ipaQTL-atlas: an atlas of intronic polyadenylation quantitative trait loci across human tissues.

Functional interpretation of disease-associated non-coding variants remains a significant challenge in the post-GWAS era. Our recent study has identified 3'UTR alternative polyadenylation (APA) quantitative trait loci (3'aQTLs) and connects APA events with QTLs as a major driver of human traits and diseases. Besides 3'UTR, APA events can also occur in intron regions, and increasing evidence has connected intronic polyadenylation with disease risk. However, systematic investigation of the roles of intronic polyadenylation in human diseases remained challenging due to the lack of a comprehensive database across a variety of human tissues. Here, we developed ipaQTL-atlas (http://bioinfo.szbl.ac.cn/ipaQTL) as the first comprehensive portal for intronic polyadenylation. The ipaQTL-atlas is based on the analysis of 15 170 RNA-seq data from 838 individuals across 49 Genotype-Tissue Expression (GTEx v8) tissues and contains ∼0.98 million SNPs associated with intronic APA events. It provides an interface for ipaQTLs search, genome browser, boxplots, and data download, as well as the visualization of GWAS and ipaQTL colocalization results. ipaQTL-atlas provides a one-stop portal to access intronic polyadenylation information and could significantly advance the discovery of APA-associated disease susceptibility genes.

Cancer-associated dynamics and potential regulators of intronic polyadenylation revealed by IPAFinder using standard RNA-seq data.

Intronic polyadenylation (IpA) usually leads to changes in the coding region of an mRNA, and its implication in diseases has been recognized, although at its very beginning status. Conveniently and accurately identifying IpA is of great importance for further evaluating its biological significance. Here, we developed IPAFinder, a bioinformatic method for the de novo identification of intronic poly(A) sites and their dynamic changes from standard RNA-seq data. Applying IPAFinder to 256 pan-cancer tumor/normal pairs across six tumor types, we discovered 490 recurrent dynamically changed IpA events, some of which are novel and derived from cancer-associated genes such as TSC1, SPERD2, and CCND2 Furthermore, IPAFinder revealed that IpA could be regulated by factors related to splicing and m6A modification. In summary, IPAFinder enables the global discovery and characterization of biologically regulated IpA with standard RNA-seq data and should reveal the biological significance of IpA in various processes.

Comprehensive characterization of somatic variants associated with intronic polyadenylation in human cancers.

Somatic single nucleotide variants (SNVs) in cancer genome affect gene expression through various mechanisms depending on their genomic location. While somatic SNVs near canonical splice sites have been reported to cause abnormal splicing of cancer-related genes, whether these SNVs can affect gene expression through other mechanisms remains an open question. Here, we analyzed RNA sequencing and exome data from 4,998 cancer patients covering ten cancer types and identified 152 somatic SNVs near splice sites that were associated with abnormal intronic polyadenylation (IPA). IPA-associated somatic variants favored the localization near the donor splice sites compared to the acceptor splice sites. A proportion of SNV-associated IPA events overlapped with premature cleavage and polyadenylation events triggered by U1 small nuclear ribonucleoproteins (snRNP) inhibition. GC content, intron length and polyadenylation signal were three genomic features that differentiated between SNV-associated IPA and intron retention. Notably, IPA-associated SNVs were enriched in tumor suppressor genes (TSGs), including the well-known TSGs such as PTEN and CDH1 with recurrent SNV-associated IPA events. Minigene assay confirmed that SNVs from PTEN, CDH1, VEGFA, GRHL2, CUL3 and WWC2 could lead to IPA. This work reveals that IPA acts as a novel mechanism explaining the functional consequence of somatic SNVs in human cancer.

Adaptive liquid lens with a tunable field of view.

In this paper, we demonstrate an adaptive liquid lens with a tunable field of view (FOV). The proposed liquid lens consists of an actuator and a lens chamber, the annular sheet is just placed on the liquid-liquid (L-L) interface in order to change the curvature and steer the tilt angle of the interface. Different from the conventional FOV adjustable lens combined with a liquid lens and a liquid prism, the proposed lens requires only one L-L interface to achieve the focal length change and FOV deflection. Moreover, the proposed lens reduces aberrations while maintaining high resolution. The experiments show that the optical power range is -27 m-1 to 30 m-1. It can realize the FOV deflection while tuning the focal length, with an angular resolution of 37"05. The proposed lens can be applied to telescopic system and microscopic system.

The landscape and clinical relevance of intronic polyadenylation in human cancers.

Intronic polyadenylation (IPA) is an RNA 3' end processing event which has been reported to play important roles in cancer development. However, the comprehensive landscape of IPA events across various cancer types is lacking. Here, we apply IPAFinder to identify and quantify IPA events in 10,383 samples covering all 33 cancer types from The Cancer Genome Atlas (TCGA) project. We identify a total of 21,835 IPA events, almost half of which are ubiquitously expressed. We identify 2761 unique dynamically changed IPA events across cancer types. Furthermore, we observe 8855 non-redundant clinically relevant IPA events, which could potentially be used as prognostic indicators. Our analysis also reveals that dynamic IPA usage within cancer signaling pathways may affect drug response. Finally, we develop a user-friendly data portal, IPACancer Atlas (http://www.tingni-lab.com/Pancan_IPA/), to search and explore IPAs in cancer.

Pan-cancer transcriptome analysis reveals widespread regulation through alternative tandem transcription initiation.

Abnormal transcription initiation from alternative first exon has been reported to promote tumorigenesis. However, the prevalence and impact of gene expression regulation mediated by alternative tandem transcription initiation were mostly unknown in cancer. Here, we developed a robust computational method to analyze alternative tandem transcription start site (TSS) usage from standard RNA sequencing data. Applying this method to pan-cancer RNA sequencing datasets, we observed widespread dysregulation of tandem TSS usage in tumors, many of which were independent of changes in overall expression level or alternative first exon usage. We showed that the dynamics of tandem TSS usage was associated with epigenomic modulation. We found that significant 5' untranslated region shortening of gene TIMM13 contributed to increased protein production, and up-regulation of TIMM13 by CRISPR-mediated transcriptional activation promoted proliferation and migration of lung cancer cells. Our findings suggest that dysregulated tandem TSS usage represents an addtional layer of cancer-associated transcriptome alterations.

The landscape of cryptic antisense transcription in human cancers reveals an oncogenic noncoding RNA in lung cancer.

Cryptic transcription initiation has been previously linked to activation of oncogenic transcripts. However, the prevalence and impact of cryptic antisense transcription from the opposite strand of protein-coding genes were mostly unknown in cancer. Applying a robust computational pipeline to publicly available transcriptome and epigenome datasets, we identified hundreds of previously unannotated cryptic antisense polyadenylated transcripts (CAPTs) that were enriched in tumor samples. We showed that the activation of cryptic antisense transcription was associated with increased chromatin accessibility and active histone marks. Accordingly, we found that many of the antisense transcripts were inducible by treatment of epigenetic drugs. Moreover, CRISPR-mediated epigenetic editing assays revealed that transcription of a noncoding RNA LRRK1-CAPT promoted LUSC cell proliferation, suggesting its oncogenic role. Our findings largely expand our understanding of cancer-associated transcription events, which may facilitate the development of novel strategies for cancer diagnosis and treatment.

Structural insights into RNase J that plays an essential role in Mycobacterium tuberculosis RNA metabolism.

Ribonucleases (RNases) are responsible for RNA metabolism. RNase J, the core enzyme of the RNA degradosome, plays an essential role in global mRNA decay. Emerging evidence showed that the RNase J of Mycobacterium tuberculosis (Mtb-RNase J) could be an excellent target for treating Mtb infection. Here, crystal structures of Mtb-RNase J in apo-state and complex with the single-strand RNA reveal the conformational change upon RNA binding and hydrolysis. Mtb-RNase J forms an active homodimer through the interactions between the ß-CASP and the ß-lactamase domain. Knockout of RNase J slows the growth rate and changes the colony morphologies and cell length in Mycobacterium smegmatis, which is restored by RNase J complementation. Finally, RNA-seq analysis shows that the knockout strain significantly changes the expression levels of 49 genes in metabolic pathways. Thus, our current study explores the structural basis of Mtb-RNase J and might provide a promising candidate in pharmacological treatment for tuberculosis.

E2F1/CKS2/PTEN signaling axis regulates malignant phenotypes in pediatric retinoblastoma.

Retinoblastoma (RB) is the most common pediatric intraocular malignancy and is a serious vision- and life-threatening disease. The biallelic mutation of the retinoblastoma gene RB1 is the initial event in the malignant transformation of RB, but the exact molecular mechanism is still unclear. E2F transcription factors can be activated by RB1 loss of function and lead to uncontrolled cell division. Among E2F family numbers, E2F1 has higher expression abundance than E2F2 and E2F3 in RB clinical samples. By integrating E2F1 ChIP-seq data, RNA-seq profiling from RB samples and RNA-seq profiling upon E2F1 knockdown, together with pathway analysis, literature searching and experimental validation, we identified Cyclin-dependent kinases regulatory subunit 2 (CKS2) as a novel regulator in regulating tumor-associated phenotypes in RB. CKS2 exhibited aberrantly higher expression in RB. Depletion of CKS2 in Y79 retinoblastoma cell line led to reduced cell proliferation, delayed DNA replication and decreased clonogenic growth. Downregulation of CKS2 also slowed tumor xenograft growth in nude mice. Importantly, reversed expression of CKS2 rescued cancer-associated phenotypes. Mechanistically, transcription factor E2F1 enhanced CKS2 expression through binding to its promoter and CKS2 regulated the cancer-associated PI3K-AKT pathway. This study discovered E2F1/CKS2/PTEN signaling axis regulates malignant phenotypes in pediatric retinoblastoma, and CKS2 may serve as a potential therapeutic target for this disease.

Lung cancer cells expressing a shortened CDK16 3'UTR escape senescence through impaired miR-485-5p targeting.

Inducing senescence in cancer cells is an emerging strategy for cancer therapy. The dysregulation and mutation of genes encoding cyclin-dependent kinases (CDKs) have been implicated in various human cancers. However, whether CDK can induce cancer cell senescence remains poorly understood. We observed that CDK16 expression was high in multiple cancer types, including lung cancer, whereas various replicative senescence models displayed low CDK16 expression. CDK16 knockdown caused senescence-associated phenotypes in lung cancer cell lines. Interestingly, the CDK16 3' UTR was shortened in cancer and lengthened in senescence models, which was regulated by alternative polyadenylation (APA). The longer 3'UTR [using the distal polyA (pA) site] generated less protein than the shorter one (using the proximal pA site). Since microRNAs (miRNAs) usually bind to the 3'UTR of target genes to suppress their expression, we investigated whether miRNAs targeting the region between the shortened and longer 3'UTR are responsible for the reduced expression. We found that miR-485-5p targeted the 3'UTR between the distal and proximal pA site and caused senescence-associated phenotypes by reducing protein production from the longer CDK16 transcript. Of note, CDK16 knockdown led to a reduced expression of MYC proto-oncogene, bHLH transcription factor (MYC) and CD274 molecule (PD-L1), which in turn enhanced the tumor-suppressive effects of senescent cancer cells. The present study discovered that CDK16, whose expression is under the regulation of APA and miR-485-5p, is a potential target for prosenescence therapy for lung cancer.

Transcription Factor ASCL1 Acts as a Novel Potential Therapeutic Target for the Treatment of the Cushing's Disease.

BACKGROUND: The pathogenesis of Cushing's disease (CD) is still not adequately understood despite the identification of somatic driver mutations in USP8, BRAF, and USP48. In this multiomics study, we combined RNA sequencing (RNA-seq) with Sanger sequencing to depict transcriptional dysregulation under different gene mutation backgrounds. Furthermore, we evaluated the potential of achaete-scute complex homolog 1 (ASCL1), a pioneer transcription factor, as a novel therapeutic target for treatment of CD and its possible downstream pathway. METHODS: RNA-seq was adopted to investigate the gene expression profile of CD, and Sanger sequencing was adopted to detect gene mutations. Bioinformatics analysis was used to depict transcriptional dysregulation under different gene mutation backgrounds. The function of ASCL1 in hormone secretion, cell proliferation, and apoptosis were studied in vitro. The effectiveness of an ASCL1 inhibitor was evaluated in primary CD cells, and the clinical relevance of ASCL1 was examined in 68 patients with CD. RNA-seq in AtT-20 cells on Ascl1 knockdown combined with published chromatin immunoprecipitation sequencing data and dual luciferase assays were used to explore downstream pathways. RESULTS: ASCL1 was exclusively overexpressed in USP8-mutant and wild-type tumors. Ascl1 promoted adrenocorticotrophin hormone overproduction and tumorigenesis and directly regulated Pomc in AtT-20 cells. An ASCL1 inhibitor presented promising efficacy in both AtT-20 and primary CD cells. ASCL1 overexpression was associated with a larger tumor volume and higher adrenocorticotrophin secretion in patients with CD. CONCLUSION: Our findings help to clarify the pathogenesis of CD and suggest that ASCL1 is a potential therapeutic target the treatment of CD. SUMMARY: The pathogenesis of Cushing's disease (CD) is still not adequately understood despite the identification of somatic driver mutations in USP8, BRAF, and USP48. Moreover, few effective medical therapies are currently available for the treatment of CD. Here, using a multiomics approach, we first report the aberrant overexpression of the transcription factor gene ASCL1 in USP8-mutant and wild-type tumors of CD. Ascl1 promoted adrenocorticotrophin hormone overproduction and tumorigenesis and directly regulated Pomc in mouse AtT-20 cells. Notably, an ASCL1 inhibitor presented promising efficacy in both AtT-20 and primary CD cells. Importantly, ASCL1 overexpression was associated with a larger tumor volume and higher adrenocorticotrophin secretion in patients with CD. Thus, our findings improve understanding of CD pathogenesis and suggest that ASCL1 is a potential therapeutic target the treatment of CD.