Doctor of Philosophy, University of North Carolina, Chapel Hill (2014)
Bachelor of Science, Fudan University (2008)
Howard Chang, Postdoctoral Faculty Sponsor
RNA has the intrinsic property to base pair, forming complex structures fundamental to its diverse functions. Here, we develop PARIS, a method based on reversible psoralen crosslinking for global mapping of RNA duplexes with near base-pair resolution in living cells. PARIS analysis in three human and mouse cell types reveals frequent long-range structures, higher-order architectures, and RNA-RNA interactions in trans across the transcriptome. PARIS determines base-pairing interactions on an individual-molecule level, revealing pervasive alternative conformations. We used PARIS-determined helices to guide phylogenetic analysis of RNA structures and discovered conserved long-range and alternative structures. XIST, a long noncoding RNA (lncRNA) essential for X chromosome inactivation, folds into evolutionarily conserved RNA structural domains that span many kilobases. XIST A-repeat forms complex inter-repeat duplexes that nucleate higher-order assembly of the key epigenetic silencing protein SPEN. PARIS is a generally applicable and versatile method that provides novel insights into the RNA structurome and interactome. VIDEO ABSTRACT.
View details for DOI 10.1016/j.cell.2016.04.028
View details for Web of Science ID 000376478200024
View details for PubMedID 27180905
We report the discovery of a class of abundant circular noncoding RNAs that are produced during metazoan tRNA splicing. These transcripts, termed tRNA intronic circular (tric)RNAs, are conserved features of animal transcriptomes. Biogenesis of tricRNAs requires anciently conserved tRNA sequence motifs and processing enzymes, and their expression is regulated in an age-dependent and tissue-specific manner. Furthermore, we exploited this biogenesis pathway to develop an in vivo expression system for generating "designer" circular RNAs in human cells. Reporter constructs expressing RNA aptamers such as Spinach and Broccoli can be used to follow the transcription and subcellular localization of tricRNAs in living cells. Owing to the superior stability of circular vs. linear RNA isoforms, this expression system has a wide range of potential applications, from basic research to pharmaceutical science.
View details for DOI 10.1261/rna.052944.115
View details for PubMedID 26194134
Pre-mRNA splicing is a critical step in eukaryotic gene expression that contributes to proteomic, cellular, and developmental complexity. Small nuclear (sn)RNAs are core spliceosomal components; however, the extent to which differential expression of snRNA isoforms regulates splicing is completely unknown. This is partly due to difficulties in the accurate analysis of the spatial and temporal expression patterns of snRNAs. Here, we use high-throughput RNA-sequencing (RNA-seq) data to profile expression of four major snRNAs throughout Drosophila development. This analysis shows that individual isoforms of each snRNA have distinct expression patterns in the embryo, larva, and pharate adult stages. Expression of these isoforms is more heterogeneous during embryogenesis; as development progresses, a single isoform from each snRNA subtype gradually dominates expression. Despite the lack of stable snRNA orthologous groups during evolution, this developmental switching of snRNA isoforms also occurs in distantly related vertebrate species, such as Xenopus, mouse, and human. Our results indicate that expression of snRNA isoforms is regulated and lays the foundation for functional studies of individual snRNA isoforms.
View details for DOI 10.1534/g3.114.015735
View details for PubMedID 25416704
Non-coding (nc)RNAs are important structural and regulatory molecules. Accurate determination of the primary sequence and secondary structure of ncRNAs is important for understanding their functions. During cDNA synthesis, RNA 3' end stem-loops can self-prime reverse transcription, creating RNA-cDNA chimeras. We found that chimeric RNA-cDNA fragments can also be detected at 5' end stem-loops, although at much lower frequency. Using the Gubler-Hoffman method, both types of chimeric fragments can be converted to cDNA during library construction, and they are readily detectable in high-throughput RNA sequencing (RNA-seq) experiments. Here, we show that these chimeric reads contain valuable information about the boundaries of ncRNAs. We developed a bioinformatic method, called Vicinal, to precisely map the ends of numerous fruitfly, mouse and human ncRNAs. Using this method, we analyzed chimeric reads from over 100 RNA-seq datasets, the results of which we make available for users to find RNAs of interest. In summary, we show that Vicinal is a useful tool for determination of the precise boundaries of uncharacterized ncRNAs, facilitating further structure/function studies.
View details for DOI 10.1093/nar/gku207
View details for Web of Science ID 000336495400006
View details for PubMedID 24623808
Reduced levels of survival motor neuron (SMN) protein lead to a neuromuscular disease called spinal muscular atrophy (SMA). Animal models of SMA recapitulate many aspects of the human disease, including locomotion and viability defects, but have thus far failed to uncover the causative link between a lack of SMN protein and neuromuscular dysfunction. While SMN is known to assemble small nuclear ribonucleoproteins (snRNPs) that catalyze pre-mRNA splicing, it remains unclear whether disruptions in splicing are etiologic for SMA. To investigate this issue, we carried out RNA deep-sequencing (RNA-seq) on age-matched Drosophila Smn-null and wild-type larvae. Comparison of genome-wide mRNA expression profiles with publicly available data sets revealed the timing of a developmental arrest in the Smn mutants. Furthermore, genome-wide differences in splicing between wild-type and Smn animals did not correlate with changes in mRNA levels. Specifically, we found that mRNA levels of genes that contain minor introns vary more over developmental time than they do between wild-type and Smn mutants. An analysis of reads mapping to minor-class intron-exon junctions revealed only small changes in the splicing of minor introns in Smn larvae, within the normal fluctuations that occur throughout development. In contrast, Smn mutants displayed a prominent increase in levels of stress-responsive transcripts, indicating a systemic response to the developmental arrest induced by loss of SMN protein. These findings not only provide important mechanistic insight into the developmental arrest displayed by Smn mutants, but also argue against a minor-intron-dependent etiology for SMA.
View details for DOI 10.1261/rna.038919.113
View details for Web of Science ID 000325813900006
View details for PubMedID 24006466
The REV3L gene, encoding the catalytic subunit of human polymerase zeta, plays a significant role in the cytotoxicity, mutagenicity, and chemoresistance of certain tumors. However, the role of REV3L in regulating the sensitivity of glioma cells to chemotherapy remains unknown. In this study, we investigated the expression of the REV3L gene in 10 normal brain specimens and 30 human glioma specimens and examined the value of REV3L as a potential modulator of cellular response to various DNA-damaging agents. Reverse transcriptase PCR/real-time PCR analysis revealed that REV3L was overexpressed in human gliomas compared with normal brain tissues. A glioma cell model with stable overexpression of REV3L was used to probe the role of REV3L in cisplatin treatment; upregulation of REV3L markedly attenuated cisplatin-induced apoptosis of the mitochondrial apoptotic pathway. We therefore assessed the REV3L-targeted treatment modality that combines suppression of REV3L expression using RNA interference (RNAi) with the cytotoxic effects of DNA-damaging agents. Downregulation of REV3L expression significantly enhanced the sensitivity of glioma cells to cisplatin, as evidenced by the increased apoptosis rate and marked alterations in the anti-apoptotic proteins B-cell lymphoma 2 (Bcl-2) and B-cell lymphoma-extra large (Bcl-xl) and proapoptotic Bcl-2-associated x protein (Bax) expression levels, and reduced mutation frequencies in surviving glioma cells. These results suggest that REV3L may potentially contribute to gliomagenesis and play a crucial role in regulating cellular response to the DNA cross-linking agent cisplatin. Our findings indicate that RNAi targeting REV3L combined with chemotherapy has synergistic therapeutic effects on glioma cells, which warrants further investigation as an effective novel therapeutic regimen for patients with this malignancy.
View details for DOI 10.1215/15228517-2009-015
View details for Web of Science ID 000272974100009
View details for PubMedID 19289490
Hepatic glucokinase (GCK) is a key enzyme in glucose utilisation. Downregulation of its activity is associated with insulin resistance and type 2 diabetes mellitus. However, it is unknown whether hepatic Gck expression is influenced by age and is involved in ageing-mediated diabetes, and whether the degree of methylation of the hepatic Gck promoter is correlated with the transcription of Gck. To address the question, we evaluated hepatic Gck transcription and promoter methylation in young (14 weeks), adult (40 weeks) and aged (80 weeks) rats.Hepatic glycogen, Gck expression and the kinase activity of GCK were measured in three age groups. The CpG methylation status was determined by both bisulphite direct sequencing and clone sequencing of the PCR amplificates of Gck promoter. The causal relationship between Gck methylation and mRNA expression was confirmed by treating rat primary hepatocytes with 5-aza-2'-deoxycytidine (5-Aza-CdR).We have shown an age-associated decline in hepatic glycogen, Gck expression levels and the kinase activity of hepatic GCK. The eleven CpG sites studied displayed age-related progressive methylation changes in hepatic Gck promoter, which were confirmed by two methods: direct and clone sequencing. After 5-Aza-CdR treatment of rat primary hepatocytes, there was a fourfold increase in Gck expression.Our results demonstrate that an age-related increase in methylation is negatively associated with hepatic Gck expression, suggesting that DNA methylation could be involved in increasing age-dependent susceptibility to hepatic insulin resistance and diabetes. Thus, the epigenetic modification of the hepatic Gck promoter may represent an important marker for diabetogenic potential during the ageing process.
View details for DOI 10.1007/s00125-008-1034-8
View details for Web of Science ID 000257848900023
View details for PubMedID 18496667