Profiling the mTOR-regulated translatome in O. dioica
In order to determine whether or not the translation of trans-spliced TOP mRNAs is regulated by mTOR in O. diocia, we profiled translation genome-wide using ribosome profiling with deep sequencing [15] in day 6 female animals that were exposed to either the mTOR inhibitor Torin 1 or DMSO control. At this developmental stage the bulk of the animal’s mass is from a single-celled coenocyst [12, 16, 17] within the ovary, the transcriptional output of which is enriched for trans-spliced transcripts [5]. In parallel, we sequenced total RNA in order to normalise ribosome protected RNA fragments (RPFs) to the abundance of mRNA transcripts (RNA), giving a measure of translational efficiency for each gene in both treatment and control conditions.
We confirmed that exposing female O. dioica to the mTOR inhibitor Torin 1, in seawater, resulted in the expected absence of phosphorylated 4E-BP1 (Fig. 1a and Additional file 1: Figure S1): phosphorylated 4E-BP1 was absent after 1.5 h of treatment, similar to what was observed in mouse embryonic fibroblast (MEF) cells [9]. We also demonstrated that the commercial antibody we used was specific to the phosphorylated form of 4E-BP1 in O. dioica, as it is in other species (Additional file 1: Figure S1E). In addition, we used polysome profiling to show a global down-regulation of translation as indicated by reduced polysome peaks (Additional file 2: Figure S2).
We measured the effect of mTOR inhibition on the translation of individual mRNAs by quantifying and precisely mapping RPFs and normalising to total RNA to obtain the translational efficiency (TE) of each mRNA. This allows the detection of mRNAs with unusually high or low ribosome density given their transcript abundance. Sequencing generated 57.9 M (vehicle control: DMSO) and 42.8 M (Torin 1) total RNA exon-mapped reads and 24.1 M (DMSO) and 1.4 M (Torin 1) RPF exon-mapped reads, across three biological replicates (Additional file 8: Table S1). By excluding genes with low read counts (see Methods) we were able to confidently assess the translational efficiency of 14,574 expressed genes out of 17,212 in the O .dioica reference genome. Our results indicate that the mTOR-regulated translatome is conserved between O. dioica and vertebrates. As expected, we found the set of genes regulated by mTOR was enriched for classical TOP mRNAs, the majority of which are trans-spliced in O. dioica.
The mTOR-regulated translatome is conserved between O. dioica and mammals and enriched for TOP mRNAs that are SL trans-spliced in O. dioica
We detected 762 genes with mRNAs that had significantly reduced translational efficiencies when mTOR was inhibited (Fig. 1b and Additional file 9: Table S2). These represent the main targets of mTOR-mediated translational control in female O. dioica. Gene ontology (GO) analysis revealed that these were enriched for functions known to be regulated by TOR signalling, including translation and translation elongation, mitotic spindle elongation, fatty acid metabolism and TOR signalling (Additional file 3: Figure S3). Importantly, these include known TOP mRNAs: 60/127 expressed O. dioica ribosomal protein mRNAs (129 ribosomal proteins are annotated in the genome) and 59/89 mRNAs with orthologs to known human TOP mRNAs [18] (mostly ribosomal protein mRNAs) were significantly down-regulated (Fig. 1c, d and Additional file 10: Table S3, Additional file 9: Table S2). As found in mammalian cells [9], histone mRNAs were amongst those resistant to Torin 1 (Fig. 1c and Additional file 9: Table S2). We validated these results by qRT-PCR, testing 10 genes with the largest translational changes, which showed no statistically-significant changes in gene expression upon Torin 1 treatment (Welch Two Sample t-test, p > 0.05). The similarity of the mTOR-dependent translatome between O. dioica and mammals [9] indicates that the targets of mTOR regulation are likely conserved between O. dioica and it’s sister group, vertebrates. The down-regulation of translation of trans-spliced TOP mRNAs confirm that they are regulated by mTOR, indicating that the SL likely replaces the role of the critical TOP motif found in TOP mRNAs of other species.
Translation of trans-spliced TOP mRNAs is regulated by mTOR
The TOP motif in vertebrate canonical TOP mRNAs (ribosomal proteins and other members of the translational apparatus) is highly conserved and required for growth-dependent translational control via mTOR signalling [7]. A 5′ TOP motif is also enriched in ribosomal protein mRNAs in the ascidian Ciona intestinalis [5, 19]. The canonical TOP motif begins with a cytosine and is followed by a stretch of 4–14 pyrimidines [20]. It was recently shown in MEFs that mTOR regulates a broader spectrum of mRNAs [9]. These are enriched for the presence of a TOP-like pyrimidine-enriched motif (a stretch of at least 5 pyrimidines within 4 nucleotides of the transcription start site (TSS)) [9]. The majority of established TOP mRNAs, including those discovered recently, are trans-spliced in O. dioica [5]. These include 103 out of all 129 (80%) annotated ribosomal proteins (127 ribosomal proteins were expressed in day 6 females), 33 out of 40 eukaryotic translation initiation factors (including 4 out of 5 that are known TOP mRNAs), eukaryotic elongation factor 1A, eukaryotic elongation factor 2, translationally controlled tumour protein (TCTP), vimentin and rack1 (Additional file 10: Table S3). All these TOP mRNAs receive the 40 nt spliced leader (SL) RNA sequence at their 5′ ends. The 5′ end of this SL sequence [21] (ACTCATCCCATTTTTGAGTCCGATTTCGATTGTCTAACAG) is pyrimidine-enriched (12 out of the first 15 nucleotides are pyrimidines), although it starts with an adenine and is interrupted by several purines. This suggests that the 5′ end of the spliced-leader may function as a TOP motif in the mTOR-mediated regulation of translation. Our data showed that the translation of trans-spliced TOP mRNAs was suppressed upon the inhibition of mTOR: 51 out of the 60 O. dioica ribosomal protein mRNAs with translation significantly repressed by mTOR inhibition are trans-spliced (Additional file 10: Table S3).
Trans-spliced transcripts dominate the primary translational response to mTOR inhibition
Trans-splicing of mRNAs is not limited to TOP mRNAs but is associated with 39% of all O. dioica genes, a subset that is enriched for a wider array of functions related to growth [5]. Of the female-expressed genes that we analysed, 43% are trans-spliced. Since the translation of trans-spliced TOP mRNAs is mediated via mTOR in O. dioica, it opens the possibility that all trans-spliced mRNAs are potential targets for growth-dependent translational control. Indeed, we found that 352/762 (46%) of transcripts with suppressed translation upon mTOR inhibition are trans-spliced, although this is not significantly more than expected given the frequency of trans-splicing. Interestingly, however, we found that mRNAs that were suppressed only at the translation level, and not at the transcription level, were enriched for trans-spliced transcripts (56% of mRNAs with translation-only suppression are trans-spliced compared to 34% of those with both translational and transcriptional responses to mTOR inhibition, Fisher’s exact test P-value = 2.97 × 10− 9) (Fig. 2b). This shows that trans-spliced transcripts dominate the primary translational response to mTOR inhibition and indicates that non-trans-spliced transcripts constitute a longer-term, secondary response involving additional, slower transcriptional adjustment of gene expression. Indeed, GO analysis of these subsets revealed that genes with a transcriptional response to mTOR inhibition were enriched for functions related to proteolysis and muscle contraction (Fig. 2a), the latter being characteristic of genes with transcription down-regulated during growth arrest in O. dioica [5].
Oocyte-stocked mRNAs are trans-spliced and translationally dormant
Surprisingly, given the clear regulation of translation of trans-spliced TOP mRNAs, trans-spliced genes in female O. dioica were, on average, more resistant to mTOR inhibition (mean log2 (ΔTE) = − 0.28) than genes that were not trans-spliced (mean log2 (ΔTE) = − 0.74) (Welch two sample t-test: t = 24.484, df = 14,384, P-value < 2.2 × 10− 16). Trans-spliced transcripts that were not suppressed had significantly lower mean translational efficiencies (mean TE = 1.12), under control conditions, than transcripts that were not trans-spliced (mean TE = 2.76) (Welch two sample t-test: t = − 9.6612, df = 11,967, P-value < 2.2 × 10− 16) (Fig. 3a) and a significantly higher mRNA abundance (Welch two sample t-test: t = 74.861, df = 12,970, P-value < 2.2 × 10− 16) (Fig. 3b). This low level of translation under normal conditions may explain why these transcripts are insensitive to translational suppression via mTOR inhibition. The high abundance but low translation of these mRNAs suggests that they are sequestered: most likely in arrested oocytes, which contain a large fraction of the total RNA pool in this stage of the female O. dioica lifecycle and where the majority of transcripts are trans-spliced [5]. Fluorescent detection of nascent protein synthesis as well as polysome profiling showed that mRNAs in oocytes are indeed dormant (Fig. 3d,e). Therefore, the majority of weakly-translated, Torin 1-resistant, trans-spliced transcripts, likely represent dormant maternal mRNAs stocked in oocytes.
We used both tiling microarray [22] and cap analysis of gene expression (CAGE) [23] data from O. dioica oocytes to determine the set of oocyte-stocked mRNAs. As expected, we found that the translational efficiency of oocyte transcripts in control animals was significantly lower than that of non-oocyte transcripts (mean oocyte log2 (TE) = − 0.80; mean non-oocyte log2 (TE) = 0.45; Welch two-sample t-test: t = − 57.494, df = 14,002, P-value < 2.2 × 10− 16) (Fig. 3c, Additional file 4: Figure S4), and the effect of Torin 1 was significantly reduced (mean oocyte log2 (Δ) = − 0.17; mean non-oocyte log2 (Δ) = − 0.97; Welch two-sample t-test: t = 43.54, df = 12,832, P-value < 2.2 × 10− 16). Importantly, we found that 80% (4639/5772) of Torin 1-resistant trans-spliced transcripts were present in the oocyte. When we removed oocyte transcripts from our analysis, we found that transcripts with suppressed translation upon mTOR inhibition were enriched for those trans-spliced with the SL (28.6% of down-regulated genes are trans-spliced compared to 17.5% of unaffected genes; Fisher’s exact test P-value = 1.26 × 10− 7). This is despite excluding most TOP mRNAs, which have transcripts present in the oocyte. We obtained similar results when excluding all transcripts with low levels of translation (DMSO log2 (TE) < 1) under control conditions (36.6% of down-regulated genes were trans-spliced compared to 22.9% of unaffected genes; Fisher’s exact test P-value = 8.4 × 10− 11). These results show that while the effect of mTOR inhibition on TOP mRNAs is clear, its full effect on trans-spliced mRNAs in general was masked by the abundance of dormant mRNAs in oocytes. Once this is accounted for our results show that mTOR-regulated mRNAs are enriched not only for trans-spliced TOP mRNAs but for trans-spliced mRNAs in general.
A TOP motif is not encoded in the genes of mTOR-regulated transcripts
We next wanted to determine whether or not a TOP-like motif is present at the 5′ ends of translation-suppressed transcripts that were not trans-spliced. We obtained transcription start sites (TSSs) at bp-resolution in female animals from CAGE data [23] and examined the 5′ sequences of all expressed transcripts. Out of 2772 robustly expressed, non-trans-spliced transcripts, only 4 had a canonical TOP motif and only 66 had 5′ pyridine-enrichment comparable to the SL sequence. A more relaxed definition of a TOP-like motif (a stretch of at least 5 pyrimidines starting within 4 nucleotides of a TSS) [9] was also only present at a low frequency (0.076); lower than in mammalian cells (0.16) [9]. We found no significant enrichment of this motif at the 5′ ends of transcripts that had suppressed translation upon mTOR inhibition in O. dioica. Since the SL has a stretch of 5 pyrimidines further downstream we also relaxed the definition of the TOP motif further by searching for a stretch of at least 5 pyrimidines within 15 nucleotides of a TSS but still found no significant enrichment in suppressed transcripts. This indicates that these transcripts are indirect targets of translational suppression resulting from a global down-regulation of translation. In further support, a GO term analysis of mTOR-regulated transcripts lacking a spliced leader revealed an enrichment of functions related to autophagy (proteolysis) and lipid catabolism whereas those that were trans-spliced were enriched for known TOP mRNA functions related to protein synthesis. These results provide further evidence that the spliced leader supplies the TOP-like motif necessary for mTOR regulation and that the primary translational response to mTOR-inhibition is dominated by the selective suppression of trans-spliced mRNAs.
Trans-spliced transcripts in C. elegans are under growth-dependent translational control
We next sought to identify trans-spliced TOP mRNAs that are under mTOR regulation in another metazoan species. C. elegans trans-splices 70% of its mRNAs to one of two pyrimidine-enriched spliced leaders [4, 24]. SL1 is associated with monocistrons and the first gene in an operon and SL2 is associated with downstream operon genes. Included amongst these are all but one ribosomal protein gene (TOP mRNAs), which are mostly trans-spliced with SL1 [5]. A genome-wide study of translation during L1 diapause exit identified ribosomal protein mRNAs as transcripts with the highest translational up-regulation [25]. While no mention of the association of these transcripts with trans-splicing was made in this study, the data nevertheless clearly showed that trans-spliced ribosomal protein (TOP) mRNAs were targets of nutrient-dependent translational-control. Furthermore, a recent study showed that trans-splicing in C. elegans enhances translational efficiency [26].
In order to establish whether or not a relationship exists between the presence of SL1 and/or SL2 at the 5′ end of an mRNA and its translational control during recovery from growth arrest, we re-analysed existing ribosome profiling and mRNA-seq data from L1 diapause exit [25] together with existing data mapping trans-splice sites genome-wide in C. elegans [24]. We used a total of 10,362 genes that could be tested for differential translational regulation and assigned a trans-splicing category with high confidence. Amongst these, we found a strong relationship between the presence of a 5′ spliced leader and translational control during L1 diapause exit in response to food availability (χ2 = 711.45, df = 4, P value < 2.2 × 10− 16) (Additional file 5: Figure S5). Amongst transcripts with up-regulated translation, 54% (786/1460) are trans-spliced to SL1 and 18% (260/1460) are trans-spliced to SL2, while 414 (28%) lack a 5′ spliced leader (Additional file 5: Figure S5). This constitutes an enrichment of trans-spliced transcripts compared to unaffected transcripts (56% of which lack a 5′ spliced leader). These results show that trans-spliced TOP mRNAs in C. elegans are also under nutrient-dependent translational control, indicating that the spliced leaders in C. elegans may be targets of mTOR.
Exit from growth arrest in O. dioica is not dependent on mTOR
Having established that trans-spliced transcripts in female O. dioica are targets of mTOR-regulated translational control and that translation of trans-spliced TOP mRNAs are up-regulated during recovery from growth arrest in C. elegans, we next wanted to assess the translational regulation of trans-spliced transcripts during recovery from growth arrest in O. dioica.
We previously proposed that translational control, rather than transcriptional control, may up-regulate trans-spliced growth-related genes during recovery [5]. To test this we performed ribosome profiling followed by deep sequencing, together with total RNA sequencing, on O. dioica during growth arrest (stasis: animals were collected on day 7, one day beyond their normal 6-day lifespan) and recovery from growth arrest (release into normal animal density).
Sequencing generated 27.0 M (stasis) and 38.6 M (release) total RNA exon-mapped reads and 1.8 M (stasis) and 1.5 M (release) RPF exon-mapped reads, across two biological replicates.
We detected 1601 genes with significantly up-regulated transcription and 638 with significantly down-regulated transcription during release from stasis. Consistent with our previous observations [5], genes that were transcriptionally up-regulated were enriched for muscle-related GO terms and trans-splicing was under-represented in this set (Additional file 6: Figure S6).
We then analysed differential translational efficiency and found 1382 genes with significantly up-regulated translational efficiency upon release from stasis and only 28 significantly down-regulated (Fig. 4). Surprisingly, we found that only 8/129 ribosomal protein mRNAs were up-regulated (Fig. 4b). Trans-spliced transcripts were not over-represented in the set of up-regulated genes and the mean change in translational efficiency was not significantly different between trans-spliced and non-trans-spliced transcripts (t-test: t = − 0.32652, df = 13,368, P value = 0.744). GO terms that were over-represented in the set of genes with up-regulated translational efficiencies included terms related to muscle contraction, hormone regulation and the cell cycle (Additional file 7: Figure S7), rather than terms typical of the mTOR-dependent translatome we identified in our mTOR-inhibition experiments.
These results show that up-regulation of nutrient-dependent growth-related genes (genes regulated by mTOR) is not the initial response to release from growth arrest in O. dioica. Supporting this, replication tracing by EdU incorporation showed that endocycling, which is suppressed during growth arrest, resumed in released animals regardless of whether or not food was available (Fig. 4c-g).