The molecular and genetic regulation of shoot branching.

The architecture of flowering plants exhibits both phenotypic diversity and plasticity, determined, in part, by the number and activity of axillary meristems and, in part, by the growth characteristics of the branches that develop from the axillary buds. The plasticity of shoot branching results from a combination of various intrinsic and genetic elements, such as number and position of nodes and type of growth phase, as well as environmental signals such as nutrient availability, light characteristics, and temperature (Napoli et al., 1998; Bennett and Leyser, 2006; Janssen et al., 2014; Teichmann and Muhr, 2015; Ueda and Yanagisawa, 2019). Axillary meristem initiation and axillary bud outgrowth are controlled by a complex and interconnected regulatory network. Although many of the genes and hormones that modulate branching patterns have been discovered and characterized through genetic and biochemical studies, there are still many gaps in our understanding of the control mechanisms at play. In this review, we will summarize our current knowledge of the control of axillary meristem initiation and outgrowth into a branch.

Flexibility of the petunia strigolactone receptor DAD2 promotes its interaction with signaling partners.

Strigolactones (SLs) are terpenoid-derived plant hormones that regulate various developmental processes, particularly shoot branching, root development, and leaf senescence. The SL receptor has an unusual mode of action. Upon binding SL, it hydrolyzes the hormone, and then covalently binds one of the hydrolytic products. These initial events enable the SL receptor DAD2 (in petunia) to interact with the F-box protein PhMAX2A of the Skp-Cullin-F-box (SCF) complex and/or a repressor of SL signaling, PhD53A. However, it remains unclear how binding and hydrolysis structurally alters the SL receptor to enable its engagement with signaling partners. Here, we used mutagenesis to alter DAD2 and affect SL hydrolysis or DAD2's ability to interact with its signaling partners. We identified three DAD2 variants whose hydrolytic activity had been separated from the receptor's interactions with PhMAX2A or PhD53A. Two variants, DAD2N242I and DAD2F135A, having substitutions in the core α/ß hydrolase-fold domain and the hairpin, exhibited hormone-independent interactions with PhMAX2A and PhD53A, respectively. Conversely, the DAD2D166A variant could not interact with PhMAX2A in the presence of SL, but its interaction with PhD53A remained unaffected. Structural analyses of DAD2N242I and DAD2D166A revealed only small differences compared with the structure of the WT receptor. Results of molecular dynamics simulations of the DAD2N242I structure suggested that increased flexibility is a likely cause for its SL-independent interaction with PhMAX2A. Our results suggest that PhMAX2A and PhD53A have distinct binding sites on the SL receptor and that its flexibility is a major determinant of its interactions with these two downstream regulators.

Genome-wide characterization of 5-hydoxymethylcytosine in melanoma reveals major differences with nevus.

Melanoma demonstrates altered patterns of DNA methylation that are associated with genetic instability and transcriptional repression of numerous genes. Active DNA demethylation is mediated by TET enzymes that catalyze conversion of 5-methylcytosine (mC) to 5-hydroxymethylcytosine (hmC). Loss of hmC occurs in melanoma and correlates with disease progression. Here we analyzed the genomic distribution of hmC along with mC in nevus and melanoma using oxidative bisulfite chemistry combined with high-density arrays. HmC was enriched relative to mC at enhancers, 5'UTR regions and CpG shores in nevus and melanoma samples, pointing to specific TET enzyme activity. The proportion of interrogated CpG sites with high hmC levels was lower in melanoma (0.54%) than in nevus (2.0%). Depletion of hmC in melanoma was evident across all chromosomes and intragenic regions, being more pronounced in metastatic than in non-metastatic tumors. The patterns of hmC distribution in melanoma samples differed significantly from those in nevus samples, exceeding differences in mC patterns. We identified specific CpG sites and regions with significantly lower hmC levels in melanoma than in nevus that might serve as diagnostic markers. Differentially hydroxymethylated regions localized to cancer-related genes, including the PTEN gene promoter, suggesting that deregulated DNA hydroxymethylation may contribute to melanoma pathogenesis.

Inhibition of strigolactone receptors by N-phenylanthranilic acid derivatives: Structural and functional insights.

The strigolactone (SL) family of plant hormones regulates a broad range of physiological processes affecting plant growth and development and also plays essential roles in controlling interactions with parasitic weeds and symbiotic fungi. Recent progress elucidating details of SL biosynthesis, signaling, and transport offers many opportunities for discovering new plant-growth regulators via chemical interference. Here, using high-throughput screening and downstream biochemical assays, we identified N-phenylanthranilic acid derivatives as potent inhibitors of the SL receptors from petunia (DAD2), rice (OsD14), and Arabidopsis (AtD14). Crystal structures of DAD2 and OsD14 in complex with inhibitors further provided detailed insights into the inhibition mechanism, and in silico modeling of 19 other plant strigolactone receptors suggested that these compounds are active across a large range of plant species. Altogether, these results provide chemical tools for investigating SL signaling and further define a framework for structure-based approaches to design and validate optimized inhibitors of SL receptors for specific plant targets.

Expression of MdCCD7 in the scion determines the extent of sylleptic branching and the primary shoot growth rate of apple trees.

Branching has a major influence on the overall shape and productivity of a plant. Strigolactones (SLs) have been identified as plant hormones that have a key role in suppressing the outgrowth of axillary meristems. CAROTENOID CLEAVAGE DIOXYGENASE (CCD) genes are integral to the biosynthesis of SLs and are well characterized in annual plants, but their role in woody perennials is relatively unknown. We identified CCD7 and CCD8 orthologues from apple and demonstrated that MdCCD7 and MdCCD8 are able to complement the Arabidopsis branching mutants max3 and max4 respectively, indicating conserved function. RNAi lines of MdCCD7 show reduced gene expression and increased branching in apple. We performed reciprocal grafting experiments with combinations of MdCCD7 RNAi and wild-type 'Royal Gala' as rootstocks and scion. Unexpectedly, wild-type roots were unable to suppress branching in MdCCD7 RNAi scions. Another key finding was that MdCCD7 RNAi scions initiated phytomers at an increased rate relative to the wild type, resulting in a greater node number and primary shoot length. We suggest that localized SL biosynthesis in the shoot, rather than roots, controls axillary bud outgrowth and shoot growth rate in apple.

Detecting PKD1 variants in polycystic kidney disease patients by single-molecule long-read sequencing.

A genetic diagnosis of autosomal-dominant polycystic kidney disease (ADPKD) is challenging due to allelic heterogeneity, high GC content, and homology of the PKD1 gene with six pseudogenes. Short-read next-generation sequencing approaches, such as whole-genome sequencing and whole-exome sequencing, often fail at reliably characterizing complex regions such as PKD1. However, long-read single-molecule sequencing has been shown to be an alternative strategy that could overcome PKD1 complexities and discriminate between homologous regions of PKD1 and its pseudogenes. In this study, we present the increased power of resolution for complex regions using long-read sequencing to characterize a cohort of 19 patients with ADPKD. Our approach provided high sensitivity in identifying PKD1 pathogenic variants, diagnosing 94.7% of the patients. We show that reliable screening of ADPKD patients in a single test without interference of PKD1 homologous sequences, commonly introduced by residual amplification of PKD1 pseudogenes, by direct long-read sequencing is now possible. This strategy can be implemented in diagnostics and is highly suitable to sequence and resolve complex genomic regions that are of clinical relevance.

Critical points for an accurate human genome analysis.

Next-generation sequencing is radically changing how DNA diagnostic laboratories operate. What started as a single-gene profession is now developing into gene panel sequencing and whole-exome and whole-genome sequencing (WES/WGS) analyses. With further advances in sequencing technology and concomitant price reductions, WGS will soon become the standard and be routinely offered. Here, we focus on the critical steps involved in performing WGS, with a particular emphasis on points where WGS differs from WES, the important variables that should be taken into account, and the quality control measures that can be taken to monitor the process. The points discussed here, combined with recent publications on guidelines for reporting variants, will facilitate the routine implementation of WGS into a diagnostic setting.

Environmental control of branching in petunia.

Plants alter their development in response to changes in their environment. This responsiveness has proven to be a successful evolutionary trait. Here, we tested the hypothesis that two key environmental factors, light and nutrition, are integrated within the axillary bud to promote or suppress the growth of the bud into a branch. Using petunia (Petunia hybrida) as a model for vegetative branching, we manipulated both light quality (as crowding and the red-to-far-red light ratio) and phosphate availability, such that the axillary bud at node 7 varied from deeply dormant to rapidly growing. In conjunction with the phenotypic characterization, we also monitored the state of the strigolactone (SL) pathway by quantifying SL-related gene transcripts. Mutants in the SL pathway inhibit but do not abolish the branching response to these environmental signals, and neither signal is dominant over the other, suggesting that the regulation of branching in response to the environment is complex. We have isolated three new putatively SL-related TCP (for Teosinte branched1, Cycloidia, and Proliferating cell factor) genes from petunia, and have identified that these TCP-type transcription factors may have roles in the SL signaling pathway both before and after the reception of the SL signal at the bud. We show that the abundance of the receptor transcript is regulated by light quality, such that axillary buds growing in added far-red light have greatly increased receptor transcript abundance. This suggests a mechanism whereby the impact of any SL signal reaching an axillary bud is modulated by the responsiveness of these cells to the signal.

FEI's direct electron detector developments: Embarking on a revolution in cryo-TEM.

In early 2011 FEI Company launched the "Falcon", its first commercial direct electron detector product intended for application in 3-D electron microscopy in the life sciences. In this paper we discuss the principle of direct electron detection and its implementation in Falcon cameras. We describe the signal formation in the sensor and its impact on the detection quantum efficiency (DQE) of the sensor. Insights into the signal formation led us to improved camera designs. Three significant improvements are discussed. (1) Back thinning of the sensor. This is implemented in the second-generation Falcon (Falcon 2), where the sensor thickness is reduced to 50 µm, and in the latest generation Falcon 3 detector with further back-thinning down to 30 µm. (2) The introduction of electron counting, a signal processing technology implemented in Falcon 3. (3) Dose fractionation mode, which allows the user to access intermediate results during the illumination of the sample.

Copy number alterations and allelic ratio in relation to recurrence of rectal cancer.

BACKGROUND: In rectal cancer, total mesorectal excision surgery combined with preoperative (chemo)radiotherapy reduces local recurrence rates but does not improve overall patient survival, a result that may be due to the harmful side effects and/or co-morbidity of preoperative treatment. New biomarkers are needed to facilitate identification of rectal cancer patients at high risk for local recurrent disease. This would allow for preoperative (chemo)radiotherapy to be restricted to high-risk patients, thereby reducing overtreatment and allowing personalized treatment protocols. We analyzed genome-wide DNA copy number (CN) and allelic alterations in 112 tumors from preoperatively untreated rectal cancer patients. Sixty-six patients with local and/or distant recurrent disease were compared to matched controls without recurrence. Results were validated in a second cohort of tumors from 95 matched rectal cancer patients. Additionally, we performed a meta-analysis that included 42 studies reporting on CN alterations in colorectal cancer and compared results to our own data. RESULTS: The genomic profiles in our study were comparable to other rectal cancer studies. Results of the meta-analysis supported the hypothesis that colon cancer and rectal cancer may be distinct disease entities. In our discovery patient study cohort, allelic retention of chromosome 7 was significantly associated with local recurrent disease. Data from the validation cohort were supportive, albeit not statistically significant, of this finding. CONCLUSIONS: We showed that retention of heterozygosity on chromosome 7 may be associated with local recurrence in rectal cancer. Further research is warranted to elucidate the mechanisms and effect of retention of chromosome 7 on the development of local recurrent disease in rectal cancer.

DWARF27 and CAROTENOID CLEAVAGE DIOXYGENASE 7 genes regulate release, germination and growth of gemma in Marchantia polymorpha.

Strigolactones (SLs), a class of carotenoid-derived hormones, play a crucial role in flowering plants by regulating underground communication with symbiotic arbuscular mycorrhizal fungi (AM) and controlling shoot and root architecture. While the functions of core SL genes have been characterized in many plants, their roles in non-tracheophyte plants like liverworts require further investigation. In this study, we employed the model liverwort species Marchantia polymorpha, which lacks detectable SL production and orthologs of key SL biosynthetic genes, including CAROTENOID CLEAVAGE DIOXYGENASE 8 (CCD8) and MORE AXILLARY GROWTH 1 (MAX1). However, it retains some SL pathway components, including DWARF27 (D27) and CCD7. To help elucidate the function of these remaining components in M. polymorpha, knockout mutants were generated for MpD27-1, MpD27-2 and MpCCD7. Phenotypic comparisons of these mutants with the wild-type control revealed a novel role for these genes in regulating the release of gemmae from the gemma cup and the germination and growth of gemmae in the dark. Mpd27-1, Mpd27-2, and Mpccd7 mutants showed lower transcript abundance of genes involved in photosynthesis, such as EARLY LIGHT INDUCED (ELI), and stress responses such as LATE EMBRYOGENESIS ABUNDANT (LEA) but exhibited higher transcript levels of ETHYLENE RESPONSE FACTORS (ERFs) and SL and carotenoid related genes, such as TERPENE SYNTHASE (TS), CCD7 and LECITHIN-RETINAL ACYL TRANSFERASE (LRAT). Furthermore, the mutants of M. polymorpha in the SL pathway exhibited increased contents of carotenoid. This unveils a previously unrecognized role for MpD27-1, MpD27-2 and MpCCD7 in controlling release, germination, and growth of gemmae in response to varying light conditions. These discoveries enhance our comprehension of the regulatory functions of SL biosynthesis genes in non-flowering plants.

Varying the expression pattern of the strigolactone receptor gene DAD2 results in phenotypes distinct from both wild type and knockout mutants.

The action of the petunia strigolactone (SL) hormone receptor DAD2 is dependent not only on its interaction with the PhMAX2A and PhD53A proteins, but also on its expression patterns within the plant. Previously, in a yeast-2-hybrid system, we showed that a series of a single and double amino acid mutants of DAD2 had altered interactions with these binding partners. In this study, we tested the mutants in two plant systems, Arabidopsis and petunia. Testing in Arabidopsis was enabled by creating a CRISPR-Cas9 knockout mutant of the Arabidopsis strigolactone receptor (AtD14). We produced SL receptor activity in both systems using wild type and mutant genes; however, the mutants had functions largely indistinguishable from those of the wild type. The expression of the wild type DAD2 from the CaMV 35S promoter in dad2 petunia produced plants neither quite like the dad2 mutant nor the V26 wild type. These plants had greater height and leaf size although branch number and the plant shape remained more like those of the mutant. These traits may be valuable in the context of a restricted area growing system such as controlled environment agriculture.

Transcription Factor Dynamics in Cross-Regulation of Plant Hormone Signaling Pathways.

Cross-regulation between hormone signaling pathways is indispensable for plant growth and development. However, the molecular mechanisms by which multiple hormones interact and co-ordinate activity need to be understood. Here, we generated a cross-regulation network explaining how hormone signals are integrated from multiple pathways in etiolated Arabidopsis (Arabidopsis thaliana) seedlings. To do so we comprehensively characterized transcription factor activity during plant hormone responses and reconstructed dynamic transcriptional regulatory models for six hormones; abscisic acid, brassinosteroid, ethylene, jasmonic acid, salicylic acid and strigolactone/karrikin. These models incorporated target data for hundreds of transcription factors and thousands of protein-protein interactions. Each hormone recruited different combinations of transcription factors, a subset of which were shared between hormones. Hub target genes existed within hormone transcriptional networks, exhibiting transcription factor activity themselves. In addition, a group of MITOGEN-ACTIVATED PROTEIN KINASES (MPKs) were identified as potential key points of cross-regulation between multiple hormones. Accordingly, the loss of function of one of these (MPK6) disrupted the global proteome, phosphoproteome and transcriptome during hormone responses. Lastly, we determined that all hormones drive substantial alternative splicing that has distinct effects on the transcriptome compared with differential gene expression, acting in early hormone responses. These results provide a comprehensive understanding of the common features of plant transcriptional regulatory pathways and how cross-regulation between hormones acts upon gene expression.

Molecular EPISTOP, a comprehensive multi-omic analysis of blood from Tuberous Sclerosis Complex infants age birth to two years.

We present a comprehensive multi-omic analysis of the EPISTOP prospective clinical trial of early intervention with vigabatrin for pre-symptomatic epilepsy treatment in Tuberous Sclerosis Complex (TSC), in which 93 infants with TSC were followed from birth to age 2 years, seeking biomarkers of epilepsy development. Vigabatrin had profound effects on many metabolites, increasing serum deoxycytidine monophosphate (dCMP) levels 52-fold. Most serum proteins and metabolites, and blood RNA species showed significant change with age. Thirty-nine proteins, metabolites, and genes showed significant differences between age-matched control and TSC infants. Six also showed a progressive difference in expression between control, TSC without epilepsy, and TSC with epilepsy groups. A multivariate approach using enrollment samples identified multiple 3-variable predictors of epilepsy, with the best having a positive predictive value of 0.987. This rich dataset will enable further discovery and analysis of developmental effects, and associations with seizure development in TSC.

Advancing 89Zr-immuno-PET in neuroscience with a bispecific anti-amyloid-beta monoclonal antibody - The choice of chelator is essential.

The accelerated approval of the monoclonal antibody (mAb) aducanumab as a treatment option for Alzheimer's Disease and the continued discussions about its efficacy have shown that a better understanding of immunotherapy for the treatment of neurodegenerative diseases is needed. 89Zr-immuno-PET could be a suitable tool to open new avenues for the diagnosis of CNS disorders, monitoring disease progression, and assessment of novel therapeutics. Herein, three different 89Zr-labeling strategies and direct radioiodination with 125I of a bispecific anti-amyloid-beta aducanumab derivate, consisting of aducanumab with a C-terminal fused anti-transferrin receptor binding single chain Fab fragment derived from 8D3 (Adu-8D3), were compared ex vivo and in vivo with regard to brain uptake and target engagement in an APP/PS1 Alzheimer's disease mouse model and wild type animals. Methods: Adu-8D3 and a negative control antibody, based on the HIV specific B12 antibody also carrying C-terminal fused 8D3 scFab (B12-8D3), were each conjugated with NCS-DFO, NCS-DFO*, or TFP-N-suc-DFO-Fe-ester, followed by radiolabeling with 89Zr. 125I was used as a substitute for 124I for labeling of both antibodies. 30 µg of radiolabeled mAb, corresponding to approximately 6 MBq 89Zr or 2.5 MBq 125I, were injected per mouse. PET imaging was performed 1, 3 and 7 days post injection (p.i.). All mice were sacrificed on day 7 p.i. and subjected to ex vivo biodistribution and brain autoradiography. Immunostaining on brain tissue was performed after autoradiography for further validation. Results: Ex vivo biodistribution revealed that the brain uptake of [89Zr]Zr-DFO*-NCS-Adu-8D3 (2.19 ±0.12 %ID/g) was as high as for its 125I-analog (2.21 ±0.15 %ID/g). [89Zr]Zr-DFO-NCS-Adu-8D3 and [89Zr]Zr-DFO-N-suc-Adu-8D3 showed significantly lower uptake (< 0.65 %ID/g), being in the same range as for the 89Zr-labeled controls (B12-8D3). Autoradiography of [89Zr]Zr-DFO*-NCS-Adu-8D3 and [125I]I-Adu-8D3 showed an amyloid-beta related granular uptake pattern of radioactivity. In contrast, the [89Zr]Zr-DFO-conjugates and the control antibody groups did not show any amyloid-beta related uptake pattern, indicating that DFO is inferior for 89Zr-immuno-PET imaging of the brain in comparison to DFO* for Adu-8D3. This was confirmed by day 7 PET images showing only amyloid-beta related brain uptake for [89Zr]Zr-DFO*-NCS-Adu-8D3. In wild type animals, such an uptake was not observed. Immunostaining showed a co-localization of all administered Adu-8D3 conjugates with amyloid-beta plaques. Conclusion: We successfully demonstrated that 89Zr-immuno-PET is suitable for imaging and quantifying amyloid-beta specific brain uptake using a bispecific aducanumab brain shuttling antibody, Adu-8D3, but only when using the novel chelator DFO*, and not DFO, for labeling with 89Zr.

CRISPRs in the human genome are differentially expressed between malignant and normal adjacent to tumor tissue.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) have been identified in bacteria, archaea and mitochondria of plants, but not in eukaryotes. Here, we report the discovery of 12,572 putative CRISPRs randomly distributed across the human chromosomes, which we termed hCRISPRs. By using available transcriptome datasets, we demonstrate that hCRISPRs are distinctively expressed as small non-coding RNAs (sncRNAs) in cell lines and human tissues. Moreover, expression patterns thereof enabled us to distinguish normal from malignant tissues. In prostate cancer, we confirmed the differential hCRISPR expression between normal adjacent and malignant primary prostate tissue by RT-qPCR and demonstrate that the SHERLOCK and DETECTR dipstick tools are suitable to detect these sncRNAs. We anticipate that the discovery of CRISPRs in the human genome can be further exploited for diagnostic purposes in cancer and other medical conditions, which certainly will lead to the development of point-of-care tests based on the differential expression of the hCRISPRs.

The Use of Differential Scanning Fluorimetry to Assess Strigolactone Receptor Function.

Differential scanning fluorimetry (DSF) is a method used for assessing the interaction of ligands with proteins. In most cases binding of a ligand to proteins tends to increase the melting temperature (Tm) of the protein involved. However, in the case of strigolactone receptors (e.g., D14, AtD14, DAD2, RMS3) from plants, the Tm tends to be reduced in the presence of strigolactones. This is likely due to increased flexibility of the receptors in the presence of hormone ligands.DSF experiments are simple, fast, amenable to high-throughput formats, and cost effective. They have therefore gained in popularity, including within the field of SL signaling. Typically in DSF the receptor protein is purified and incubated with the ligand (strigolactone, agonist, or antagonist) and a (fluorescent) reporter dye. The mixture is then placed in a quantitative PCR instrument and subjected to an increasing temperature gradient. Changes in fluorescence are recorded along the gradient, as the dye interacts with unfolded portions of the protein becoming accessible when the protein "melts". Differences in the temperature at which the protein unfolds in the absence and in the presence of the ligand are interpreted as indicating interactions between the ligand and the receptor.

Gene Regulation Network Analysis on Human Prostate Orthografts Highlights a Potential Role for the JMJD6 Regulon in Clinical Prostate Cancer.

BACKGROUND: Prostate cancer (PCa) is the second most common tumour diagnosed in men. Tumoral heterogeneity in PCa creates a significant challenge to develop robust prognostic markers and novel targets for therapy. An analysis of gene regulatory networks (GRNs) in PCa may provide insight into progressive PCa. Herein, we exploited a graph-based enrichment score to integrate data from GRNs identified in preclinical prostate orthografts and differentially expressed genes in clinical resected PCa. We identified active regulons (transcriptional regulators and their targeted genes) associated with PCa recurrence following radical prostatectomy. METHODS: The expression of known transcription factors and co-factors was analysed in a panel of prostate orthografts (n = 18). We searched for genes (as part of individual GRNs) predicted to be regulated by the highest number of transcriptional factors. Using differentially expressed gene analysis (on a per sample basis) coupled with gene graph enrichment analysis, we identified candidate genes and associated GRNs in PCa within the UTA cohort, with the most enriched regulon being JMJD6, which was further validated in two additional cohorts, namely EMC and ICGC cohorts. Cox regression analysis was performed to evaluate the association of the JMJD6 regulon activity with disease-free survival time in the three clinical cohorts as well as compared to three published prognostic gene signatures (TMCC11, BROMO-10 and HYPOXIA-28). RESULTS: 1308 regulons were correlated to transcriptomic data from the three clinical prostatectomy cohorts. The JMJD6 regulon was identified as the top enriched regulon in the UTA cohort and again validated in the EMC cohort as the top-ranking regulon. In both UTA and EMC cohorts, the JMJD6 regulon was significantly associated with cancer recurrence. Active JMJD6 regulon also correlated with disease recurrence in the ICGC cohort. Furthermore, Kaplan-Meier analysis confirmed shorter time to recurrence in patients with active JMJD6 regulon for all three clinical cohorts (UTA, EMC and ICGC), which was not the case for three published prognostic gene signatures (TMCC11, BROMO-10 and HYPOXIA-28). In multivariate analysis, the JMJD6 regulon status significantly predicted disease recurrence in the UTA and EMC, but not ICGC datasets, while none of the three published signatures significantly prognosticate for cancer recurrence. CONCLUSIONS: We have characterised gene regulatory networks from preclinical prostate orthografts and applied transcriptomic data from three clinical cohorts to evaluate the prognostic potential of the JMJD6 regulon.

Fusion transcripts and their genomic breakpoints in polyadenylated and ribosomal RNA-minus RNA sequencing data.

BACKGROUND: Fusion genes are typically identified by RNA sequencing (RNA-seq) without elucidating the causal genomic breakpoints. However, non-poly(A)-enriched RNA-seq contains large proportions of intronic reads that also span genomic breakpoints. RESULTS: We have developed an algorithm, Dr. Disco, that searches for fusion transcripts by taking an entire reference genome into account as search space. This includes exons but also introns, intergenic regions, and sequences that do not meet splice junction motifs. Using 1,275 RNA-seq samples, we investigated to what extent genomic breakpoints can be extracted from RNA-seq data and their implications regarding poly(A)-enriched and ribosomal RNA-minus RNA-seq data. Comparison with whole-genome sequencing data revealed that most genomic breakpoints are not, or minimally, transcribed while, in contrast, the genomic breakpoints of all 32 TMPRSS2-ERG-positive tumours were present at RNA level. We also revealed tumours in which the ERG breakpoint was located before ERG, which co-existed with additional deletions and messenger RNA that incorporated intergenic cryptic exons. In breast cancer we identified rearrangement hot spots near CCND1 and in glioma near CDK4 and MDM2 and could directly associate this with increased expression. Furthermore, in all datasets we find fusions to intergenic regions, often spanning multiple cryptic exons that potentially encode neo-antigens. Thus, fusion transcripts other than classical gene-to-gene fusions are prominently present and can be identified using RNA-seq. CONCLUSION: By using the full potential of non-poly(A)-enriched RNA-seq data, sophisticated analysis can reliably identify expressed genomic breakpoints and their transcriptional effects.