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Regulation of transcript structure generates transcript diversity and plays an important role in human disease 1 – 7 . The advent of long-read sequencing technologies offers the opportunity to study the role of genetic variation in transcript structure 8 – 16 . In this Article, we present a large human long-read RNA-seq dataset using the Oxford Nanopore Technologies platform from 88 samples from Genotype-Tissue Expression (GTEx) tissues and cell lines, complementing the GTEx resource. We identified just over 70,000 novel transcripts for annotated genes, and validated the protein expression of 10% of novel transcripts. We developed a new computational package, LORALS, to analyse the genetic effects of rare and common variants on the transcriptome by allele-specific analysis of long reads. We characterized allele-specific expression and transcript structure events, providing new insights into the specific transcript alterations caused by common and rare genetic variants and highlighting the resolution gained from long-read data. We were able to perturb the transcript structure upon knockdown of PTBP1, an RNA binding protein that mediates splicing, thereby finding genetic regulatory effects that are modified by the cellular environment. Finally, we used this dataset to enhance variant interpretation and study rare variants leading to aberrant splicing patterns. To understand the contribution of variants to transcript expression regulation, long-read transcriptome data are generated from the GTEx resource, and a new software package to perform allele-specific analysis is developed.

Parkinson’s disease is characterized by loss of dopamine neurons in the substantia nigra 1 . Similar to other major neurodegenerative disorders, there are no disease-modifying treatments for Parkinson’s disease. While most treatment strategies aim to prevent neuronal loss or protect vulnerable neuronal circuits, a potential alternative is to replace lost neurons to reconstruct disrupted circuits 2 . Here we report an efficient one-step conversion of isolated mouse and human astrocytes to functional neurons by depleting the RNA-binding protein PTB (also known as PTBP1). Applying this approach to the mouse brain, we demonstrate progressive conversion of astrocytes to new neurons that innervate into and repopulate endogenous neural circuits. Astrocytes from different brain regions are converted to different neuronal subtypes. Using a chemically induced model of Parkinson’s disease in mouse, we show conversion of midbrain astrocytes to dopaminergic neurons, which provide axons to reconstruct the nigrostriatal circuit. Notably, re-innervation of striatum is accompanied by restoration of dopamine levels and rescue of motor deficits. A similar reversal of disease phenotype is also accomplished by converting astrocytes to neurons using antisense oligonucleotides to transiently suppress PTB. These findings identify a potentially powerful and clinically feasible approach to treating neurodegeneration by replacing lost neurons. Depletion of the RNA-binding protein PTB (also known as PTBP1) in astrocytes reprograms these cells to become functional neurons and, in a mouse model of Parkinson’s disease, reverses the disease phenotype.

Single-cell transcriptomics analyses of cell intermediates during the reprogramming from fibroblast to cardiomyocyte were used to reconstruct the reprogramming trajectory and to uncover intermediate cell populations, gene pathways and regulators involved in this process. Fibroblast splicing factor To elucidate the mechanistic underpinnings of fibroblasts reprogramming to cardiomyocytes, Li Qian and colleagues have used a single-cell RNA sequencing approach. They find that the initial steps that drive the global expression changes that are critical for reprogramming encompass the downregulation of factors involved in mRNA processing and splicing, and in particular the splicing factor Ptbp1. Downregulation of Ptbp1 is essential for cells to adopt a cardiac-specific splicing pattern. The approach also led to the identification of surface markers that allow enrichment of induced cardiomyocytes during reprogramming. Direct lineage conversion offers a new strategy for tissue regeneration and disease modelling. Despite recent success in directly reprogramming fibroblasts into various cell types, the precise changes that occur as fibroblasts progressively convert to the target cell fates remain unclear. The inherent heterogeneity and asynchronous nature of the reprogramming process renders it difficult to study this process using bulk genomic techniques. Here we used single-cell RNA sequencing to overcome this limitation and analysed global transcriptome changes at early stages during the reprogramming of mouse fibroblasts into induced cardiomyocytes (iCMs) 1 , 2 , 3 , 4 . Using unsupervised dimensionality reduction and clustering algorithms, we identified molecularly distinct subpopulations of cells during reprogramming. We also constructed routes of iCM formation, and delineated the relationship between cell proliferation and iCM induction. Further analysis of global gene expression changes during reprogramming revealed unexpected downregulation of factors involved in mRNA processing and splicing. Detailed functional analysis of the top candidate splicing factor, Ptbp1, revealed that it is a critical barrier for the acquisition of cardiomyocyte-specific splicing patterns in fibroblasts. Concomitantly, Ptbp1 depletion promoted cardiac transcriptome acquisition and increased iCM reprogramming efficiency. Additional quantitative analysis of our dataset revealed a strong correlation between the expression of each reprogramming factor and the progress of individual cells through the reprogramming process, and led to the discovery of new surface markers for the enrichment of iCMs. In summary, our single-cell transcriptomics approaches enabled us to reconstruct the reprogramming trajectory and to uncover intermediate cell populations, gene pathways and regulators involved in iCM induction.

Although N4-acetylcytidine (ac4C) modification affects the stability and translation of mRNA, it is unknown whether it exists in noncoding RNAs, and its biological function is unclear. Here, nucleotide-resolution method for profiling CTC-490G23.2 ac4C sites and gain- and loss-of-function experiments revealed that N-acetyltransferase 10 (NAT10) is responsible for ac4C modification of long noncoding RNAs (lncRNAs). NAT10-mediated ac4C modification leads to the stabilization and overexpression of lncRNA CTC-490G23.2 in primary esophageal squamous cell carcinoma (ESCC) and its further upregulation in metastatic tissues. CTC-490G23.2 significantly promotes cancer invasion and metastasis in vitro and in vivo. Mechanistically, CTC-490G23.2 acts as a scaffold to increase the binding of CD44 pre-mRNA to polypyrimidine tract-binding protein 1 (PTBP1), resulting in a oncogenic splicing switch from the standard isoform CD44s to the variant isoform CD44v(8-10). CD44v(8-10), but not CD44s, binds to and increases the protein stability of vimentin. Expression levels of CTC-490G23.2 and CD44v(8-10) can predict poor prognosis in cancer patients. Furthermore, the antisense oligonucleotide (ASO)/SV40-LAH4-L1 peptide self-assembled nanocomplexes targeting CTC490G23.2 exerts a significantly suppressive effect on cancer metastasis. The outcome of this study will provide new mechanistic insight into the ac4C modification of lncRNAs and useful clues for the development of novel systemic therapies and prognostic biomarkers.

Long noncoding RNAs (lncRNAs) play crucial roles in tumor progression and are aberrantly expressed in various cancers. However, the functional roles of lncRNAs in breast cancer remain largely unknown. Based on public databases and integrating bioinformatics analyses, the overexpression of lncRNA BCRT1 in breast cancer tissues was detected and further validated in a cohort of breast cancer tissues. The effects of lncRNA BCRT1 on proliferation, migration, invasion and macrophage polarization were determined by in vitro and in vivo experiments. Luciferase reporter assay and RNA immunoprecipitation (RIP) were carried out to reveal the interaction between lncRNA BCRT1, miR-1303, and PTBP3. Chromatin immunoprecipitation (ChIP) and RT-PCR were used to evaluate the regulatory effect of hypoxia-inducible factor-1α (HIF-1α) on lncRNA BCRT1. LncRNA BCRT1 was significantly upregulated in breast cancer tissues, which was correlated with poor prognosis in breast cancer patients. LncRNA BCRT1 knockdown remarkably suppressed tumor growth and metastasis in vitro and in vivo. Mechanistically, lncRNA BCRT1 could competitively bind with miR-1303 to prevent the degradation of its target gene PTBP3, which acts as a tumor-promoter in breast cancer. LncRNA BCRT1 overexpression could promote M2 polarization of macrophages, mediated by exosomes, which further accelerated breast cancer progression. Furthermore, lncRNA BCRT1 was upregulated in response to hypoxia, which was attributed to the binding of HIF-1α to HREs in the lncRNA BCRT1 promoter. Collectively, these results reveal a novel HIF-1α/lncRNA BCRT1/miR-1303/PTBP3 pathway for breast cancer progression and suggest that lncRNA BCRT1 might be a potential biomarker and therapeutic target for breast cancer.

Terminal oligopyrimidine (TOP) motifs are sequences at the 5′ ends of mRNAs that link their translation to the mTOR Complex 1 (mTORC1) nutrient-sensing signaling pathway. They are commonly regarded as discrete elements that reside on ∼100 mRNAs that mostly encode translation factors. However, the full spectrum of TOP sequences and their prevalence throughout the transcriptome remain unclear, primarily because of uncertainty over the mechanism that detects them. Here, we globally analyzed translation targets of La-related protein 1 (LARP1), an RNA-binding protein and mTORC1 effector that has been shown to repress TOP mRNA translation in a few specific cases. We establish that LARP1 is the primary translation regulator of mRNAs with classical TOP motifs genome-wide, and also that these motifs are extreme instances of a broader continuum of regulatory sequences. We identify the features of TOP sequences that determine their potency and quantify these as a metric that accurately predicts mTORC1/LARP1 regulation called a TOPscore. Analysis of TOPscores across the transcriptomes of 16 mammalian tissues defines a constitutive “core” set of TOP mRNAs, but also identifies tissue-specific TOP mRNAs produced via alternative transcription initiation sites. These results establish the central role of LARP1 in TOP mRNA regulation on a transcriptome scale and show how it connects mTORC1 to a tunable and dynamic program of gene expression that is tailored to specific biological contexts.

Polypyrimidine tract-binding protein 1 (PTBP1) is a member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family, which plays a key role in alternative splicing of precursor mRNA and RNA metabolism. PTBP1 is universally expressed in various tissues and binds to multiple downstream transcripts to interfere with physiological and pathological processes such as the tumor growth, body metabolism, cardiovascular homeostasis, and central nervous system damage, showing great prospects in many fields. The function of PTBP1 involves the regulation and interaction of various upstream molecules, including circular RNAs (circRNAs), microRNAs (miRNAs) and long non-coding RNAs (lncRNAs). These regulatory systems are inseparable from the development and treatment of diseases. Here, we review the latest knowledge regarding the structure and molecular functions of PTBP1 and summarize its functions and mechanisms of PTBP1 in various diseases, including controversial studies. Furthermore, we recommend future studies on PTBP1 and discuss the prospects of targeting PTBP1 in new clinical therapeutic approaches.

Background Hypoxic tumors are refractory to DNA damage drugs. However, the underlying mechanism has yet to be elucidated. We aimed to identify lncRNAs that upregulated under hypoxia and their effects on colorectal cancer (CRC). Methods CRC cells were treated with 1% O 2 to identify lncRNAs that upregulated under hypoxia. We integrated these lncRNAs with RNA-seq of 4 paired CRC tissues and TCGA data to get candidate lncRNAs. Multiple in vitro and in vivo assays were used to explore the role of LUCAT1 in CRC. Results We identified a hypoxia-induced lncRNA LUCAT1 that facilitated the growth of CRC cells and contributed to drug resistance of CRC cells both in vitro and in vivo. Mechanically, LUCAT1 interacts with polypyrimidine tract binding protein 1 (PTBP1) in CRC cells, facilitates the association of a set of DNA damage related genes with PTBP1, thus resulting in altered alternative splicing of these genes. Moreover, ectopic expression of PTBP1 in CRC cells with knockdown of LUCAT1 abrogated the effects induced by LUCAT1 knockdown. Chemotherapeutics drug combined with LUCAT1 knockdown via antisense oligonucleotides (ASO) would get a better outcome in vivo, compared with group treated with chemotherapeutic drug only. Notably, LUCAT1 is upregulated in CRC tissues, compared to adjacent normal tissues; and CRC patients with higher LUCAT1 have a worse prognosis and poorly responded to chemotherapy in the clinic. Conclusions Our data suggested CRC cells utilizes LUCAT1 to develop resistance to DNA damage drugs, and disrupting the LUCAT1/PTBP1 axis might be a promising therapeutic strategy for refractory hypoxic tumors.

BackgroundGastric cancer (GC) tumorigenesis and treatment failure are caused by cancer stem cells. Polypyrimidine tract binding protein 1 (PTBP1) was shown to be involved in the development of embryonic stem cells and is now being considered as a therapeutic target for tumour progression and stem-cell characteristics.MethodsPTBP1 expression in GC samples was detected using tissue microarrays. Proliferation, colony formation, spheroid formation and stem-cell analysis were used to examine PTBP1’s role in tumorigenesis and stem-cell maintenance. In AGS and HGC-27 cells with or without PTBP1 deficiency, ubiquitin-related protein expression and co-precipitation assays were performed.ResultsWe identified that PTBP1 was aberrantly highly expressed and represented a novel prognostic factor in GC patients. PTBP1 maintained the tumorigenic activity and stem-cell characteristics of GC in vitro and in vivo. PTBP1 directly interacts with c-Myc and stabilises its protein levels by preventing its proteasomal degradation. This is mediated by upregulating the ubiquitin-specific proteases USP28 and limiting FBW7-mediated ubiquitination of c-Myc. Moreover, the depletion of PTBP1-caused tumour regression was significantly compromised by exogenous c-Myc expression.ConclusionsBy preserving the stability of c-Myc through the ubiquitin–proteasome pathway, the oncogene PTBP1 supports stem-cell-like phenotypes of GC and is involved in GC progression.

Alternative splicing, a fundamental step in gene expression, is deregulated in many diseases. Splicing factors (SFs), which regulate this process, are up- or down regulated or mutated in several diseases including cancer. To date, there are no inhibitors that directly inhibit the activity of SFs. We designed decoy oligonucleotides, composed of several repeats of a RNA motif, which is recognized by a single SF. Here we show that decoy oligonucleotides targeting splicing factors RBFOX1/2, SRSF1 and PTBP1, can specifically bind to their respective SFs and inhibit their splicing and biological activities both in vitro and in vivo. These decoy oligonucleotides present an approach to specifically downregulate SF activity in conditions where SFs are either up-regulated or hyperactive. Alternative splicing, critical for gene expression, is deregulated in many diseases. Here the authors develop decoy oligonucleotides to specifically downregulate splicing factors activity.