Diversity of Translation Start Sites May Define Increased Complexity of the Human Short ORFeome

Our previous proteomics analysis of small proteins expressed in human K562 cells provided the first direct evidence of translation of upstream ORFs in human full-length cDNAs (Oyama, M., Itagaki, C., Hata, H., Suzuki, Y., Izumi, T., Natsume, T., Isobe, T., and Sugano, S. (2004) Analysis of small human proteins reveals the translation of upstream open reading frames of mRNAs. Genome Res. 14, 2048–2052). In the present study, we performed an in-depth proteomics analysis of human K562 and HEK293 cells using a two-dimensional nano-liquid chromatography-tandem mass spectrometry system. The results led to the identification of eight protein-coding regions besides 197 small proteins with a theoretical mass less than 20 kDa that were already annotated coding sequences in the curated mRNA database. In addition to the upstream ORFs in the presumed 5′-untranslated regions of mRNAs, bioinformatics analysis based on accumulated 5′-end cDNA sequence data provided evidence of novel short coding regions that were likely to be translated from the upstream non-AUG start site or from the new short transcript variants generated by utilization of downstream alternative promoters. Protein expression analysis of the GRINL1A gene revealed that translation from the most upstream start site occurred on the minor alternative splicing transcript, whereas this initiation site was not utilized on the major mRNA, resulting in translation of the downstream ORF from the second initiation codon. These findings reveal a novel post-transcriptional system that can augment the human proteome via the alternative use of diverse translation start sites coupled with transcriptional regulation through alternative promoters or splicing, leading to increased complexity of short protein-coding regions defined by the human transcriptome. Our previous proteomics analysis of small proteins expressed in human K562 cells provided the first direct evidence of translation of upstream ORFs in human full-length cDNAs (Oyama, M., Itagaki, C., Hata, H., Suzuki, Y., Izumi, T., Natsume, T., Isobe, T., and Sugano, S. (2004) Analysis of small human proteins reveals the translation of upstream open reading frames of mRNAs. Genome Res. 14, 2048–2052). In the present study, we performed an in-depth proteomics analysis of human K562 and HEK293 cells using a two-dimensional nano-liquid chromatography-tandem mass spectrometry system. The results led to the identification of eight protein-coding regions besides 197 small proteins with a theoretical mass less than 20 kDa that were already annotated coding sequences in the curated mRNA database. In addition to the upstream ORFs in the presumed 5′-untranslated regions of mRNAs, bioinformatics analysis based on accumulated 5′-end cDNA sequence data provided evidence of novel short coding regions that were likely to be translated from the upstream non-AUG start site or from the new short transcript variants generated by utilization of downstream alternative promoters. Protein expression analysis of the GRINL1A gene revealed that translation from the most upstream start site occurred on the minor alternative splicing transcript, whereas this initiation site was not utilized on the major mRNA, resulting in translation of the downstream ORF from the second initiation codon. These findings reveal a novel post-transcriptional system that can augment the human proteome via the alternative use of diverse translation start sites coupled with transcriptional regulation through alternative promoters or splicing, leading to increased complexity of short protein-coding regions defined by the human transcriptome. According to a widely accepted model of translation in eukaryotes, a 40 S ribosomal subunit is first recruited to the cap structure of mRNA and linearly scans the 5′-untranslated region (UTR) 1The abbreviations used are: UTR, untranslated region; CDS, coding sequence; 2D, two-dimensional; DBTSS, DataBase of Transcriptional Start Sites; HEK, human embryonic kidney; aa, amino acids. 1The abbreviations used are: UTR, untranslated region; CDS, coding sequence; 2D, two-dimensional; DBTSS, DataBase of Transcriptional Start Sites; HEK, human embryonic kidney; aa, amino acids. for the initiator ATG. When it recognizes the translation initiation site, it pauses until a large 60 S subunit joins, and the complete ribosomal complex starts translation (1Kozak M. The scanning model for translation: an update.J. Cell Biol. 1989; 108: 229-241Crossref PubMed Scopus (2803) Google Scholar). Thus, the most “upstream” ORF of the mRNA should be translated according to this model especially when there is a good context around its ATG codon (2Kozak M. Initiation of translation in prokaryotes and eukaryotes.Gene (Amst.). 1999; 234: 187-208Crossref PubMed Scopus (1126) Google Scholar). Surprisingly large scale sequence analyses focusing on the 5′-UTRs of human full-length cDNA sequences showed that 41–49% of these cDNAs had at least one ATG codon upstream of the presumed coding sequence (3Peri S. Pandey A. A reassessment of the translation initiation codon in vertebrates.Trends Genet. 2001; 17: 685-687Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 4Yamashita R. Suzuki Y. Nakai K. Sugano S. Small open reading frames in 5′ untranslated regions of mRNAs.C. R. Biol. 2003; 326: 987-991Crossref PubMed Scopus (21) Google Scholar). This means that there are potential coding regions in the 5′-UTRs of many genes if this classical model indeed represents a general mechanism of translation initiation. However, there have been only a limited number of reports on mammalian genes that investigated translation of the upstream ORFs (5Iacono M. Mignone F. Pesole G. uAUG and uORFs in human and rodent 5′untranslated mRNAs.Gene (Amst.). 2005; 349: 97-105Crossref PubMed Scopus (185) Google Scholar). These previous studies referred to the upstream short ORF as a regulator of the translation of each downstream coding sequence (CDS) in which cases there was no direct evidence of translation of the upstream ORF in vivo (6Morris D.R. Geballe A.P. Upstream open reading frames as regulators of mRNA translation.Mol. Cell. Biol. 2000; 20: 8635-8642Crossref PubMed Scopus (563) Google Scholar, 7Meijer H.A. Thomas A.A. Control of eukaryotic protein synthesis by upstream open reading frames in the 5′-untranslated region of an mRNA.Biochem. J. 2002; 367: 1-11Crossref PubMed Scopus (249) Google Scholar). Our recent proteomics analysis of small proteins expressed in human leukemia K562 cells led to identification of novel peptides corresponding to the upstream short ORFs in four kinds of human full-length cDNAs (8Oyama M. Itagaki C. Hata H. Suzuki Y. Izumi T. Natsume T. Isobe T. Sugano S. Analysis of small human proteins reveals the translation of upstream open reading frames of mRNAs.Genome Res. 2004; 14: 2048-2052Crossref PubMed Scopus (79) Google Scholar). That study clearly provided direct evidence of the translation of the upstream ORFs in the presumed 5′-UTRs within human cells. To make a more comprehensive search for unpredicted short coding sequences from human mRNAs, we have constructed an automated two-dimensional (2D) nano-LC system coupled with a high resolution hybrid tandem mass spectrometer. Separation of peptide mixtures through on-line multidimensional LC enables us to perform large scale identification of highly complex biological samples, such as cell lysates (9Washburn M.P. Wolters D. Yates III, J.R. Large-scale analysis of the yeast proteome by multidimensional protein identification technology.Nat. Biotechnol. 2001; 19: 242-247Crossref PubMed Scopus (4062) Google Scholar, 10Mawuenyega K.G. Kaji H. Yamuchi Y. Shinkawa T. Saito H. Taoka M. Takahashi N. Isobe T. Large-scale identification of Caenorhabditis elegans proteins by multidimensional liquid chromatography-tandem mass spectrometry.J. Proteome Res. 2003; 2: 23-35Crossref PubMed Scopus (115) Google Scholar, 11Taoka M. Yamauchi Y. Shinkawa T. Kaji H. Motohashi W. Nakayama H. Takahashi N. Isobe T. Only a small subset of the horizontally transferred chromosomal genes in Esch e richia coli are translated into proteins.Mol. Cell. Proteomics. 2004; 3: 780-787Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). In the present study, we applied this technology for shotgun identification of the components in small protein-enriched fractions prepared from human cultured cells. We carried out analyses of small proteins expressed in human K562 and HEK293 cells and mapped the regions for all the identified peptides on RefSeq human mRNAs, which represent curated non-redundant transcripts (12Pruitt K.D. Tatusova T. Maglott D.R. NCBI Reference Sequence (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteins.Nucleic Acids Res. 2005; 33: D501-D504Crossref PubMed Scopus (1286) Google Scholar) based on large scale collection of human full-length cDNA sequences (13Wiemann S. Weil B. Wellenreuther R. Gassenhuber J. Glassl S. Ansorge W. 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Nagai K. et al.Complete sequencing and characterization of 21,243 full-length human cDNAs.Nat. Genet. 2004; 36: 40-45Crossref PubMed Scopus (719) Google Scholar). Here we describe the locations of short coding regions besides the RefSeq CDSs and analyze the diversity and the utilization of their probable translation start sites. Human erythroleukemia K562 cells were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum. HEK293 cells were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. After harvesting, the cells were washed three times with PBS. For proteasome inhibition analysis, HEK293 cells were labeled with stable isotopes of arginine according to a previous study (17Blagoev B. Ong S.E. Kratchmarova I. Mann M. Temporal analysis of phosphotyrosine-de pend ent signaling networks by quantitative proteomics.Nat. Biotechnol. 2004; 22: 1139-1145Crossref PubMed Scopus (585) Google Scholar). HEK293 cells labeled with either l-arginine, l-[U-13C6,14N4]arginine, or l-[U-13C6,15N4]arginine (2 × 10-cm dishes per condition) were treated with 20 μm MG-132 (Calbiochem) for 0, 2, and 5 h, respectively. After washing with PBS, the harvested cells were all combined for subsequent quantitative analysis. A small protein-enriched fraction was prepared from the harvested cells by acid extraction as described previously (8Oyama M. Itagaki C. Hata H. Suzuki Y. Izumi T. Natsume T. Isobe T. Sugano S. Analysis of small human proteins reveals the translation of upstream open reading frames of mRNAs.Genome Res. 2004; 14: 2048-2052Crossref PubMed Scopus (79) Google Scholar). Sixty micrograms of the protein-enriched fraction was digested overnight at 37 °C with 150 pmol of trypsin in 25 mm Tris-HCl (pH 8.8) and desalted using a ZipTip (C18; Millipore). The digested peptide mixture was then analyzed on an automated 2D LC-MS/MS system. After applying the peptide mixture to an strong cation exchange column (500-μm inner diameter × 35 mm long), elution was done with discrete step gradients of ammonium acetate from 0 to 400 mm (0, 5, and 400 at a of by each step were on a column inner diameter × mm and desalted with of the peptides was done on a column inner diameter × mm with using a direct LC system The peptides were with a of acid at a of and into a tandem mass The were the RefSeq for protein and mRNA database (12Pruitt K.D. Tatusova T. Maglott D.R. NCBI Reference Sequence (RefSeq): a curated non-redundant sequence database of genomes, transcripts and proteins.Nucleic Acids Res. 2005; 33: D501-D504Crossref PubMed Scopus (1286) Google Scholar) using the with the peptide mass 500 Only the peptide sequences that peptide identification with a were in this to the search results for novel we performed to the peptides with a sequence of at least four amino acids. The RefSeq were from the NCBI site The of the identified proteins was according to the RefSeq as of For proteasome inhibition analysis, a for mass analysis was prepared from HEK293 cells in the of the identified was performed using which we have for based on LC-MS/MS data A. M. M. H. K. Sugano S. T. S. an for based on LC-MS/MS proteome data and its to the analysis of PubMed Scopus Google Scholar). of the GRINL1A cDNAs from the 5′-end in to the of the downstream ORF was and 5′ to the in the as described previously S. M. to the of large of open reading frames or 2000; PubMed Google Scholar). The were by sequence analysis, and HEK293 cells were with each using HEK293 cells were then in mm Tris-HCl at 150 mm mm supplemented with mixture and by for at at For of proteins by the GRINL1A transcript we generated the upstream ORF and the downstream ORF of the transcript are from were in using a peptide from to of the upstream ORF or from to of the downstream ORF by by human cells were as described and of each cell was performed using with ORF or ORF For the analysis of the expressed from the cDNA 150 of each was analyzed with mouse by The lysates were analyzed with mouse as a protein To out a proteomics analysis of small proteins expressed in human we prepared a small protein-enriched fraction for mass analysis by acid After 60 of the acid was digested with the peptide mixture was analyzed using a 2D system. For the prepared from human K562 and HEK293 automated 2D LC of the peptides led to the of and respectively. a of a database search the RefSeq human protein database of peptides were identified from the of human K562 and peptides were identified from that of HEK293 cells. The identified peptides were to and protein respectively. and proteins with a theoretical mass of less than 20 resulting in 197 non-redundant short proteins to search for protein-coding regions besides the annotated CDSs in the RefSeq human the data were all three reading frames of RefSeq of a peptides were identified from regions besides the annotated CDSs peptides were mapped on four upstream ORFs in the 5′-UTRs of the corresponding mRNAs, three peptides were in downstream short ORFs in the The peptides were from reading frames with no initiator ATG in the corresponding and sequences besides each identified by RefSeq human data is based on the sequence of the corresponding RefSeq mRNA of the the identified peptide and the corresponding RefSeq The peptides identified from each cell are in a new a data is based on the sequence of the corresponding RefSeq mRNA of the the identified peptide and the corresponding RefSeq The peptides identified from each cell are large scale analyses of 5′-end sequences of human cDNAs have provided in-depth for the transcriptional start sites of human on a scale Y. H. T. J. J. Hata H. T. T. T. S. K. Y. Y. A. Sugano S. transcriptional initiation revealed by of mRNA start 2001; 2: PubMed Scopus Google Scholar, K. Wakamatsu A. Suzuki Y. T. Nishikawa T. R. J. M. K. H. S. Sugiyama T. Saito K. Y. Irie R. N. T. R. K. T. H. M. K. S. T. A. T. Nagai K. H. Nakai K. T. Sugano S. of transcriptional identification and characterization of alternative promoters of human Res. PubMed Scopus Google Scholar, A. B. S. K. J. C. R. T. T. S. M. Y. H. C. M. H. K. S. A. S. F. Suzuki H. C. M. J. Y. analysis of mammalian and Genet. PubMed Scopus Google Scholar). To the probable translation start sites of the novel protein-coding regions with no initiator ATG in the corresponding RefSeq and we analyzed their 5′-end sequence data accumulated in the DataBase of Transcriptional Start in which we have 5′-end sequence data of human cDNAs from kinds of cDNA R. Suzuki Y. H. K. Nakai K. Sugano S. DataBase of Human Start Acids Res. PubMed Scopus Google Scholar). the transcripts of and most of the 5′-end sequence data showed the structure as the corresponding RefSeq transcript with diverse transcriptional start sites as previously Y. H. T. J. J. Hata H. T. T. T. S. K. Y. Y. A. Sugano S. transcriptional initiation revealed by of mRNA start 2001; 2: PubMed Scopus Google Scholar). the no ATG codon upstream of the novel coding the of their non-AUG translation start sites. the 5′-end structure of the transcripts accumulated in previous studies that of a or start codon by a strong context M. the of the scanning mechanism for initiation of (Amst.). 2002; PubMed Scopus Google the only codon by a at and a at was to be a translation start site in the corresponding transcripts expressed in K562 cells For the upstream codon that context was to be a novel translation start site in the reading as the identified coding region not in we identified novel protein-coding regions downstream of the annotated in the RefSeq transcripts of and scale identification of start sites of human genes provided evidence of utilization of alternative promoters at the K. Wakamatsu A. Suzuki Y. T. Nishikawa T. R. J. M. K. H. S. Sugiyama T. Saito K. Y. Irie R. N. T. R. K. T. H. M. K. S. T. A. T. Nagai K. H. Nakai K. T. Sugano S. of transcriptional identification and characterization of alternative promoters of human Res. PubMed Scopus Google Scholar, A. B. S. K. J. C. R. T. T. S. M. Y. H. C. M. H. K. S. A. S. F. Suzuki H. C. M. J. Y. analysis of mammalian and Genet. PubMed Scopus Google Scholar). For the analysis of the accumulated 5′-end cDNA sequence data in provided evidence of short transcript variants the identified coding region in the ORF The curated that was from the short transcript showed and with the translated of the mouse cDNA gene protein on this novel there was only one 5′-end cDNA data for the sequence data at it a short transcript that was to be generated from the downstream transcriptional start site in the of the RefSeq These results that translation of these small proteins was likely to from the novel translation start sites on the short transcript variants generated through their downstream alternative promoters. In the of the GRINL1A recent analysis of its transcript revealed that this gene a of transcript variants B. L. The human GRINL1A gene a complex an of gene in 2004; PubMed Scopus Google Scholar). The peptides identified in analysis to three kinds of splicing variants expression was by the which were and the most upstream ORFs by the downstream ORFs as as and aa, respectively. is generated by the use of the alternative site of the first an ORF with the and amino acid sequences as the most upstream ORF of These transcript variants the 5′-end structure in which the first ATG is in a good whereas the second the (2Kozak M. Initiation of translation in prokaryotes and eukaryotes.Gene (Amst.). 1999; 234: 187-208Crossref PubMed Scopus (1126) Google Scholar) analysis showed expression of variants and in human as as human K562 cells and HEK293 cells not we performed analysis of their translated in Our the of the proteins by the upstream ORF of and the downstream ORF of in the human cells investigated HEK293 cells that were with cDNA corresponding to GRINL1A transcript generated the by the upstream ORF as as the downstream ORF translation of this upstream ORF was not performed in the human cells investigated These results alternative utilization of translation start sites of GRINL1A splicing variants to proteins within the cells. Our large scale proteomics analysis of small proteins expressed in human K562 and HEK293 cells us to novel short CDSs from more diverse regions of human than we had In addition to novel upstream ORFs with the corresponding initiator ATG codon study revealed translation of the presumed 5′-UTRs of and the of a ATG start site were previous studies that showed translation initiation at an upstream non-AUG codon such as or to protein (2Kozak M. Initiation of translation in prokaryotes and eukaryotes.Gene (Amst.). 1999; 234: 187-208Crossref PubMed Scopus (1126) Google Scholar, M. the of the scanning mechanism for initiation of (Amst.). 2002; PubMed Scopus Google Scholar). Our results that non-AUG start be for to short proteins that are of the analysis led to identification of the translated from the downstream short ORFs in the presumed of and analyses of transcriptional start sites of mammalian genes have revealed the of new transcripts generated through the use of alternative promoters in the CDSs or of already transcripts A. B. S. K. J. C. R. T. T. S. M. Y. H. C. M. H. K. S. A. S. F. Suzuki H. C. M. J. Y. analysis of mammalian and Genet. PubMed Scopus Google Scholar). short transcripts generated through downstream promoters are to be potential transcripts A. B. S. K. J. C. R. T. T. S. M. Y. H. C. M. H. K. S. A. S. F. Suzuki H. C. M. J. Y. analysis of mammalian and Genet. PubMed Scopus Google Scholar, S. Y. T. K. M. M. H. Suzuki M. J. Suzuki H. Y. C. M. T. J. J. L. S. Y. A. S. B. C. Genome Genome in the mammalian 2005; PubMed Scopus Google results the that an large number of small proteins be by such short transcripts In addition to through alternative splicing to of an analysis of the human GRINL1A transcripts B. L. The human GRINL1A gene a complex an of gene in 2004; PubMed Scopus Google Scholar) revealed corresponding to the identified peptides and described in the sequence of their first ATG that the of the upstream ORF in the GRINL1A transcript and an on translation initiation of the downstream analysis of human the of an protein that was to be by the downstream ORF of the GRINL1A transcript of the corresponding cDNA translation of this downstream ORF the of the upstream ORF and the that to a protein from the ORFs O. S. I. in gene Cell. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). results showed that the upstream first ATG was as a translation start site on transcript the ATG codon was by on in HEK293 and K562 cells is that of the translation start sites of these GRINL1A variants is by scanning or pend ent translation H.A. Thomas A.A. Control of eukaryotic protein synthesis by upstream open reading frames in the 5′-untranslated region of an mRNA.Biochem. J. 2002; 367: 1-11Crossref PubMed Scopus (249) Google Scholar, M. and regulation of gene 2004; PubMed Scopus Google Scholar). that recent analysis of 5′-end cDNA sequences showed high diversity of the first of human genes as as their transcriptional start sites on a scale K. Wakamatsu A. Suzuki Y. T. Nishikawa T. R. J. M. K. H. S. Sugiyama T. Saito K. Y. Irie R. N. T. R. K. T. H. M. K. S. T. A. T. Nagai K. H. Nakai K. T. Sugano S. of transcriptional identification and characterization of alternative promoters of human Res. PubMed Scopus Google it is that utilization of translation start sites on diverse first the complexity of short protein-coding regions that are upstream of each major analysis of the mechanism that the of proteins from the to the for the utilization of translation start sites on the of mRNAs. Our results revealed evidence for a novel of complex regulation that led to the of a of short proteins that have not been in gene This study, in the identification of only a small number of novel short protein-coding sequences from already the system is to be for the of a of short we the of proteasome inhibition to the diversity of unknown translated expressed in HEK293 cells. proteins such as protein were accumulated by proteasome S.F. G. identification of proteins from human cells 2005; PubMed Scopus Google there were no novel proteins that accumulated and were identified in analysis, that translation of short ORFs not within the cells in sequence analysis of the upstream ORF of showed and with the mouse on this In the upstream ORF of was highly with and of the novel ORFs identified in analysis have in their amino acid analysis is to reveal the of these translated of a comprehensive mRNA transcripts generated by the use of alternative promoters or alternative splicing be to of a of unpredicted short CDSs based on The of data on short coding regions in the of human in with analysis of the regulation of each in a of a highly translation system for the human We K. for in the LC system. We are to T. and A. Saito for large scale proteomics We are to for reading of the with