Comparative genomic analysis of novel conserved peptide upstream open reading frames in Drosophila melanogaster and other dipteran species

Background Upstream open reading frames (uORFs) are elements found in the 5'-region of an mRNA transcript, capable of regulating protein production of the largest, or major ORF (mORF), and impacting organismal development and growth in fungi, plants, and animals. In Drosophila, approximately 40% of transcripts contain upstream start codons (uAUGs) but there is little evidence that these are translated and affect their associated mORF. Results Analyzing 19,389 Drosophila melanogaster transcript annotations and 666,153 dipteran EST sequences we have identified 44 putative conserved peptide uORFs (CPuORFs) in Drosophila melanogaster that show evidence of negative selection, and therefore are likely to be translated. Transcripts with CPuORFs constitute approximately 0.3% of the total number of transcripts, a similar frequency to the Arabidopsis genome, and have a mean length of 70 amino acids, much larger than the mean length of plant CPuORFs (40 amino acids). There is a statistically significant clustering of CPuORFs at cytological band 57 (p = 10-5), a phenomenon that has never been described for uORFs. Based on GO term and Interpro domain analyses, genes in the uORF dataset show a higher frequency of ORFs implicated in mitochondrial import than the genome-wide frequency (p < 0.01) as well as methyltransferases (p < 0.02). Conclusion Based on these data, it is clear that Drosophila contain putative CPuORFs at frequencies similar to those found in plants. They are distinguished, however, by the type of mORF they tend to associate with, Drosophila CPuORFs preferentially occurring in transcripts encoding mitochondrial proteins and methyltransferases. This provides a basis for the study of CPuORFs and their putative regulatory role in mitochondrial function and disease.


Background
It is becoming increasingly clear that controlling protein levels post-transcriptionally is an important mechanism for growth and development in eukaryotic cells. Upstream start codons (uAUGs), AUGs found 5' of the longest, or major, open reading frame (mORF), occur in 20-50% of eukaryotic mRNAs of a given genome [1][2][3][4][5]. When translation is initiated at a uAUG, these upstream ORFs (uORFs) can affect the protein level of the mORF with serious biological consequences. uORFs can regulate mORF protein production in response to starvation conditions [6], polyamine concentrations [7,8], and sucrose levels in the cell [9]. For example, the yeast General Control Nondepressible 4 (GCN4) transcript contains multiple uORFs that differentially regulate the protein level of a transcription factor-encoding mORF under starvation and non-starvation conditions. In turn, the protein produced from the mORF, the GCN4 protein, is essential to the transcriptional activation of some 40 genes involved in amino acid biosynthesis [6]. Because uORFs influence the levels of mORF protein, it is not surprising that disruption of the uAUG can lead to human disease such as thrombocythemia [10], a disease which is thought to arise as a result of increased mORF protein product, thrombopoietin (TPO). In addition, uAUGs occur in transcripts coding for oncogenes more frequently than other mammalian transcripts [11]. Indeed, oncogenes Mdm2 [12], her-2 [13], MYEOV [14], Bcl-2 [15], and SCL [16], all contain uORFs that affect the level of oncoproteins produced.
Potentially thousands of genes are regulated via uORFs, but there are no demonstrated examples of uORFs affecting mORF protein production in Drosophila or other insect species. Several uORF-containing genes have been well studied in fungi, plants, and mammals [17] and genomewide searches of conserved uORFs have been conducted using fungal, mammalian and plant transcripts [4,[18][19][20][21].
Given the examples found in other eukaryotic species, it is plausible that uORFs fill a regulatory role in the arthropod lineage as well.
There is some evidence that regulatory uORFs may occur in insect species. Firstly, a Drosophila gene coding for a putative mannosyl transferase contains a uORF-mORF pair that seems to be evolutionarily conserved in insects [19]. Secondly, there are several examples of Drosophila dicistronic transcripts in which the first open reading frame could be regulatory to the second [22][23][24]. However, polycistronic transcripts do not all code for putative uORFs; many transcripts defined as polycistronic are initially transcribed as pre-mRNA with two or more ORFs, but are subsequently processed into separate monocistronic transcripts [25]. For this reason, we prefer to use the terminology 'uORF' to refer to an ORF (a) which is upstream of a mORF on a single mature mRNA, and (b) which is itself translated as a polypeptide distinct from protein translated from a mORF. In addition, polycistronic transcripts that are not processed into separate mRNA molecules are at times part of this uORF/mORF classification. The computational identification of dicistronic transcripts by Misra et al [22] resulted in the reannotation of 31 gene models, some of which may contain conserved uORF-mORF pairs. However, their search was limited to polycistronic transcripts with ORFs greater than 50 a.a., and it is known that uORF peptides as short as 6 a.a. can regulate mORF translation in mammals [26].
Their analysis also discarded overlapping ORFs, some of which are important for the regulation of mORFs [27].
To identify transcripts with uORFs that are likely to be translated, we took a comparative genomics approach using D. melanogaster transcript annotations, Anopheles gambiae transcript annotations, and dipteran expressed sequence tags (ESTs). Using this approach, we determined the prevalence, diversity, and genomic clustering of CPuORFs under negative selection in dipteran genomes and compared these findings to those reported for the plant lineage.

Identification of conserved peptide uORFs in D. melanogaster
To determine the prevalence of uORFs most likely to be translated, Drosophila melanogaster release 4.3 transcript sequences (19,389) were used to identify the largest, or major, ORF (mORF). Of these, 13,746 contain unique Flybase gene numbers, 5,851 of which contain one or more AUGs upstream of the mORF. This suggests that 43% of Drosophila mORF proteins could be affected in their expression level by translated uORFs. Our calculated percentage is slightly lower than previously reported Drosophila uAUG frequencies [2], but this discrepancy can be explained by the smaller dataset used in the previous study.
Putative dipteran homologs were found by comparing D. melanogaster mORFs to 666,153 NCBI ESTs using tBLASTn. Many of the EST sequences contained truncated uORF and mORF sequences, therefore the search was limited to species that diverged from D. melanogaster more than 15 Mya (non-melanogaster group species; AAA: 12 Drosophila Genomes Website) [28,29], to increase detection of negative selection acting on short protein sequences. For each pair of homologs, global alignment of uORFs identified candidate CPuORFs and K a /K s ratios were used to further verify evolutionary conservation of the uORF amino acid sequence. In addition, Flybase transcript annotations were used to discard any genes in which the putative CPuORF was fused to the mORF in any given transcript splice variant.
Another indicator of translation is start codon context. Based on nucleotide frequencies of sequences surrounding mORFs, it is predicted that the Drosophila optimal consensus sequence is CAaaAUGg [2,30], but no functional experiments have been conducted in insects to validate the strength of this initation context. Therefore, although the predominant CPuORF start context (AAaaAUGa) seems to be weaker than the predominant mORF context, it remains to be determined whether ribosomes initiate efficiently at the uORF AUG. It is also quite likely that initiation of some CPuORFs is dependent upon cellular conditions, as has been shown in various genes [6,31], leading to regulation of mORF protein levels.
A number of uORF-mORF pairs were used as positive controls for the modified uORF-Finder program. In a previous study, CG9865 was shown to contain a putative uORF-mORF pair that has been conserved among distantly related insect species [19]. This gene was identified by our analysis, therefore validating our approach. Drosophila Tat-like (DTL), a gene containing a uORF with amino acid similarity in D. melanogaster and D. pseudoobscura [24] was also found by the uORF-Finder program. A third gene identified by our analysis, CG10238, is a bicistronic transcript encoding the small and large subunit of Molybdopterin synthase 2 (MOCS2) [23]. It is well conserved across distantly related eukaryotic species (see Additional File 2). In addition, 5 of the 31 dicistronic genes described by Misra et al [22] were shown to contain CPuORFs (Table 2; denoted by Misra and colleagues as CG33071ORFA-CG33071ORFB, Tim9b-CG12788, CG33009ORFA-CG33009ORFB, CG33005ORFA-CG33005ORFB, and snapin-CG9960, but subsequently renamed CG33713-CG33714, CG12788-CG17767, CG33671-CG33672, CG33786-CG33785, and CG9960-CG9958, respectively). Many of the dicistronic transcripts identified by Misra et al [22] are transcripts with ORF pairs that are not well conserved among the Drosophila species. For example, the mei217-mei218uAUG is not conserved in any of the 11 other sequenced Drosophila genomes (UCSC D. melanogaster genome browser) [32], therefore it is not surprising that a number of the dicistronic genes were not identified by the uORF-Finder program. Additionally, it is likely that neither the D. melanogaster annotations nor the dipteran ESTs are representative of the complete transcript population within each species due to the incomplete annotation of 5' transcription start sites [33], and incomplete coverage of the genomes by ESTs.
Initially, 41 genes and 43 uORFs showed evidence of mild to strong purifying selection (K a /K s ratio significantly < 1), and an additional gene with one uORF was detected dur-ing subsequent duplication analysis (see below). The proportion of genes in the Drosophila genome showing evidence of CPuORFs is approximately 0.3% (42 genes out of 14,040 genes), which is similar to the frequency predicted for the Arabidopsis genome (0.4-0.5%) [19]. The present study likely underestimates the prevalence of CPuORFs due to incomplete EST resources and potentially misannotated 5' regions in D. melanogaster.
Consistent with calculated K a /K s values, the majority of CPuORFs with a low K a /K s ratio are present in lineages beyond the Drosophilidae (Table 1) and therefore have been conserved more than 40 My (Assembly/Alignment/ Annotation of 12 Drosophila species) [28,29]. Those uORFs that exhibit a low K a /K s ratio but are only found within Drosophila species may represent uORFs that have recently emerged within the Drosophila lineage but are nonetheless under mild to strong selection pressures.

Insect CPuORFs are longer in average length than plant CPuORFs
Two studies have shown that the length of a uORF can influence the ability of a ribosome to reinitiate scanning and translation initiation at a mORF [34,35]. The plant and mammalian cell systems used in these studies show that reinitiation at a downstream AUG is generally more efficient in the presence of shorter uORFs, and in plant protoplasts reinitiation drops sharply in constructs containing uORFs longer than 34 amino acids. Both studies were carried out using viral components, and as such it is not clear whether these observations extend to mRNAs in a native eukaryotic cellular environment. Nonetheless, uORF length could play an important role in the regulation of mORFs, therefore we analyzed Drosophila CPuORFs in terms of their amino acid lengths. Initial characterization of the 44 putative CPuORFs under negative selection reveals a wide distribution of lengths, ranging from 15 to 179 amino acids (Table 2, Figure 1A).
To date, most, if not all, functionally characterized uORFs are smaller than 100 amino acids, but more than one fourth (12/44) of D. melanogaster CPuORFs are above this size. In general, Drosophila CPuORFs seem to be larger than those found in plants. While 83% of Arabidopsis CPuORFs are between 21 and 60 amino acids in length (mean of 40 amino acids ± 16 standard deviation; Figure  1B), the Drosophila uORF length distribution peaks between 41 and 80 amino acids (mean of 76 amino acids ± 44; Figure 1A). These plant and insect datasets were not generated by comparing species with the same evolutionary distance but a more convincing comparison can be made by analyzing uORFs that have been conserved over more than 200 My: between Arabidopsis and rice, and between Drosophila and non-Brachycera lineages (e.g. Anopheles). The Arabidopsis distribution peak remains essentially unchanged under these restrictions (mean of 39 amino acids ± 13), whereas the distribution of Drosophila uORFs peaks at an even greater length, 81-100 amino acids (mean of 92 amino acids ± 29; Figure 1C). Longer uORF lengths in Drosophila may reflect a need for stronger suppression of mORF translation than in plants, consistent with the observations of the above-mentioned cell culture studies. Alternatively, insect cells may exhibit more efficient reinitiation resulting in a requirement for longer uORFs to attenuate mORF translation.
Conserved peptide uORF length distribution   (Table 1) and compared to a random distribution (Methods). uORF frequencies were not statistically different from a randomly generated dataset except for a cluster of 6 uORFs residing on band 57 (p-value = 10 -5 ), five of which fall on a much smaller segment of the chromosome, band 57F. Upon closer examination, some of these uORFs may have arisen as a result of tandem duplications; one uORF found in the CG30290 transcript as well as two uORFs found in the CG9865 transcript (uORF1 and uORF3) all contain twin CX 9 C motifs. Interestingly, the observed clustering is not dependent upon the putative duplication events of CX 9 C motif-containing uORFs. Eliminating the duplication bias by collapsing CX 9 C-containing uORFs to one representative, clustering is still statistically significant, with 4 uORFs on cytological band 57 (p-value = 0.004) and 3 uORFs on band 57F (p-value = 0.0002). Therefore, the data suggest that there is a preponderance of both clustering and duplicate retention of uORFs on band 57. Clustering at this region could be an indicator of chromatin interactions at this site that could mediate CPuORF regulation.
The twin CX 9 C motif is an integral part of coiled-coil helix, coiled-coil helix (CHCH) domains, a domain previously implicated in uORF-mORF associations in group 8 plant uORFs [19]. In fact, the group 8-like Drosophila uORF member described in the plant study is uORF3 of CG9865. It is interesting to note that the plant group 8 uORF has consistently lost its duplicate copy during both recent and ancient polyploidy events whereas the Drosophila group 8 putative homologue may be retaining its duplicates. Different duplication retention histories could indicate that twin CX 9 C motif-containing ORFs play different roles in plants and animals.

CPuORF-mORF pair duplicate retention is low within Drosophila melanogaster
To determine whether there has been retention of uORF-mORF pair duplicates within the Drosophila genome itself, the 41 mORFs with strongly conserved uORFs were compared to the D. melanogaster transcriptome. A single gene, CG17325 showed evidence of a duplicate copy, CG10570, in which the uORF-mORF pair is conserved (See Additional File 3). CG10570 was not detected by our program due to the short length of its mORF (< 100 amino acids), therefore this gene was added to our list of CPuORFs following our duplication analysis (Tables 1  and 2). CG17325 and CG10570 reside adjacent to one another on chromosome 2, band 37A4-A5, and are transcribed on opposite strands away from one another. The close proximity of the genes suggests a segmental duplication gave rise to the two genes, both of which are conserved throughout the Drosophila lineage and exhibit a K a / K s ratio < 0.28 (Table 1). This duplication presumably occurred more than 40 Mya since both loci are present in D. melanogaster, D. grimshawi, and D. virilis. Unlike the extensive uORF-mORF duplication retention history of the Arabidopsis genome, CG17325 and CG10570 were the only example of gene duplicate retention in Drosophila.

GO term and protein domain analysis suggest a link between CPuORFs and both mitochondrial proteins and methyltransferases
Further differences between plant and insect CPuORFs were observed following gene ontology (GO) term analysis. GO term frequencies in the D. melanogaster genome were compared to frequencies in the insect uORF dataset to look for overrepresentation of terms. P-values were determined using the Bonferroni correction method, a method that accounts for multiple comparisons and calculates a conservative p-value. Also, the recent tandem duplicate (see above) was not included in the analysis to eliminate bias from recent duplication events. Because GO terms have been assigned to all ORFs found in bicistronic transcripts, GO terms were extracted for both uORF and mORF gene identifiers, designated hereafter as the uORF dataset (41 mORFs and 7 uORFs). This analysis differs from previous analyses in plants; it not only identifies 1) classes of mORF proteins that tend to associate with CPuORFs, but it also identifies 2) ORFs that preferentially associate with other ORFs on a single transcript. In plants, a large proportion of CPuORFs associate with mORFs encoding transcription factors, however this trend was not observed in insects. Instead, mORF proteins showing evidence of N-methyltransferase activity (GO term for CG9666 and CG9960 mORFs; Table 3) tend to associate with CPuORFs (p = 0.02). This methyltransferase activity may act on DNA or RNA, since both types of Interpro domains are overrepresented in these two genes.
Additionally, overrepresentation of GO term 'protein import into the mitochondrial inner membrane' is driven by two proteins in the Drosophila uORF dataset, CG9878 (Translocase of inner membrane 10, Tim10) and CG17767 (Tim9b), which contain the Interpro Zn-finger Tim10/ DDP-type domain (p = 0.01). Unlike the overrepresented methyltransferase domain, the Tim10/DDP-type domain is not limited to the mORFs, but appears in either the uORF or mORF, demonstrating that these ORFs show a preference for associating with other ORFs in a transcript. Specifically, Tim10 is encoded by the mORF of its transcript while Tim9b is encoded by the uORF. This does not imply that Tim9b does not act as a regulatory uORF, however. Tim9b may act both as a chaperone in the intermembrane space, as well as a regulatory element controlling the translation of its associated mORF.
In support of a model in which mitochondrial proteins preferentially associate with other ORFs on a single transcript, a further connection to the mitochondrial inner membrane is found when examining other genes in the uORF dataset. The CG8026 mORF encodes a putative mitochondrial folate transport protein [37,38] (Table 4). Interestingly, this trend may extend to the mammalian lineage, exemplified by the human Uncoupling protein 2 (UCP2) mORF, a putative inner mitochondrial membrane transporter. The UCP2 mORF is not only associated with what appears to be a CPuORF, but it is regulated by its uORF in a glutamine-dependent manner [39]. B-cell lymphoma 2 (BCL-2) is another mammalian oncogene that produces a protein from its mORF, BCL-2, which is localized to mitochondria [40] and is associated with a functional uORF [15].
Other Drosophila genes also have potential links to the mitochondrion, such as CG18624, a putative NADH dehydrogenase that is predicted to act in mitochondrial electron transport (Table 4). Also, uORF1 of CG9865 is a putative homolog of p8Mature T-Cell Proliferation 1 (p8MTCP1), an ORF that is transcribed on the same mRNA as p13MTCP1, is targeted to mitochondria [41], and may play a role in oncogenesis [42,43]. CG9865 uORF1 has a twin CX 9 C motif, as do p8MTCP1 and other proteins targeted to mitochondria, namely yeast proteins Mitochondrial Ribosomal Protein 10 (Mrp10p) [44], Cytochrome Oxidase 19 (Cox19p) [45], Cytochrome Oxidase 17 (Cox17p) [46], and Mitochondrial intermembrane space Import and Assembly 40 (Mia40p) [47]. In humans, the twin CX 9 C motif found in Mia40p is required for import and stable accumulation of Mia40 in the intermembrane space [48]. Several genes in the uORF dataset contain ORFs with CX 9 C motifs, such as uORFs 1 and 3 of CG9865, the uORFs of CG30290 and CG9288, and the mORF of CG7950 (See Additional File 2). These open reading frames could be interacting with other ORFs on the same transcript to target them to the mitochondria or to form a stabilizing protein complex.
It is possible that these ORF associations are vestiges of ancient prokaryotic operons that originated in the mitochondrion and were transferred to the nuclear genome over time. This hypothesis runs counter to the prevailing thought that mitochondrial proteins involved in transport are generally of eukayotic origin [49]. Regardless of their origin, nuclear ORFs coding for mitochondrial proteins may maintain an association with other ORFs on a single transcript over long periods of evolutionary time for several reasons. Both ORFs may be co-regulated at the transcriptional level and be required at similar times in development, thus providing more efficient transcription of DNA. Alternatively, the uORF may be regulating expression of the mORF with important biological consequences. These possibilities are not mutually exclusive and further experimentation will be required to determine whether this energy-producing organelle is influenced by the translational regulation of uORF-mORF pairs on single transcripts.
Interestingly, the trend in animal mitochondrial ORFs was not observed in plants. Instead, plant uORFs tend to associate with mORFs encoding transcription factors [19]. Perhaps these unique characteristics reflect fundamental differences in the two eukaryotic lineages. Despite their differences, plants and animals both seem to contain uORF-mORF pairs involved in a wide range of biochemical and regulatory pathways (Table 4). There is some evidence in the literature that transcripts with uORFs can occur in similar biochemical pathways, such as genes affecting the polyamine biochemical pathway [50], but this is the exception rather than the rule and no additional examples have been born out by our analyses. To facilitate future studies of these elements, all CPuORF annotations will be submitted to Flybase.

Conclusion
The identification and characterization of putative CPuORFs has established a knowledge base from which many hypotheses have been generated and can now be  tested. CPuORFs in dipterans show similarities to their plant counterparts in terms of their prevalence within the genome and diversity of sequence, but differ in their greater average length, their genome clustering, and their preferential association with methyltransferases. In addition, the present analysis has shown a significant correlation between mitochondrially-targeted proteins and transcripts containing uORFs, an observation that could lead to important discoveries impacting our understanding of human disease. Given the wealth of genetic tools available in Drosophila, this model system is ideally suited to the basic understanding of uORF-containing transcripts and post-transcriptional regulation.  [28,29], their transcript sequences are of limited use in detecting strong negative selection over short sequence lengths due to the accumulation of few synonymous and non-synonymous substitutions. Therefore these species were excluded from this first comparison, as were D. melanogaster ESTs.

Identification of conserved peptide uORFs
Comparative analysis of D. melanogaster and A. gambiae sequences was performed using uORF-Finder [19], a program that identifies the longest open reading frame of a transcript in the first species (defined as the mORF), finds the putative homolog in the second species, and aligns all open reading frames upstream of these homologs to identify putatively conserved uORFs. uORF-Finder was designed to compare full-length cDNA sequences from two species, therefore to accommodate a D. melanogaster full-length transcript-to-dipteran EST comparison, the program was modified and putative homologs in the ESTs were identified using the first 100 amino acids of the D. melanogaster mORFs. uORF size was also limited to 200 amino acids (no additional uORFs were found when uORF size was limited to 300 a.a.  uORF start and stop codons [32]. Any putative uORF sequences that showed evidence of in-frame fusion with the mORF on the UCSC browser (in an alternative splice form, for example) were not included in the final list of CPuORF-containing transcripts.

Calculation of K a /K s
The K a /K s ratio was determined using pairwise_kaks.PLS (version 1.7) [54] and is derived from the highest scoring BLAST homolog in the D. melanogaster-dipteran high scoring pairs. Both the approximate method (option -kaks yn00) and the maximum likelihood method (-kaks codeml) were used. Only the approximate method calculation is reported in Table 1 due to the typically short evolutionary distance between the organisms found in the highest scoring BLAST pairs. The Nei-Gojobori p-distance model was used to test for purifying selection (Null hypothesis K a = K s ; alternate hypothesis K a <K s ). MEGA4 default settings were used to run codon-based Z-test analyses [55] on highest scoring BLAST homologs.

Cytological distribution of uORFs
To determine whether the 44 uORFs were randomly distributed along the Drosophila chromosomes relative to annotated transcript positions, a perl script was written to generate a random distribution of 44 positions along the chromosomes. Cytological positions for each CG gene identifier were extracted from D. melanogaster release 4.3 gene annotations [51], from which 44 positions were randomly chosen. This ensured that clustering would not simply reflect gene rich regions. The number of 'hits' within a given cytological band were tallied, and the entire process was iterated 30,000 times, providing a random distribution of 'hits' at any given band when 44 positions were picked across the entire genome. The random distributions were then used to provide a p-value for the observed number of uORFs within a given cytological band.

Gene Ontology, Pfam domain, and Interpro domain retrieval and analysis
Over-and under-representation of Gene Ontology (GO) terms in the uORF dataset (41 mORFs and 7 uORFs with associated GO terms) versus the D. melanogaster genome was determined using Genemerge v.