Emergence of the Diversified Short ORFeome by Mass Spectrometry-Based Proteomics

Abstract

In proteomics analyses, protein identification by mass spectrometry (MS) is usually performed using protein sequence databases such as RefSeq (NCBI; http://www.ncbi.nlm.nih.gov/RefSeq/), UniProt (http://www.uniprot.org/) or IPI (http://www.ebi.ac.uk/IPI/IPIhelp.html). Because these databases usually target the longest (main) open reading frame (ORF) in the corresponding mRNA sequence, whether shorter ORFs on the same mRNA are actually translated still shrouds in mystery. In the first place, it had been considered that almost all eukaryotic mRNAs contains only one ORF and functions as monocistronic mRNAs. It is now known, however, that some eukaryotic mRNAs had multiple ORFs, which are recognized as polycistronic mRNAs. One of the wellknown extra ORFs is an upstream ORF (uORF) and it functions as regulators of mRNA translation (Diba et al., 2001; Geballe & Morris, 1994; Morris & Geballe, 2000; Vilela & McCarthy, 2003; Zhang & Dietrich, 2005). For getting clues to the mystery of diversified short ORFs, full-length mRNA sequence databases with complete 5‘-untranslated regions (5‘-UTRs) were essentially needed (Morris & Geballe, 2000; Suzuki et al., 2001). The oligo-capping method was developed to construct full-length cDNA libraries (Maruyama & Sugano, 1994) and the corresponding sequence were stored into the database called DBTSS (DataBase of Transcriptional Start Site; http://dbtss.hgc.jp/) (Suzuki et al., 1997, 2002, 2004; Tsuchihara et al., 2009; Wakaguri et al., 2008; Yamashita et al., 2006). Comparing the dataset of DBTSS with the corresponding RefSeq entries, it was found that about 50 % of the RefSeq entries had at least one upstream ATG (uATG) except the functional ATG initiator codon (Yamashita et al., 2003). Although it had been suggested that upstream AUGs (uAUGs) and uORFs play important roles for translation of the main ORF, none of the proteins from these uORFs was detected in biological experiments in vivo. Our previous proteomics analysis focused on small proteins revealed the first evidence of the existence of four novel small proteins translated from uORFs in vivo using highly sensitive nanoflow liquid chromatography (LC) coupled with the electrospray ionization-tandem mass spectrometry (ESI-MS/MS) system (Oyama et al., 2004). Large-scale analysis based on in-depth separation by two-dimensional LC also led to the identification of additional eight novel small proteins not only from uORFs but also from downstream ORFs and one of them was found to be translated from a non-AUG initiator codon (Oyama et al., 2007). Finding of these novel small proteins indicate the possibility of diverse control mechanisms of translation initiation. In this chapter, we first introduce widely-recognized mechanism of translation initiation and functional roles of uORF in translational regulation. We then review how we identified novel small proteins with MS and lastly discuss the progress of bioinformatical analyses for elucidating the diversification of short coding regions defined by the transcriptome.