Background: 5-Methylcytosine (m 5 C) is a prevalent base modification in tRNA and rRNA but it also occurs more broadly in the transcriptome, including in mRNA, where it serves incompletely understood molecular functions. In pursuit of potential links of m 5 C with mRNA translation, we performed polysome profiling of human HeLa cell lysates and subjected RNA from resultant fractions to efficient bisulfite conversion followed by RNA sequencing (bsRNA-seq). Bioinformatic filters for rigorous site calling were devised to reduce technical noise.Results: We obtained~1000 candidate m 5 C sites in the wider transcriptome, most of which were found in mRNA. Multiple novel sites were validated by amplicon-specific bsRNA-seq in independent samples of either human HeLa, LNCaP and PrEC cells. Furthermore, RNAi-mediated depletion of either the NSUN2 or TRDMT1 m 5 C:RNA methyltransferases showed a clear dependence on NSUN2 for the majority of tested sites in both mRNAs and noncoding RNAs. Candidate m 5 C sites in mRNAs are enriched in 5′UTRs and near start codons and are embedded in a local context reminiscent of the NSUN2-dependent m 5 C sites found in the variable loop of tRNA. Analysing mRNA sites across the polysome profile revealed that modification levels, at bulk and for many individual sites, were inversely correlated with ribosome association. Conclusions: Our findings emphasise the major role of NSUN2 in placing the m 5 C mark transcriptome-wide. We further present evidence that substantiates a functional interdependence of cytosine methylation level with mRNA translation. Additionally, we identify several compelling candidate sites for future mechanistic analysis.

Graphical abstract

Extracellular vesicles (EVs) are small membranous vesicles that contain an abundant cargo of different RNA species with specialized functions and clinical implications. Here, we introduce an updated online database (http://www.exoRBase.org), exoRBase 2.0, which is a repository of EV long RNAs (termed exLRs) derived from RNA-seq data analyses of diverse human body fluids. In exoRBase 2.0, the number of exLRs has increased to 19 643 messenger RNAs (mRNAs), 15 645 long non-coding RNAs (lncRNAs) and 79 084 circular RNAs (circRNAs) obtained from ∼1000 human blood, urine, cerebrospinal fluid (CSF) and bile samples. Importantly, exoRBase 2.0 not only integrates and compares exLR expression profiles but also visualizes the pathway-level functional changes and the heterogeneity of origins of circulating EVs in the context of different physiological and pathological conditions. Our database provides an attractive platform for the identification of novel exLR signatures from human biofluids that will aid in the discovery of new circulating biomarkers to improve disease diagnosis and therapy.

Abstract5-methylcytosine (m5C) is a prevalent base modification in tRNA and rRNA but it also occurs more broadly in the transcriptome, including in mRNA, where it serves incompletely understood molecular functions. In pursuit of potential links of m5C with mRNA translation, we performed polysome profiling of human HeLa cell lysates and subjected RNA from resultant fractions to efficient bisulfite conversion followed by RNA sequencing (bsRNA-seq). Bioinformatic filters for rigorous site calling were devised to reduce technical noise. We obtained ∼1,000 candidate m5C sites in the wider transcriptome, most of which were found in mRNA. Multiple novel sites were validated by amplicon-specific bsRNA-seq in independent samples of either human HeLa, LNCaP and PrEC cells. Furthermore, RNAi-mediated depletion of either the NSUN2 or TRDMT1 m5C:RNA methyltransferases showed a clear dependence on NSUN2 for the majority of tested sites in both mRNAs and noncoding RNAs. Candidate m5C sites in mRNAs are enriched in 5’UTRs and near start codons, and are commonly embedded in a local context reminiscent of the NSUN2-dependent m5C sites found in the variable loop of tRNA. Analysing mRNA sites across the polysome profile revealed that modification levels, at bulk and for many individual sites, were inversely correlated with ribosome association. Altogether, these findings emphasise the major role of NSUN2 in making this mark transcriptome-wide and further substantiate a functional interdependence of cytosine methylation level with mRNA translation.

No abstract

The transcription variation, leading to various forms of transcripts and protein diversity, remains largely unexplored in triple-negative breast cancers (TNBCs). Here, we presented a comprehensive analysis of RNA splicing in breast cancer to illustrate the biological function and clinical implications of tumor-specific transcripts (TSTs) arising from these splicing junctions. Aberrant RNA splicing or TSTs were frequently harbored in TNBC and were correlated with a poor outcome. We discovered a tumor-specific splicing variant of macrophage receptor with collagenous structure–TST (MARCO-TST), which was distinguished from myeloid cell-specific wild-type MARCO. MARCO-TST expression was associated with poor outcomes in TNBC patients and could promote tumor progression in vitro and in vivo. Mechanically, MARCO-TST interacted with PLOD2 and enhanced the stability of HIF-1α, which resulted in the metabolic dysregulation of TNBC to form a hypoxic tumor microenvironment. MARCO-TST was initiated from a de novo alternative transcription initiation site that was activated by a superenhancer. Tumors with MARCO-TST expression conferred greater sensitivity to bromodomain and extraterminal protein inhibitors. This treatment strategy was further validated in patient-derived organoids. In conclusion, our results revealed the transcription variation landscape of TNBC, highlighting MARCO-TST as a crucial oncogenic transcript and therapeutic target.

No abstract

Background Prostate cancer (PCa) is a major type of cancer in man worldwide. Androgen deprivation therapy (ADT) and the next‐generation androgen receptor (AR) pathway inhibitors have acquired great success in treating PCa. However, patients treated with ADT or AR targeted therapy are inevitably developing into castration‐resistant prostate cancer (CRPC) or becoming drug resistance. The role of mRNA 5‐methylcytosine (m5C) modification in cancers is largely unknown. This study aimed to explore the role of the m5C methyltransferase NSUN2 in Prostate cancer (PCa). Methods The expression of NSUN2 and its clinicopathological impact were evaluated in PCa cohorts. The effect of NSUN2 on the biological characteristics of PCa cells was investigated on the basis of gain‐offunction and loss‐of‐function analyses. Subcutaneous models further uncovered the role of NSUN2 in tumor growth. Epi‐transcriptome assays with RNA bisulfite sequencing (RNA‐BisSeq) analysis and in vitro enzyme reaction assays were performed to validate the targeted effect of NSUN2 on AR. AR‐binding sites in the NSUN2 promoter were investigated by ChIP and luciferase assays to uncover the interplay between NSUN2 and AR signaling. RIP‐qPCR and EMSA methods were performed to confirm that YBX1 binds to AR m 5 C sites. Results NSUN2 is highly expressed in PCa and predicts poor outcome. NSUN2 plays roles as a PCa oncogene both in vitro and in vivo. Depletion of NSUN2 results in decreased expression and activities of AR, including AR‐V7. Mechanistically, NSUN2 posttranscriptionally stabilized AR by cluster m 5 C modification in a m5CYBX1‐dependent manner. Strikingly, treatment with enzalutamide, an effective AR inhibitor, reduces NSUN2 expression and decreases the m5C modification level in prostate cancer cells. Finally, we found that AR transcriptionally regulates NSUN2. Conclusion NSUN2 stabilizes AR mRNA through cluster 5‐methylcytosine modification and activates a positive feedback loop to promote prostate cancer.