Different regions of oral squamous cell carcinoma (OSCC) have particular histopathological and molecular characteristics limiting the standard tumor−node−metastasis prognosis classification. Therefore, defining biological signatures that allow assessing the prognostic outcomes for OSCC patients would be of great clinical significance. Using histopathology-guided discovery proteomics, we analyze neoplastic islands and stroma from the invasive tumor front (ITF) and inner tumor to identify differentially expressed proteins. Potential signature proteins are prioritized and further investigated by immunohistochemistry (IHC) and targeted proteomics. IHC indicates low expression of cystatin-B in neoplastic islands from the ITF as an independent marker for local recurrence. Targeted proteomics analysis of the prioritized proteins in saliva, combined with machine-learning methods, highlights a peptide-based signature as the most powerful predictor to distinguish patients with and without lymph node metastasis. In summary, we identify a robust signature, which may enhance prognostic decisions in OSCC and better guide treatment to reduce tumor recurrence or lymph node metastasis.

The factors involved in rRNA processing in eukaryotes assemble cotranscriptionally onto the nascent prerRNAs and include endonucleases, exonucleases, RNA helicases, GTPases, modifying enzymes and snoRNPs (small nucleolar ribonucleoproteins). The precursor of three of the four eukaryotic mature rRNAs contains the rRNA sequences flanked by two internal (ITS1 and ITS2) and two external (5¢-ETS and 3¢-ETS) spacer sequences that are removed during processing [1,2]. The pre-rRNA is first assembled into a 90S particle that contains U3 snoRNP and 40S subunit-processing factors [3,4]. The early pre-rRNA endonucleolytic cleavages at sites A 0 , A 1 and A 2 occur within the 90S particles [3,5]. A 2 cleavage releases the first pre60S particle, which differs in composition from the known 90S particle. Pre60S particles contain 27S rRNA, ribosomal L proteins and many nonribosomal proteins [6].As they mature, pre60S particles migrate from the nucleolus to the nucleoplasm and their content of nonribosomal factors changes [7,8]. Nip7p was among the proteins identified in the early pre60S particle [6][7][8], and has been shown to participate in the processing of 27S pre-rRNA to the formation of 25S [9]. Interestingly, Nip7p also binds the exosome subunit Rrp43p [10]. The exosome complex is responsible for the degradation of the excised 5¢-ETS and for the 3¢)5¢ exonucleolytic processing of 7S pre-rRNA to form the mature 5.8S rRNA. The exosome is also involved in the processing of snoRNAs and in mRNA degradation [11][12][13].During processing, pre-rRNA undergoes covalent modifications that include isomerization of some uridines into pseudouridines and addition of methyl groups to specific nucleotides, mainly at the 2¢-O posi- In eukaryotes, pre-rRNA processing depends on a large number of nonribosomal trans-acting factors that form large and intriguingly organized complexes. A novel nucleolar protein, Nop53p, was isolated by using Nop17p as bait in the yeast two-hybrid system. Nop53p also interacts with a second nucleolar protein, Nip7p. A carbon source-conditional strain with the NOP53 coding sequence under the control of the GAL1 promoter did not grow in glucose-containing medium, showing the phenotype of an essential gene. Under nonpermissive conditions, the conditional mutant strain showed rRNA biosynthesis defects, leading to an accumulation of the 27S and 7S pre-rRNAs and depletion of the mature 25S and 5.8S mature rRNAs. Nop53p did not interact with any of the exosome subunits in the yeast twohybrid system, but its depletion affects the exosome function. In pull-down assays, protein A-tagged Nop53p coprecipitated the 27S and 7S pre-rRNAs, and His-Nop53p also bound directly 5.8S rRNA in vitro, which is consistent with a role for Nop53p in pre-rRNA processing.Abbreviations ETS, external transcribed spacer; b-Gal, b-galactosidase; GFP, green fluorescent protein; GST, glutathione S-transferase; ITS, internal transcribed spacer; RFP, red fluorescent protein; snoRNP, small nucleolar ribonucleoprotein.

Synthesis of mature ribosomal subunits in yeast involves many steps of rRNA processing, directed by at least 180 factors that include proteins and snoRNP complexes. The protein factors include rRNA-modifying enzymes, endonucleases, exonucleases, RNA helicases, GTPases and snoRNA-associated proteins [1,2]. Three of the rRNAs (18S, 5.8S and 25S) are transcribed as a 35S precursor, which undergoes a series of processing reactions, including endo-and exonucleolytic cleavage and nucleotide modifications. Some of the processing factors and ribosomal proteins assemble into the complex early during transcription [3][4][5][6], leading to formation of various pre-ribosomal particles, the first of which is the 90S complex [7,8]. Most of the factors forming the 90S complex are involved in processing of 18S rRNA, or are part of the 40S ribosome subunits [7,8].Co-purification of proteins and mass spectrometry studies have identified many of the factors involved in rRNA processing, such as the small ribosomal subunit (SSU) complex processome and Dim2p [9,10]. The processing factors of the large ribosomal subunit bind later during transcription of the 35S pre-rRNA, or after the early cleavages at sites A 0 , A 1 and A 2 that separate the pre-40S and pre-60S complexes [8,11,12], and include some of the large ribosomal subunit proteins, as well as 27S processing factors [11]. As some ribosomal proteins bind early during rRNA transcription, they also play an important role in rRNA processing. Rpl3p and the IPI complex have recently been shown to be involved in cleavages at ITS2, and their depletion leads to accumulation of the pre-rRNAs 35S and 27S, and a decrease in mature 25S levels [2,9]. In eukaryotes, pre-rRNA processing depends on a large number of nonribosomal trans-acting factors that form intriguingly organized complexes. One of the early stages of pre-rRNA processing includes formation of the two intermediate complexes pre-40S and pre-60S, which then form the mature ribosome subunits. Each of these complexes contains specific prerRNAs, ribosomal proteins and processing factors. The yeast nucleolar protein Nop53p has previously been identified in the pre-60S complex and shown to affect pre-rRNA processing by directly binding to 5.8S rRNA, and to interact with Nop17p and Nip7p, which are also involved in this process. Here we show that Nop53p binds 5.8S rRNA co-transcriptionally through its N-terminal region, and that this protein portion can also partially complement growth of the conditional mutant strain Dnop53 ⁄ GAL::-NOP53. Nop53p interacts with Rrp6p and activates the exosome in vitro. These results indicate that Nop53p may recruit the exosome to 7S pre-rRNA for processing. Consistent with this observation and similar to the observed in exosome mutants, depletion of Nop53p leads to accumulation of polyadenylated pre-rRNAs.Abbreviations ETS, external transcribed spacer; IPI, involved in processing of ITS2; ITS2, internal transcribed spacer 2; LSU, large ribosomal subunit; snoRNP, small nucleolar ribonucleoprotein; SSU...