5′-C transcripts enriched in poorly differentiated, proliferative cancers

Tumorigenesis is often marked by increased activity of mRNA translation machinery, encoded by transcripts bearing the 5′TOP motif and transcribed from YC initiators6,7,8,9,10,11,26. Since transcripts initiating from YC dinucleotides are pervasive across the genome23, we reasoned that 5′TOP mRNAs in tumor development may be extended to YC RNAs produced in dual-initiating genes, owing to a potential shared targeting mechanism. To test this hypothesis, we measured 5′-C RNA content in various types of tumors representing different phases of tumorigenesis.

YR and YC initiation (Fig. 1a) can be distinguished by 5′ end detection at nucleotide resolution by cap analysis of gene expression sequencing (CAGE-seq)23. CAGE-seq detects altered transcription initiation patterns in cancer as demonstrated by the promoter of GUCD1 (encoding guanylyl cyclase domain containing 1), a gene associated with enhanced cell proliferation and invasion of cancer cells27,28. CAGE-seq analysis in published FANTOM5 datasets29,30 revealed it to be a dual-initiator promoter (DIP) in healthy colon, with both YC and YR initiation present. In CRC samples, however, the YC component of expression is dramatically enhanced at the expense of the YR component (Fig. 1b), while overall expression is moderately changed. The dynamic shift in initiation site usage is invisible by traditional RNA-seq transcriptomics, although a slight elongation of reads mapping to the first exon occurs (Fig. 1b).

We then asked about the pattern of 5′-C transcript abundance in other DIPs by surveying FANTOM5 CAGE-seq data from 59 datasets, representing 17 cancer and matched healthy tissues (Supplementary Table 1). We identified CAGE transcription start sites (CTSS) that clustered into 17,480 consensus clusters between samples, representing promoter regions as previously described31. CTSS within consensus clusters were segregated into YR and YC classes with tag per million (TPM) values calculated. Dual-initiating promoters were identified (n = 3,475) with consensus clusters of >1 TPM for both 5′-R transcription and 5′-C transcription in the majority of datasets (30 datasets). The ratio of YC to YR transcription was calculated for these DIPs across the datasets and compared between matched cancer and healthy samples. This analysis revealed that the majority (22 out of 30) of cancer cell types had significantly enhanced YC transcription within DIPs (YC-enriched tumors). There was considerable heterogeneity between cancer types, with four cancers with significantly depleted 5′-C transcript usage relative to their matched healthy cell types (YC-depleted tumors) and four cancers with no significant change (YC-neutral tumors; Fig. 2a).

Fig. 2: 5′-C transcripts are most enriched in poorly differentiated and proliferative cancer types.

Dual initiators (promoters with >1 TPM for both YC and YR transcription in the majority of datasets (>30 out of 59)) were identified across all selected FANTOM5 cancer and healthy tissue CAGE datasets (n = 3,475). The relative expression of the YC versus YR component of transcription for each dual initiator was calculated and compared between matched cancer and healthy tissues. a, Table of cancer samples ordered by mean log2 fold change (log2(FC)) in the ratio of YC to YR transcription of dual-initiator promoters between cancer and matched healthy tissue. P value (paired two-tailed t-test) of this expression change is also shown (ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; full list of P values available in the Source data). This table is color-coded to show cancers where YC transcription at dual initiators is significantly enriched (orange), unchanged (gray) or depleted (green) relative to the matched healthy tissues. b, The differentiation status of each cancer sample was identified where publicly available. They were then separated into undifferentiated, moderately differentiated and well-differentiated cancer types, and the distribution of each sample’s mean log2(FC) in YC:YR transcription (as calculated in a) was plotted for each group. The distribution of TP53-mutant samples is also shown by the color of each plot point. c,d, Biological process ontology of genes specifically upregulated in YC-enriched cancers (n = 132) (c) and YC-depleted cancers (n = 144) (d). FDR, false discovery rate.

Source data

To identify features linked to enriched YC initiation, we assessed publicly available profiling data for well-characterized tumor cell lines (Supplementary Table 2). Segregating the tumors by differentiation status showed a clear contrast in the average YC usage of DIPs between poorly differentiated (100% YC-enriched) and well-differentiated (86% YC-depleted) tumors (chi-squared test, P < 0.001) (Fig. 2b). Of note, tumor protein P53 (TP53) mutation status also trended with YC-enriched cancers, with mutation-bearing tumors representing 48%, 25% and 20% of YC-enriched, YC-neutral and YC-depleted tumors, respectively (chi-squared test, P = 0.42) (Fig. 2b and Supplementary Table 2). To further mutational associations, data were extracted from the Cancer Cell Line Encyclopedia32, with coverage for 24 of 30 lines. This revealed that mutations in KIAA0586, CLCN3, ZNF22, MRPS16, DLEU7, TBX2, MKKS, DVL1 and ADGRG7 were all significantly associated with YC-depleted cancers, while USH2A mutations were significantly associated with YC-enriched cancers (Supplementary Table 3).

To further explore the factors segregating the YC-enriched versus YC-depleted cancer types, we identified genes with expression directly correlated with YC. We identified genes with an average of greater than twofold enrichment in expression in YC-enriched cancers versus matched healthy tissues and a greater than twofold depletion in expression in YC-depleted cancers versus matched healthy tissues (Supplementary Table 4). Almost half of these genes (22 out of 52) were associated with differentiation or stem cell character (Supplementary Table 4), suggesting a link between tumor differentiation status and YC–YR transcription initiation choice. This is exemplified by the dual-initiating oncogene ABI1 (encoding Abl Interactor 1), a component of the WAVE complex33. In healthy bronchial epithelial cells, and in the well-differentiated lung cancer line (PC9), its expression was predominantly from YR initiation sites. In the undifferentiated lung cancer line (A549), however, initiation switched dramatically towards a dominant YC transcription initiation site (Extended Data Fig. 1a).

Next, we sought to identify genes specifically upregulated in YC-enriched versus YC-depleted cancers. We identified genes consistently upregulated in YC-enriched cancers over matched healthy tissues (enriched by more than twofold in >75% of samples) and unchanged or depleted in all YC-depleted cancers and vice versa. This analysis identified 132 and 144 genes specific to YC-enriched or YC-depleted cancers, respectively. Gene ontology analysis revealed cell cycle control and proliferation genes upregulated in YC-enriched cancers (Fig. 2c) in contrast to cell migration in YC-depleted cancers (Fig. 2d). To further investigate this finding, we correlated YC dynamics with published cell doubling rate and metastatic association (Extended Data Fig. 1b,c) in cancer cell lines. There was no association with doubling rate, but a non-significant enrichment for cancers from patients with metastasis at collection in the YC-depleted cohort was found (metastasis reported in 36.3%, 0% and 75% of YC-enriched, YC-neutral and YC-depleted cancers, respectively; chi-squared test, P = 0.16).

We asked which DIPs showed a cancer cell type-defining switch in YC:YR TSS usage. We calculated the average YC:YR ratio for each DIP within each cohort, selecting promoters in which the YC:YR ratio dynamically changed from the highest value in YC-enriched cancers, to an intermediate value in YC-neutral cancers, to the lowest value in YC-depleted cancers. A total of 422 genes showed a cohort-dependent trajectory of YC:YR transcription initiation levels (Extended Data Fig. 1d and Supplementary Data. 1) with significant enrichment for chromatin and organelle organizational genes, in line with the proliferative activity of YC-enriched cancers, as well as genes regulated by the PI3K–Akt–mTOR and Myc regulatory axis (0.03–0.05 false discovery rate) (Extended Data Fig. 1e). Both these pathways regulate 5′TOP-containing ribosome gene transcripts and ribosome biogenesis18,19, and our findings suggest their wider role in regulating YC:YR ratios and differential RNA metabolism from dual-initiating promoters.

Taking these findings together, a segregation of transcripts between YC-enriched and YC-depleted cancers was seen, with enrichment of YC initiation correlated with poorly differentiated, proliferative (and potentially TP53-mutated) cancer subtypes. Intriguingly, these factors have all been associated with radiotherapy response34,35,36,37 and raise the potential for TSS as a prognostic indicator of tumor subtypes and therapy response.

Enriched YC initiation marks radiotherapy-responsive CRC tumors

To investigate whether 5′-C transcript abundance influences radiotherapy response, we focused on CRC, one of the YC-enriched cancer types (Fig. 2a). We performed CAGE-seq on treatment-responsive and non-responsive CRC formalin-fixed paraffin-embedded (FFPE) pre-treatment biopsy specimens (Supplementary Table 5). Samples were selected based on the response of the donor tumors to a standard course of neoadjuvant chemo-radiotherapy (45–50 Gy over 32–39 weeks, alongside capecitabine/5-FU) with either robust tumor regression (M1) or no response (M4–M5) (Extended Data Fig. 3a and Supplementary Table 5). Four responsive and four non-responsive tumor samples were sequenced using FFPEcap-seq38 and mapped to the GRCh38/hg38 genome. Around 55–60% of CTSS mapped to promoter regions, as expected for FFPEcap-seq38 (Extended Data Fig. 2a and Supplementary Table 6). Biological replicates of responsive and non-responsive CRC tumors were merged, and total expression from all CTSS with YR or YC initiation was compared between the cohorts (Fig. 3a). The YC:YR ratio was significantly (P < 0.001) shifted, with YC initiation enriched in tumors responsive to chemo-radiotherapy.

Fig. 3: Enriched YC initiation marks radiotherapy-responsive CRC tumors.

a, Bar graph of the total expression from all CTSS within consensus clusters, initiating with YR or YC dinucleotides, between the responsive and non-responsive CRC clinical tumor cohorts (chi-squared, P = 0.0001). b,c, Dual-initiating promoters were identified as before (n = 186). The proportion of transcription initiating in each dual-initiator promoter, from the YC and YR sites, was quantified for each sample and compared between them on a per-promoter basis. b, Frequency distribution graph showing the degree of expression change of the YC component (normalized to YR) of each dual promoter between responsive and non-responsive CRC clinical tumor samples (paired two-tailed t-test, P = 0.023). c, Frequency distribution graph as in b but with each expression component (YR and YC) separately analyzed (paired two-tailed t-test, *P = 0.013). d, Plot of relative survival of the five CRC organoid cultures under study, between those irradiated with 25 Gy versus 0 Gy (n = 3 independent experiments, data are presented as mean values ± s.e.m.). e, Bar graphs of the total expression from CTSS in dual-initiator consensus clusters (left, n = 6,292) and all other consensus clusters (right, n = 12,428), initiating with YR or YC dinucleotides, for each CRC organoid sample (chi-squared test, P < 0.0001 for both dual and other promoters). f, Dual-initiator promoters were identified as before (n = 6,292), and the YC:YR expression ratio was calculated for each in all organoid samples and divided by the average YC:YR ratio for that promoter. The frequency distribution of these values, illustrating the YC:YR ratio of transcription for all dual initiators, between samples is shown. g, UCSC Genome Browser view of CAGE tracks from a representative dual-initiator gene, SND1 (staphylococcal nuclease and tudor domain containing 1), showing a dynamic switch from YC-predominant transcription in radiotherapy-responsive CRC organoids (CRC1) to YR-predominant transcription in radiotherapy-non-responsive organoids (CRC5), with balanced transcriptional output from YR and YC components in the moderately responsive CRC organoid (CRC3). h, Bright-field images showing the morphology and doubling times of the five CRC organoid lines under investigation. White arrows, cysts; red arrows, crypts; scale bar, 100 µm. Doubling time analysis is based on measurements from three independent experiments.

Source data

To understand the source of this change, we calculated the YC:YR ratio dynamics of DIPs, grouped CTSS into 3,472 consensus clusters between samples, identified 186 DIPs and compared the YC:YR ratio of DIPs between the responsive and non-responsive cohorts (Fig. 3b). A significant (P = 0.02) enrichment in the YC:YR ratio was found. To determine the contribution of both types of initiation site selection, we compared 5′-C and 5′-R transcript levels separately between the responsive and non-responsive cohorts (Fig. 3c). 5′-C transcripts were specifically enriched in the responsive cohort, while the 5′-R component was modestly depleted (Fig. 3c). This suggests that the YC:YR change in DIPs was due primarily to enrichment of YC initiation in chemo-radiotherapy-responsive tumors and that the total gene expression variation was due to selective differential metabolism of 5′-C transcripts.

It is possible that differences in initiation site usage were due to potentially unequal RNA degradation in FFPE archived CRC clinical samples. Additionally, FFPEcap-seq is less efficient in identifying DIPs than traditional CAGE (186 DIPs (5.3% of all promoters) versus 3,475 DIPs (19.9% of all promoters) in FANTOM5 data). Therefore, we aimed to confirm our observations on CRC organoid samples by CAGE-seq (CRC1–CRC5) (Supplementary Table 5).

We characterized the radiotherapy responsiveness of these organoids upon exposure to 25 Gy of irradiation over 5 days, followed by a 5-day recovery period to allow the physiological and transcriptomic effects of irradiation to register in the samples, mimicking the standard short-course radiotherapy protocol given clinically in rectal cancer39,40. This analysis revealed significant differences in the radiotherapy responsiveness between the organoid lines, with two showing a robust ~95% reduction in viability (CRC1 and CRC2) compared to untreated samples, one with moderate response (70% viability reduction, CRC3) and two with little response (36% and 17.5% viability reduction, CRC4 and CRC5, respectively) (Fig. 3d). This finding allowed us to investigate differences in promoter usage across organoids with a range of radiosensitivities.

As before, CAGE reads were mapped and CTSS assigned, with ~90% of CTSS mapping to the promoter region of genes (Extended Data Fig. 2a and Supplementary Table 6). The CTSS were clustered into 18,713 consensus clusters. Notably, this revealed that the YC:YR ratio was directly correlated with radiotherapy response, displaying a continuous gradient from 33–36% of transcripts starting with C in the most responsive organoid samples (CRC1 and CRC2) to 15% in the least responsive sample (CRC5) (Extended Data Figs. 2b and 3b), in agreement with the clinical samples (Fig. 3a). To explore whether this dynamic was a property of altered use of DIPs rather than a global transcript-level change, we identified DIPs as before (n = 6,285, 34% of consensus clusters) and analyzed CTSS expression from all DIPs versus all other promoters (n = 12,428) (Fig. 3e). This analysis revealed that (1) the radiotherapy response-associated YC:YR dynamic was significant in both dual-initiating and non-dual-initiating promoters; (2) other promoters almost exclusively generate YR transcripts, with promoters generating only YC-initiating transcripts a very rare event (six consistently across all CRC organoid samples and ~30 per sample); (3) the change in the YC:YR ratios in DIPs was predominantly due to altered 5′-C transcript levels (in agreement with Fig. 3c and Extended Data Fig. 3c); and (4) the vast majority (~83%) of transcripts in the CRC organoid samples emanated from DIPs. Further to this finding, frequency distribution analysis of YC:YR ratios within DIPs again showed enrichment in the YC content in radiotherapy-responsive samples relative to non-responsive samples (Fig. 3f and Extended Data Fig. 3c), in agreement with clinical samples (Fig. 3b,c). To determine the contribution of YC enrichment and YR depletion within DIPs, we compared the expression of YC transcription and YR transcription separately between organoids (Extended Data Fig. 3d,e). This analysis revealed that the global YC:YR ratio shift between radiotherapy-responsive and non-responsive organoids was predominantly driven by DIPs in which 5′-C transcripts were enriched and 5′-R transcripts were unchanged (1,479 DIPs) in the organoid cohort but with a significant minority of cases in which 5′-R transcripts were depleted and 5′-C transcripts were unchanged (790 DIPs) (Extended Data Fig. 3d,e).

This dynamic radiotherapy response-associated shift in the YC:YR ratio is well demonstrated by the oncogene SND1 (staphylococcal nuclease and tudor domain containing 1), a gene associated with cancer proliferation, angiogenesis, metastasis and the stress response (reviewed in ref. 41). This dual-initiating promoter displayed a significant change in its TSS usage between responsive, moderately responsive and non-responsive organoids, transitioning from predominantly YC TSS in responsive organoids to balanced transcription in moderately responsive organoids and YR-predominant TSS in non-responsive organoids (Fig. 3g).

As discussed, the investigation of mRNA metabolism previously focused on 79 human ribosomal genes and a small set of translation-associated genes bearing 5′TOP motifs5. To investigate the radiotherapy responsiveness of YC-initiating transcripts in these genes, we extracted a definitive gene list from ref. 5, identified genes with sufficient expression (>5 TPM) across all CRC organoid samples and quantified their TSS levels (Supplementary Table 7). This analysis revealed that all of these genes have DIPs, although for the majority, YC was the predominant initiation class, as expected; the majority of these genes showed radiotherapy responsiveness dynamics in overall gene expression level, but particularly in the 5′-C transcript content with relatively little change in the 5′-R transcript content, in agreement with the data displayed in Fig. 3. Intriguingly, a few showed a reverse dynamic in 5′-C transcript content (YC transcription enriched in the non-responsive cohort), namely RPL27, eiF3F, RPS19, RPS14, RPL7A and RPL41, suggesting that depletion of 5′-C transcript content in non-responsive CRCs was not concurrent with a complete loss of ribosome and translation machinery transcripts but rather a potential switch of TSS (Supplementary Table 7).

The overall dynamics displayed by the majority of known 5′TOP genes, together with the examples in Figs. 1b and 3g and Extended Data Fig. 1a, demonstrated that a large number of DIPs have the capacity to transition their initiation between YC and YR depending on context. This pervasive shift in TSS dynamics represents a hitherto unexplored level of transcript regulation with the potential to impact the post-transcriptional fate of these genes6,7,8,9,10,11. The striking disparity in YC usage in DIPs between responsive and non-responsive CRC tumors and organoids (Fig. 3b–g) suggests that the YC to YR TSS switch could be crucial to our understanding of the differing dynamics between responsive and non-responsive CRC tumors. To identify in which genes these transitions were occurring, we identified 807 DIPs in which YC:YR ratios directly correlated with radiosensitivity (Extended Data Fig. 3f and Supplementary Data 2), with ontology significantly associated with ribosomal, metabolic and biosynthetic processes (Extended Data Fig. 3g,h).

Physiological assessment of the CRC organoids revealed an association between YC enrichment (and radiosensitivity) and faster proliferation rate (Fig. 3h and Extended Data Fig. 3i), in agreement with the pan-cancer association between YC enrichment and proliferation (Fig. 2c). Furthermore, there was a clear distinction in morphological features among the five organoids. The radiotherapy-resistant lines displayed crypt-like structures within the organoids, reflective of a well-differentiated identity similar to healthy colon organoids (Fig. 3h). The YC-enriched radiotherapy-responsive lines, on the other hand, were cystic in morphology, with no cellular segregation or polarization, previously shown to represent a failure to form crypt-like structures42,43 (Fig. 3h). This, too, aligns with the pan-cancer analysis shown in Fig. 2, suggesting a link between YC enrichment, cell proliferation and an undifferentiated tumor identity.

Irradiation depletes YC transcription in responsive organoids

Motivated by the observation of enrichment of initiation of YC transcripts in radiotherapy-responsive CRC tumors, we asked about the potential effect on 5′-C transcript abundance of radiotherapy itself. We challenged the CRC organoid lines with 25 Gy of radiation and performed CAGE-seq. We compared the relative frequency of YC and YR initiation between irradiated and control samples for each organoid (Fig. 4a and Extended Data Fig. 4a). This analysis revealed that YC initiation was depleted relative to YR initiation upon irradiation and that the extent of depletion was again correlated with radiotherapy responsiveness, with a 45%, 28.4% and 4.9% reduction in YC content in responsive (CRC1 and CRC2), moderately responsive (CRC3) and non-responsive (CRC4 and CRC5) organoids, respectively (Fig. 4a and Extended Data Fig. 4a). This global reduction in 5′-C transcripts was again replicated in previously identified DIPs, where YC:YR frequency distribution analysis revealed a depletion in the YC content of DIPs, most marked in radiotherapy-responsive organoids (Fig. 4b).

Fig. 4: Radiotherapy-responsive modulation of YC transcription initiation correlates with CRC clinical response.

a, Bar graph showing the proportion of CTSS in dual-initiator consensus clusters, initiating with YR or YC dinucleotides, in responsive (average of CRC1 and CRC2), moderately responsive (CRC3) and non-responsive (average of CRC4 and CRC5) organoids treated with 0 Gy (control) or 25 Gy (irradiated) irradiation treatment (chi-square analysis, P = 0.0001). b, Frequency distribution graph showing the degree of expression change of the YC component (normalized to YR) of each dual promoter upon irradiation in responsive (average of CRC1 and CRC2), moderately responsive (CRC3) and non-responsive (average of CRC4 and CRC5) organoids. c, UCSC Genome Browser view of an irradiation-responsive dual promoter (C9orf85). This promoter shows a clear loss of the YC component upon irradiation in responsive tumors (CRC1) and a relative lack of the YC component in moderately responsive (CRC3) and non-responsive (CRC5) organoid samples.

Source data

This YC:YR shift of transcripts was demonstrated well by the dual-initiating promoter of the C9orf85 gene, linked to cellular differentiation44 (Fig. 4c). The most abundant transcript isoform in radiotherapy-responsive tumors arose from a YC initiator, in contrast to dominant YR initiation in the moderately responsive and non-responsive cohorts, in line with the dynamic highlighted in Fig. 3. Upon irradiation, however, the 5′-C transcript isoform was specifically depleted in the responsive cohort but unchanged in the moderately responsive cohort and slightly enriched in the non-responsive cohort (Fig. 4c). Besides this example, we surveyed irradiation-associated TSS usage of known 5′TOP-bearing genes. We revisited the 5′TOP gene list (Supplementary Table 7) and compared relative expression between irradiation and control samples for each organoid (Supplementary Table 8). This analysis revealed that the majority of these genes displayed irradiation response-dependent depletion of overall expression but particularly the YC content (Supplementary Table 8).

We then asked which DIPs showed a radiotherapy-responsive transition in TSS usage (YC:YR ratio most depleted upon irradiation in responsive samples to least depleted or enriched in non-responsive samples) (Extended Data Fig. 4b and Supplementary Data 3). This analysis identified 411 genes with gene ontologies associated with translation, biosynthetic and metabolic processes (Extended Data Fig. 4c,d), similar to the 807 radiosensitivity trajectory genes identified in Extended Data Fig. 3g.

YC-initiating transcripts share radiotherapy-responsive dynamics

Besides the well-known dependence on the mTOR pathway of 5′TOP mRNAs, a recent study25 revealed that shorter polypyrimidine stretches could permit mTOR regulation through LARP1-dependant pathways. To investigate whether the radiotherapy-responsive dynamics seen in 5′-C transcripts were due to the differential processing of canonical 5′TOP mRNAs or a more general 5′-C-associated phenomenon, we segregated YC-initiating transcripts into TOP (5 pyrimidines), TOP-deg (4/5 pyrimidines) and YC-other (≤3/5 pyrimidines) transcripts based on their 5′ ends (Fig. 5a). We first surveyed the proportion of DIPs with each of these YC classes (>1 TPM), performing Venn intersection analysis for each 5′-C class (Fig. 5b). This analysis revealed that YC-other transcripts represented the most abundant class, being present (>1 TPM) in 5,747 DIPs (91%), and there was a high degree of overlap between classes, with 4,487 (71%) DIPs containing at least two classes and 2,038 (32%) DIPs containing all three classes. This represents a dramatic increase in the number of genes with 5′TOP-containing transcripts over the <100 5′TOP-containing ribosomal genes5.

Fig. 5: TOP-, TOP-deg- and YC-other-initiating transcripts share radiotherapy-responsive dynamics.

a, Illustration of the selection criteria for transcripts identified to initiate with a YR, TOP, TOP-deg or YC-other 5′ initiation makeup. b, The YC components of all dual initiators were subdivided into TOP, TOP-deg and YC-other forms as illustrated in a, and the number of previously identified DIPs containing >1 TPM of each YC form was calculated; Venn diagram shows the intersection of genes containing >1 TPM of each YC form (generated using Academo software). c, The ratio of each YC form to YR transcription in each dual initiator was calculated and divided by the average ratio for that promoter. The frequency distribution of these values, illustrating TOP:YR, TOP-deg:YR and YC-other:YR ratios of transcription for all dual initiators, between responsive (average of CRC1 and CRC2 (gold)) and non-responsive (average of CRC4 and CRC5 (dark blue)) organoid samples is shown. d, Analogous to c, but comparing the change in dual-initiator TOP, TOP-deg and YC-other component expression upon irradiation between responsive (average of CRC1 and CRC2 (gold)) and non-responsive (average of CRC4 and CRC5 (dark blue)) organoid samples.

We next asked what part each 5′-C transcript subtype played in radiotherapy response dynamics. We analyzed the change in YC:YR ratio between organoid samples and, upon irradiation, for each YC subclass separately (Fig. 5c,d). All three classes showed the same dynamic separation between the responsive, moderately responsive and non-responsive cohorts. Intriguingly, the extent of separation varied between YC subtypes, with TOP and TOP-deg forms better separating responsive organoids from moderately responsive and non-responsive organoids, while the YC-other subtype was superior for stratifying between the moderately responsive and non-responsive cohorts (Fig. 5c,d).

This finding is noteworthy, as the YC-other transcript format represents a hitherto unexplored transcript type, with no recognized 5′TOP motif imbuing post-transcriptional regulatory properties. However, these YC-other transcripts appeared to show very similar dynamics to those of transcripts containing TOP and/or TOP-deg motifs. One possibility is that the shared dynamics seen in YC-other and TOP or TOP-deg transcripts may be due to downstream 5′TOP motifs in YC-other transcripts (previously identified as TOP-like15), permitting post-transcriptional co-regulation with 5′TOP transcripts. Therefore, we investigated the radiotherapy-responsive dynamics of YC-other-initiating transcripts with and without 5′TOP transcripts co-expressed in DIPs (Extended Data Fig.