Hundreds of putatively functional small open reading frames in Drosophila

Abstract

BackgroundThe relationship between DNA sequence and encoded information is still an unsolved puzzle. The number of protein-coding genes in higher eukaryotes identified by genome projects is lower than was expected, while a considerable amount of putatively non-coding transcription has been detected. Functional small open reading frames (smORFs) are known to exist in several organisms. However, coding sequence detection methods are biased against detecting such very short open reading frames. Thus, a substantial number of non-canonical coding regions encoding short peptides might await characterization.ResultsUsing bio-informatics methods, we have searched for smORFs of less than 100 amino acids in the putatively non-coding euchromatic DNA of Drosophila melanogaster, and initially identified nearly 600,000 of them. We have studied the pattern of conservation of these smORFs as coding entities between D. melanogaster and Drosophila pseudoobscura, their presence in syntenic and in transcribed regions of the genome, and their ratio of conservative versus non-conservative nucleotide changes. For negative controls, we compared the results with those obtained using random short sequences, while a positive control was provided by smORFs validated by proteomics data.ConclusionsThe combination of these analyses led us to postulate the existence of at least 401 functional smORFs in Drosophila, with the possibility that as many as 4,561 such functional smORFs may exist.

Unexpected selection to retain high GC content and splicing enhancers within exons of multiexonic lncRNA loci

“…Indeed, a number of proposed lncRNA loci, at least in Drosophila (Ladoukakis et al 2011), zebrafish, and human (Bazzini et al 2014), may encode small open-reading frames (smORFs <100 amino acids). Because of the high GC content of protein-coding exons, we compared the nucleotide content of these smORFs to the remaining intergenic lnRNAs.…”

The developmental proteome of Drosophila melanogaster

“…Recently, there has been an increased interest in small proteins and translated small ORFs (smORFs) with up to 100 amino acids (aa) (Ramamurthi and Storz 2014), as their protein-coding potential is difficult to assess bioinformatically (Ladoukakis et al 2011). These small proteins localize to specific subcellular compartments and perform cellular functions as any other protein (Magny et al 2013).…”

New Peptides Under the s(ORF)ace of the Genome

“…One example is the Sarcolamban (Scl) gene [48], previously annotated as a non-coding gene [49], but identified by a bioinformatics approach as a potential functional smORF in Drosophila (Table 1; [17]). Scl encodes for two 28 and 29aa transmembrane related-micropeptides which act as inhibitors of SERCA calcium pump and regulate heart muscle contraction (Figure 2A; [48]).…”

“…Similarly, the Drosophila hemotin (hemo) gene was identified as a putative functional smORF by a computational study [17], and subsequently, its translation supported by ribosome profiling and proteomics studies [32,53] (Table 1). hemo is expressed in hemocytes (Drosophila macrophages) where it regulates endosomal maturation, and phagocytosis, by inhibiting the activity of phosphotidyl-inositol kinases through an interaction with 14-3-3z ( Figure 2B; [54]).…”

“…Generally, these methods were based on the analysis of sequence-composition frequencies ( Figure 1A; see sORFfinder and CRITICA in BOX1), and/or on the evaluation of: a) the conservation of candidate smORF sequences in related species using pair-wise alignment-based tools ( Figure 1B; BLAST [13]), and b) of their purifying selection (conservation of the aa relative to nt sequence) [14]. These initial studies identified several hundreds, and even thousands of putatively functional novel smORFs in the genomes of yeast, plants, flies, and mice [15][16][17][18][19], generally representing about 3-5% of the annotated genes in these organisms (Table 1).…”