dataesr/scientific-paragraphs-categorization huggingface dataset:
Similarly, the detection of a high proportion of sequences related to triacylglycerol catabolism, a skin lipid critically involved in epidermal permeability, suggests its utilization as a carbon source and triacylglycerol catabolism could also provide a significant contribution to the lipases produced by the skin for preventing lipid accumulation as in the ichthyosis pathology  [16]. The role of the microbiota in regulating another critical healthy state parameter (skin acidity), which controls the permeability barrier homeostasis, is also highlighted by numerous functional subsystems associated with acid resistance detected in the databases. For instance, acidification ecosystem preservation could explain the bacterial adaptive strategy of using the butanediolic fermentation as deduced from detection of alpha acetolactate and acetoin butanediol metabolism genes for transforming pyruvate into the final product (2,3-butanediol) rather than a mixed acid fermentation.
The predominance of genes involved in the arginine deiminase metabolism  [17]  in the metagenome datasets confirms the tolerance of bacteria to skin acidity  [15]. The skin metagenome analysis also brings new clues about the extensive spread of antibiotic resistance genes among bacteria. Within the human skin Staphylococcus populations of the two individuals, various Staphylococci seem to be intrinsically resistant to methicillin (fig. 3 ), although neither of the two individuals had recent contact with a methicillin-rich environment (hospitalization and/or methicillin treatment). Moreover, the level of teicoplanin and bacitracin resistance genes was particularly high in the sequence datasets. Finally, metagenomic analysis also contributes to the raising new and unexpected questions regarding bacteria adaptation and interactions with the human host.
A fully mature mRNA is usually associated to a reference open reading frame encoding a single protein. Yet, mature mRNAs contain unconventional alternative open reading frames (AltORFs) located in untranslated regions (UTRs) or overlapping the reference ORFs (RefORFs) in non-canonical +2 and +3 reading frames. Although recent ribosome profiling and footprinting approaches have suggested the significant use of unconventional translation initiation sites in mammals, direct evidence of large-scale alternative protein expression at the proteome level is still lacking. To determine the contribution of alternative proteins to the human proteome, we generated a database of predicted human AltORFs revealing a new proteome mainly composed of small proteins with a median length of 57 amino acids, compared to 344 amino acids for the reference proteome.
Introduction The proteome impacts all aspects of health and disease and deciphering the human proteome represents an important challenge in the post-genomic era. A typical fully processed mRNA includes one RefORF and is associated with a reference protein (Fig. 1A ). Reference proteins populate current protein databases used to support research in life sciences. For example, protein databases are central to the success of mass spectrometrybased protein identification for the discovery of expression and interaction proteomics, and of biomarkers  [1]. Two cellular mechanisms have evolved to increase proteomic diversity by encoding more than one protein per gene, increasing the diversity of the transcriptome or producing more than one protein from a single transcript. Transcriptome diversity  [2]  is achieved by utilization of alternative promoters  [3] , reiterative transcription  [4] , or post-transcriptional processing, including alternative splicing  [5] , alternative polyadenylation  [6]  and RNA editing  [7].
These studies demonstrate that the human proteome is more complex than previously appreciated and suggest that there are many more alternative proteins that remain undiscovered. Paradoxically, several databases predicting AltORFs in eukaryotes are available  [21]. Yet, most of these databases did not actually predict the rare examples of AltORFs translation products documented in humans  [11, 12, 14218]. Most importantly these databases did not include AltORFs present within UTRs. To address this issue, we generated a database of predicted AltORFs present in mature human mRNAs. Here, we provide evidence that AltORFs located within UTRs or overlapping the RefORF in a different reading frame are translated and significantly contribute to the human proteome. We demonstrate that basic transfection of cDNAs containing a RefORF and an AltORF results in coexpression of the reference and alternative proteins.
AltORFs database Few databases predicting alternative ORFs (AltORFs) in eukaryotes are available. Yet, most of these databases did not actually predict the very rare examples of out-of-frame alternative translation products documented in humans. Most importantly these databases did not include AltORFs present within UTRs. Other criteria included in previous predictions 2conservation among species, presence of an optimal Kozak context around the initiator AUG codon, location within the reference coding sequence2 were not taken into account in this study to reduce biases in prediction and generate a comprehensive database. The AltORF database for each species was built as previously described  [21]  with minor modifications to the pipeline of Perl scripts that populate a MySQL database, to retain AltORFs regardless of the Kozak context or the location on the mRNA.
Clones These clones were generated to verify translation initiation at predicted AUG codons, to confirm the coexpression of reference and alternative proteins from the same mRNA, and to visualize the expression of alternative proteins using a GFP tag. Predicted AltORFs with initiation and stop codons in 59UTRs. SLC35A4 and ZNF83 (GenBank mRNA entries NM_080670 and NM_001105552, respectively): gBlocks (IDT) gene fragments containing the 59UTR sequence up to the last coding nucleotide of their respective AltORF were designed and inserted into the BamHI restriction site of pEGFP-N1 using the Gibson assembly kit (New England Biolabs) according to the manufacturer's protocol. Predicted AltORF overlapping the 59UTR and the reference CDS. IDH3B (NM_174856). This construct was synthesized as gBlocks gene fragments (IDT).
The resulting PCR fragments were inserted into the BamHI site of pcDNA HA-EGFP+2. BDRKB2 was PCR amplified from a cDNA containing plasmid (clone SC119794, OriGene), and the resulting PCR fragments were inserted into the BamHI site of pcDNA HA- EGFP+3. Mutation of the predicted alternative initiation ATG codons to AAG was performed with complementary oligonucleotides containing the mutation, using the QuikChange kit (Stratagene). Each construct contained the endogenous Kozak sequence (positions 23 to +4) around both the RefORF and AltORF putative initiation codons. 59 UTR regions were present in the constructs only when the corresponding AltORF was at least partially contained within this region. The AltMRVI1 EGFP fusion was obtained by inserting a gBlock gene fragment containing the AltMRVI1 coding sequence (GenBank mRNA entry NM_001100167, nucleotides 5403 to 5688) into the BamHI digested pEGFP-N1 plasmid.
LC-MS/MS analyses of lung tissue, cerebrospinal fluid, urine, plasma and serum Raw data were obtained from the PeptideAtlas online repository  [30]  under the following accession numbers: lung (PAe001771), cerebrospinal fluid (PAe001777), urine (PAe000761 and PAe000763), plasma (PAe000846), serum (A: PAe000135 ; B: PAe000347 ; C: PAe000281 ; D: PAe000331, PAe000332, PAe000333, PAe000334, PAe000335, PAe000337, PAe000338).
Bioinformatics analysis Quantitation was performed using the program MaxQuant version 1. 2. 2. 5  [31, 32]. The derived peak list generated by Quant. exe (the first part of MaxQuant) was searched using Andromeda as the database search engine for peptide identifications against the human GenBank protein entries (release 37) containing 37,390 proteins, to which the 83,886 predicted alternative proteins and 175 commonly observed contaminants and all the reversed sequences had been added. The first search mass tolerance was set to 20 p. p. m. and main search mass tolerance was 6 p. p. m. Enzyme was set to trypsin/p with 2 missed cleavages. Carbamidomethylation of cysteine was searched as a fixed modification, whereas N-acetyl protein and oxidation of methionine were searched as variable modifications.
For the serum sample PAe000347, asparagine deamidation was added as a fixed modification, and glutamine deamidation as a variable modification. For analysis of fractionated HeLa cells, colon cell lines, colon tissue, ,10 kDa HeLa cells proteins, and paraffin-embedded human tissues, identification was set to a false discovery rate of 5%, determined by the use of a reverse database (similar results were obtained by using a randomly generated database). To achieve reliable identifications, only the proteins associated with a PEP value of less than 0. 05, and identified with at least one unique peptide were retained. Indeed, the detection of more than one tryptic fragment for small proteins is unlikely  [19] , though we detected at least two tryptic fragments for 148 alternative proteins (Table  S4 ).
For analysis of lung tissue and fluids, identification was set to a false discovery rate of 1%, with at least one unique peptide. Protein isoforms and proteins that cannot be distinguished based on the peptides identified are grouped and displayed on a single line, but with a single GenBank accession number (Tables S12S3). Accession numbers for detected alternative proteins are provided in Table  S4  and are publicly available on the nucleotide database of the European Bioinformatics Institute website (http:// www. ebi. ac. uk/). The conservation analyses of alternative and reference proteins were carried out with BLASTP  [33]  (version 2. 2. 27+). The AltORFs databases and reference databases from different species were searched against the corresponding human database with an expectation (E) value cutoff of # 10 24.
Results To generate our database, we defined AltORFs as ORFs located in a non-canonical reading frame of the RefORF, in the 59 and 39UTR regions of an mRNA, or partially overlapping with both the RefORF and an UTR region (Fig. 1A ). We based our prediction algorithm on characteristics of known AltORFs (AUG as TIS, alternative stop codon different from the RefORF stop codon), and added a size cut-off of 40 codons to keep the database to a reasonable list of polypeptides readily analysable by LC-MS/ MS or detectable by SDS-PAGE (Database S1; Fig. 1B ). Criteria included in previous predictions 2conservation among species, presence of an optimal Kozak context around the initiator AUG codon, location within the reference coding sequence2 were not taken into account in this study because experimental evidence indicate that these criteria are not necessarily required for an AltORF to be expressed  [11, 12].
Our database predicts 83,886 unique AltORFs with a minimum size of 40 codons (Fig. 1B ). Most predicted AltORFs overlap RefORFs (41. 09%) or populate 39UTRs (46. 55%) (Fig. 1C ). The majority of mRNAs (87. 58%) have at least one predicted AltORF (Fig. 1D ), and there is an average of 3. 88 AltORFs for each mRNA. These proportions are in agreement with the number of detectable translation initiation sites (TIS) determined by ribosome profiling  [34, 35]. Most predicted AltORFs have less than 100 codons, and the median alternative protein length is 57 amino acids, compared to 344 for the conventional proteome (Fig. 1E ). Using this novel alternative protein database as well as GenBank protein entries, we analyzed a HeLa cells proteomic data set we had previously generated by LC-MS/MS  [22]. A total of 68,035 peptides from 5,558 reference proteins and 280 peptides from 129 alternative proteins were identified (Table  1 ; Table  S1 ; Fig.
We completed these proteomic studies with human fluids, including cerebrospinal fluid, urine, plasma, and serum, identifying 16, 47, 90, and 928 alternative proteins in each fluid respectively (Table  1 ; Table  S3 ). Strikingly, alternative proteins represent approximately 55% of the proteins identified in plasma and serum (Table  1 ). Overall, we detected a total of 1,259 alternative proteins (Table  1 ), and 47 were expressed in different cell lines and/or tissues (Table  S4 ). In accordance with the scanning model of translation initiation, we used the first AUG rule in order to predict the TIS of AltORFs present in our database. Since other non-AUG codons can be used as TIS  [34] , we tested the reliability of our TIS prediction for the alternative proteins previously detected by two independent methods.
First, the detection of N-acetylated peptides, a modification specific to protein N-termini  [38] , in 889 out of the 1,259 total alternative proteins detected throughout our different LC-MS/MS experiments allowed us to determine that in most cases (886/889), the alternative TIS predicted in our database was correct (Table  S4 ). Second, we randomly selected and tested the co-expression of 6 alternative proteins and their corresponding reference proteins from the 129 alternative proteins detected by LC-MS/MS in the fractionated HeLa cells lysate. A strategy based on the transfection of constructs with two tags, an HA tag in frame with the reference protein and a GFP tag in frame with the alternative protein, was used to report the co-expression of both proteins in transfected cells (Fig. 3 ).
The corresponding alternative proteins were all detected by both western blot and GFP fluorescence. Importantly, inactivating mutations (AUG to AAG) of the predicted alternative TIS significantly reduced their expression (Fig. 3 ). Transfection of cDNAs in cultured cells is a routine technique in most laboratories. The possible unnoticed co-expression of an alternative protein with the reference protein could be a major issue, as 67. 36% of human protein coding genes are predicted to have at least one AltORF contained within the RefORF (Fig. 4A ). We selected 6 well studied RefORFs from the AltORFs database, including the tumor suppressor p53 (Fig. 4C ). The strategy to detect the co-expression of reference and alternative proteins is shown in Fig. 4B. After transfection, we determined that each cDNA led to the constitutive co-expression of the alternative and reference proteins as observed by western blot and fluorescence (Fig.
One sequence was 100% identical to an alternative protein from our database whose AltORF is located in the 39UTR of the mRNA produced from the MRVI1 gene (Fig. 5A ). AltMRVI1 EGFP was cloned and transfected into HeLa cells. Similar to BRCA1, AltMRVI1 EGFP localized to the nucleus (Fig. 5B ). We confirmed the interaction between BRCA1 and AltMRVI1 EGFP by co-immunoprecipitation (Fig. 5C ). Thus, AltMRVI1 is possibly a novel BRCA1 interacting protein that was already identified by Y2H, but mistakenly rejected as a false positive hit. To assess the evolutionary conservation of predicted alternative proteins, we generated a database of AltORFs present in mature mRNAs from different eukaryote species (Databases S22S8), and predicted 5019 distinct alternative proteins in Saccharomyces cerevisiae, 35,532 in Caenorhabditis elegans, 38,248 in Drosophila melanogaster, 52,454 in Xenopus tropicalis, 82,305 in Mus musculus, 57,492 in Bos taurus, and 79,874 in Pan troglodytes (Fig.
Discussion The potential of eukaryotic genomes for encoding alternative proteins from non-canonical open reading frames is well known and was recently featured in ribosome profiling studies  [34, 41, 42 ]. Yet, a large scale approach allowing the detection of the corresponding alternative proteins was lacking. In this study, we have generated a database of predicted alternative protein-coding ORFs with a minimum length of 40 codons present in human mRNAs. The data presented here indicate an average of 3. 8 AltORFs per human mRNA with a median length of 57 amino acids. Using this database, 1259 human alternative proteins were detected by mass spectrometry in the present study, 3 of which (accession numbers HF548059, HF547970, HF548029) were previously detected  [19, 43, 44]. This result strongly supports the hypothesis that the complexity of the proteome has been underestimated and alternative translation initiation already well characterized in viruses cannot be ignored in humans.
There is also evidence that an upstream ORF not only regulates the expression of a downstream RefORF by interfering in cis with canonical AUG recognition by scanning ribosomes, but also reduces the translational efficiency of the RefORF in trans  [45, 50]. AltORFs translation products could also be of particular importance during the thymal selection of T lymphocytes, serving as ''cryptic T-cell epitopes''. In some cases, this has been shown to lead to the selection of lymphocytes with antiviral or antitumor activities  [51]. Our databases of AltORFs will be useful to identify genes containing multiple protein coding ORFs and to unravel their functions. This is particularly important in experimental settings where gene expression studies (cDNA transfection, knock-down, transgenes, and gene therapy) could result in the expression or down regulation of a reference protein and an unnoticed alternative protein, leading to confounding results  [12].
Another striking example is the co-expression of therapeutic transgenes and their associated alternative proteins, which elicit a cytotoxic T lymphocyte response  [52]. Thus, transgene sequences should be carefully examined for possible AltORFs to decrease potential adverse immune responses during therapeutic gene transfer. We also propose that proteins recalcitrant to mass spectrometry identification or proteins with no sequence homology with the conventional proteome identified in large-scale cDNAs screens should be revisited with the AltORFs databases. Additionally, large fundamental and clinical proteomic studies using organs and tissues would likely benefit from the AltORFs database to achieve complete catalogs of proteins in different tissues  [53, 54]. The presence of a large fraction of alternative proteins in plasma and serum is particularly interesting as there is a constant need for biomarkers to identify a variety of disorders at an early stage  [55257].
The reason why so many alternative proteins are secreted is currently unknown. We did not find any enrichment for classical export signal peptides (not shown), and their secretion mechanism remains to be investigated. As for any databases, the AltORFs database has some limitations. Although AUG remains the main translation initiation site, recent ribosome profiling studies clearly indicate the use of non-AUG start sites  [34]. Yet, we did not take into account non-AUG initiation sites as an accurate prediction method for such functional translation start sites is not yet available  [58]. Although there is strong evidence that short peptides are also translated  [19] , we introduced a cut-off of 40 amino acids to discard from our databases polypeptides shorter than 40 residues, which are less readily detected by conventional mass spectrometry approaches, and to keep the database to a reasonable size.
Finally, we used the NCBI reference sequence database (RefSeq) as a source for RNA transcripts. This non-redundant and well-annotated database is fairly conservative, and thus is a quality source for identifying candidate AltORFs, but it would be interesting to compare with other databases (e. g. Gencode) to verify if different mRNA isoforms could also serve as template for the expression of corresponding alternative proteins. Nevertheless, the location of AltORfs is comparable with the distribution obtained in a peptidomic study of small ORFs encoded polypeptides, with the exception of AltORFs located in 59UTRs  [19]. For these AltORFs, the apparent discrepancy probably results from our prediction of AltORFs initiating at AUG sites only  [35]. In conclusion, we have provided compelling evidence that alternative proteins significantly contribute to the human proteome by identifying 1259 new proteins and many more will likely be detected in further MS experiments.
(XLSX) Database S2 A database of alternative ORFs in Pan troglodytes. For each AltORF, the gene name and accession number of the mRNA in which it is encoded is provided in the first tab. Other information can also be found for both the reference ORF and the alternative ORF, including the reading frame and the coordinates of the start and stop codon (with respect to the first nucleotide of the mRNA). The predicted amino acid sequence of the alternative protein is also indicated. The second tab displays the results of alternative protein conservation analysis against human. The gene name and accession number for chimpanzee and human homolog proteins are provided, as well as standard BLASTP output, and similarity percentage. (XLSX) Database S3 A database of alternative ORFs in Mus musculus. For each AltORF, the gene name and accession number of the mRNA in which it is encoded is provided in the first tab.
Other information can also be found for both the reference ORF and the alternative ORF, including the reading frame and the coordinates of the start and stop codon (with respect to the first nucleotide of the mRNA). The predicted amino acid sequence of the alternative protein is also indicated. The second tab displays the results of alternative protein conservation analysis against human. The gene name and accession number for mouse and human homolog proteins are provided, as well as standard BLASTP output, and similarity percentage.
(XLSX) Database S4 A database of alternative ORFs in Bos taurus. For each AltORF, the gene name and accession number of the mRNA in which it is encoded is provided in the first tab. Other information can also be found for both the reference ORF and the alternative ORF, including the reading frame and the coordinates of the start and stop codon (with respect to the first nucleotide of the mRNA). The predicted amino acid sequence of the alternative protein is also indicated. The second tab displays the results of alternative protein conservation analysis against human. The gene name and accession number for cow and human homolog proteins are provided, as well as standard BLASTP output, and similarity percentage.
(XLSX) Database S5 A database of alternative ORFs in Xenopus tropicalis. For each AltORF, the gene name and accession number of the mRNA in which it is encoded is provided in the first tab. Other information can also be found for both the reference ORF and the alternative ORF, including the reading frame and the coordinates of the start and stop codon (with respect to the first nucleotide of the mRNA). The predicted amino acid sequence of the alternative protein is also indicated. The second tab displays the results of alternative protein conservation analysis against human. The gene name and accession number for frog and human homolog proteins are provided, as well as standard BLASTP output, and similarity percentage. (XLSX) Database S6 A database of alternative ORFs in Drosophila melanogaster.
For each AltORF, the gene name and accession number of the mRNA in which it is encoded is provided in the first tab. Other information can also be found for both the reference ORF and the alternative ORF, including the reading frame and the coordinates of the start and stop codon (with respect to the first nucleotide of the mRNA). The predicted amino acid sequence of the alternative protein is also indicated. The second tab displays the results of alternative protein conservation analysis against human. The gene name and accession number for fly and human homolog proteins are provided, as well as standard BLASTP output, and similarity percentage.