytes?. Immunity. 1998, 8: 135-141. 10.1016/S1074-7613(00)80466-5.PubMedView ArticleGoogle Scholar
- Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P: Molecular Biology of the Cell. 2002, New York: Garland Science, 4Google Scholar
- Bregeon D, Doddridge ZA, You HJ, Weiss B, Doetsch PW: Transcriptional mutagenesis induced by uracil and 8-oxoguanine in Escherichia coli. Mol Cell. 2003, 12: 959-970. 10.1016/S1097-2765(03)00360-5.PubMedView ArticleGoogle Scholar
- Wagner E, Lykke-Andersen J: mRNA surveillance: the perfect persist. J Cell Sci. 2002, 115: 3033-3038.PubMedGoogle Scholar
- Vincent MC, Pujo AL, Olivier D, Calvas P: Screening for PAX6 gene mutations is consistent with haploinsufficiency as the main mechanism leading to various ocular defects. Eur J Hum Genet. 2003, 11: 163-169. 10.1038/sj.ejhg.5200940.PubMedView ArticleGoogle Scholar
- Kerr TP, Sewry CA, Robb SA, Roberts RG: Long mutant dystrophins and variable phenotypes: evasion of nonsense-mediated decay?. Hum Genet. 2001, 109: 402-407. 10.1007/s004390100598.PubMedView ArticleGoogle Scholar
- Hutchinson S, Furger A, Halliday D, Judge DP, Jefferson A, Dietz HC, Firth H, Handford PA: Allelic variation in normal human FBN1 expression in a family with Marfan syndrome: a potential modifier of phenotype?. Hum Mol Genet. 2003, 12: 2269-2276. 10.1093/hmg/ddg241.PubMedView ArticleGoogle Scholar
- Lamba JK, Adachi M, Sun D, Tammur J, Schuetz EG, Allikmets R, Schuetz JD: Nonsense mediated decay downregulates conserved alternatively spliced ABCC4 transcripts bearing nonsense codons. Hum Mol Genet. 2003, 12: 99-109. 10.1093/hmg/ddg011.PubMedView ArticleGoogle Scholar
- Sureau A, Gattoni R, Dooghe Y, Stevenin J, Soret J: SC35 autoregulates its expression by promoting splicing events that destabilize its mRNAs. EMBO J. 2001, 20: 1785-1796. 10.1093/emboj/20.7.1785.PubMedPubMed CentralView ArticleGoogle Scholar
- Wilson GM, Sun Y, Sellers J, Lu H, Penkar N, Dillard G, Brewer G: Regulation of AUF1 expression via conserved alternatively spliced elements in the 3' untranslated region. Mol Cell Biol. 1999, 19: 4056-4064.PubMedPubMed CentralView ArticleGoogle Scholar
- Mitrovich QM, Anderson P: Unproductively spliced ribosomal protein mRNAs are natural targets of mRNA surveillance in C. elegans. Genes Dev. 2000, 14: 2173-2184. 10.1101/gad.819900.PubMedPubMed CentralView ArticleGoogle Scholar
- Morrison M, Harris KS, Roth MB: smg mutants affect the expression of alternatively spliced SR protein mRNAs in Caenorhabditis elegans. Proc Natl Acad Sci USA. 1997, 94: 9782-9785. 10.1073/pnas.94.18.9782.PubMedPubMed CentralView ArticleGoogle Scholar
- Lelivelt MJ, Culbertson MR: Yeast Upf proteins required for RNA surveillance affect global expression of the yeast transcriptome. Mol Cell Biol. 1999, 19: 6710-6719.PubMedPubMed CentralView ArticleGoogle Scholar
- Le Guiner C, Gesnel MC, Breathnach R: TIA-1 or TIAR is required for DT40 cell viability. J Biol Chem. 2003, 278: 10465-10476. 10.1074/jbc.M212378200.PubMedView ArticleGoogle Scholar
- Wollerton MC, Gooding C, Wagner EJ, Garcia-Blanco MA, Smith CWJ: Autoregulation of polypyrimidine tract binding protein by alternative splicing leading to nonsense-mediated decay. Molecular Cell. 2004, 13: 91-100. 10.1016/S1097-2765(03)00502-1.PubMedView ArticleGoogle Scholar
- Lewis BP, Green RE, Brenner SE: Evidence for the widespread coupling of alternative splicing and nonsense-mediated mRNA decay in humans. Proc Natl Acad Sci USA. 2003, 100: 189-192. 10.1073/pnas.0136770100.PubMedPubMed CentralView ArticleGoogle Scholar
- Green RE, Lewis BP, Hillman RT, Blanchette M, Lareau LF, Garnett AT, Rio DC, Brenner SE: Widespread predicted nonsense-mediated mRNA decay of alternatively-spliced transcripts of human normal and disease genes. Bioinformatics. 2003, 19 (Suppl 1): I118-I121. 10.1093/bioinformatics/btg1015.PubMedView ArticleGoogle Scholar
- Medghalchi SM, Frischmeyer PA, Mendell JT, Kelly AG, Lawler AM, Dietz HC: Rent1, a trans-effector of nonsense-mediated mRNA decay, is essential for mammalian embryonic viability. Hum Mol Genet. 2001, 10: 99-105. 10.1093/hmg/10.2.99.PubMedView ArticleGoogle Scholar
- Boeckmann B, Bairoch A, Apweiler R, Blatter MC, Estreicher A, Gasteiger E, Martin MJ, Michoud K, O'Donovan C, Phan I, et al: The SWISS-PROT protein knowledgebase and its supplement TrEMBL in 2003. Nucleic Acids Res. 2003, 31: 365-370. 10.1093/nar/gkg095.PubMedPubMed CentralView ArticleGoogle Scholar
- Benson DA, Karsch-Mizrachi I, Lipman DJ, Ostell J, Wheeler DL: GenBank. Nucleic Acids Res. 2003, 31: 23-27. 10.1093/nar/gkg057.PubMedPubMed CentralView ArticleGoogle Scholar
- Pruitt KD, Maglott DR: RefSeq and LocusLink: NCBI gene-centered resources. Nucleic Acids Res. 2001, 29: 137-140. 10.1093/nar/29.1.137.PubMedPubMed CentralView ArticleGoogle Scholar
- Wheelan SJ, Church DM, Ostell JM: Spidey: a tool for mRNA-to-genomic alignments. Genome Res. 2001, 11: 1952-1957.PubMedPubMed CentralGoogle Scholar
- Horikawa Y, Oda N, Cox NJ, Li X, Orho-Melander M, Hara M, Hinokio Y, Lindner TH, Mashima H, Schwarz PE, et al: Genetic variation in the gene encoding calpain-10 is associated with type 2 diabetes mellitus. Nat Genet. 2000, 26: 163-175. 10.1038/79876.PubMedView ArticleGoogle Scholar
- Baier LJ, Permana PA, Yang X, Pratley RE, Hanson RL, Shen GQ, Mott D, Knowler WC, Cox NJ, Horikawa Y, et al: A calpain-10 gene polymorphism is associated with reduced muscle mRNA levels and insulin resistance. J Clin Invest. 2000, 106: R69-R73.PubMedPubMed CentralView ArticleGoogle Scholar
- Yang X, Pratley RE, Baier LJ, Horikawa Y, Bell GI, Bogardus C, Permana PA: Reduced skeletal muscle calpain-10 transcript level is due to a cumulative decrease in major isoforms. Mol Genet Metab. 2001, 73: 111-113. 10.1006/mgme.2001.3171.PubMedView ArticleGoogle Scholar
- Duncan PI, Stojdl DF, Marius RM, Bell JC: In vivo regulation of alternative pre-mRNA splicing by the Clk1 protein kinase. Mol Cell Biol. 1997, 17: 5996-6001.PubMedPubMed CentralView ArticleGoogle Scholar
- Duncan PI, Stojdl DF, Marius RM, Scheit KH, Bell JC: The Clk2 and Clk3 dual-specificity protein kinases regulate the intranuclear distribution of SR proteins and influence pre-mRNA splicing. Exp Cell Res. 1998, 241: 300-308. 10.1006/excr.1998.4083.PubMedView ArticleGoogle Scholar
- Menegay HJ, Myers MP, Moeslein FM, Landreth GE: Biochemical characterization and localization of the dual specificity kinase CLK1. J Cell Sci. 2000, 113: 3241-3253.PubMedGoogle Scholar
- Hanes J, von der Kammer H, Klaudiny J, Scheit KH: Characterization by cDNA cloning of two new human protein kinases. Evidence by sequence comparison of a new family of mammalian protein kinases. J Mol Biol. 1994, 244: 665-672. 10.1006/jmbi.1994.1763.PubMedView ArticleGoogle Scholar
- Modrek B, Lee CJ: Alternative splicing in the human, mouse and rat genomes is associated with an increased frequency of exon creation and/or loss. Nat Genet. 2003, 34: 177-180. 10.1038/ng1159.PubMedView ArticleGoogle Scholar
- Carter MS, Doskow J, Morris P, Li S, Nhim RP, Sandstedt S, Wilkinson MF: A regulatory mechanism that detects premature nonsense codons in T-cell receptor transcripts in vivo is reversed by protein synthesis inhibitors in vitro. J Biol Chem. 1995, 270: 28995-29003. 10.1074/jbc.270.48.28995.PubMedView ArticleGoogle Scholar
- Noensie EN, Dietz HC: A strategy for disease gene identification through nonsense-mediated mRNA decay inhibition. Nat Biotechnol. 2001, 19: 434-439. 10.1038/88099.PubMedView ArticleGoogle Scholar
- Lei XD, Chapman B, Hankinson O: Loss of cyp1a1 messenger rna expression due to nonsense-mediated decay. Mol Pharmacol. 2001, 60: 388-393.PubMedGoogle Scholar
- Thome M, Tschopp J: Regulation of lymphocyte proliferation and death by FLIP. Nat Rev Immunol. 2001, 1: 50-58. 10.1038/35095508.PubMedView ArticleGoogle Scholar
- Screaton GR, Xu XN, Olsen AL, Cowper AE, Tan R, McMichael AJ, Bell JI: LARD: a new lymphoid-specific death domain containing receptor regulated by alternative pre-mRNA splicing. Proc Natl Acad Sci USA. 1997, 94: 4615-4619. 10.1073/pnas.94.9.4615.PubMedPubMed CentralView ArticleGoogle Scholar
- Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment search tool. J Mol Biol. 1990, 215: 403-410. 10.1006/jmbi.1990.9999.PubMedView ArticleGoogle Scholar
- Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, Devon K, Dewar K, Doyle M, FitzHugh W, et al: Initial sequencing and analysis of the human genome. Nature. 2001, 409: 860-921. 10.1038/35057062.PubMedView ArticleGoogle Scholar
- Abril JF, Guigo R: gff2ps: visualizing genomic annotations. Bioinformatics. 2000, 16: 743-744. 10.1093/bioinformatics/16.8.743.PubMedView ArticleGoogle Scholar
- Myers EW, Miller W: Approximate matching of regular expressions. Bull Math Biol. 1989, 51: 5-37. 10.1016/S0092-8240(89)80046-1.PubMedView ArticleGoogle Scholar
- Boguski MS, Lowe TM, Tolstoshev CM: dbEST - database for "expressed sequence tags". Nat Genet. 1993, 4: 332-333. 10.1038/ng0893-332.PubMedView ArticleGoogle Scholar
- WU-BLAST archives. [http://blast.wustl.edu]
- Database of predicted C. intestinalis peptides. [ftp://ftp.jgi-psf.org/pub/JGI_data/Ciona/v1.0/ciona.prot.fasta.gz]
- Dehal P, Satou Y, Campbell RK, Chapman J, Degnan B, De Tomaso A, Davidson B, Di Gregorio A, Gelpke M, Goodstein DM, et al: The draft genome of Ciona intestinalis: insights into chordate and vertebrate origins. Science. 2002, 298: 2157-2167. 10.1126/science.1080049.PubMedView ArticleGoogle Scholar
- HMMER: sequence analysis using profile hidden Markov models. [http://hmmer.wustl.edu]
- Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994, 22: 4673-4680.PubMedPubMed CentralView ArticleGoogle Scholar
- Adachi J, Hasegawa M: MOLPHY version 2.3: Programs for Molecular Phylogenetics Based on Maximum Likelihood. 1996, Tokyo: Institute of Statistical MathematicsGoogle Scholar
- Database of predicted C. intestinalis transcripts. [ftp://ftp.jgi-psf.org/pub/JGI_data/Ciona/v1.0/ciona.mrna.fasta.gz]
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