Ling-Ling Zheng

deepBase v2.0: identification, expression, evolution and function of small RNAs, LncRNAs and circular RNAs from deep-sequencing data

Small non-coding RNAs (e.g. miRNAs) and long non-coding RNAs (e.g. lincRNAs and circRNAs) are emerging as key regulators of various cellular processes. However, only a very small fraction of these enigmatic RNAs have been well functionally characterized. In this study, we describe deepBase v2.0 (http://biocenter.sysu.edu.cn/deepBase/), an updated platform, to decode evolution, expression patterns and functions of diverse ncRNAs across 19 species. deepBase v2.0 has been updated to provide the most comprehensive collection of ncRNA-derived small RNAs generated from 588 sRNA-Seq datasets. Moreover, we developed a pipeline named lncSeeker to identify 176 680 high-confidence lncRNAs from 14 species. Temporal and spatial expression patterns of various ncRNAs were profiled. We identified approximately 24 280 primate-specific, 5193 rodent-specific lncRNAs, and 55 highly conserved lncRNA orthologs between human and zebrafish. We annotated 14 867 human circRNAs, 1260 of which are orthologous to mouse circRNAs. By combining expression profiles and functional genomic annotations, we developed lncFunction web-server to predict the function of lncRNAs based on protein-lncRNA co-expression networks. This study is expected to provide considerable resources to facilitate future experimental studies and to uncover ncRNA functions.

Pseudogene-derived small interference RNAs regulate gene expression in African Trypanosoma brucei

Pseudogenes have been shown to acquire unique regulatory roles from more and more organisms. We report the observation of a cluster of siRNAs derived from pseudogenes of African Trypanosoma brucei using high through-put analysis. We show that these pseudogene-derived siRNAs suppress gene expression through RNA interference. The discovery that siRNAs may originate from pseudogenes and regulate gene expression in a unicellular eukaryote provides insights into the functional roles of pseudogenes and into the origin of noncoding small RNAs.

Pseudogenes are not pseudo any more.

Recent significant progress toward understanding the function of pseudogenes in protozoa (Trypanosoma brucei), metazoa (mouse) and plants, make it pertinent to provide a brief overview on what has been learned about this fascinating subject. We discuss the regulatory mechanisms of pseudogenes at the post-transcriptional level and advance new ideas toward understanding the evolution of these, sometimes called “garbage genes” or “junk DNA,” seeking to stimulate the interest of scientists and additional research on the subject. We hope this point-of-view can be helpful to scientists working or seeking to work on these and related issues.

Discovery of Protein-lncRNA Interactions by Integrating Large-Scale CLIP-Seq and RNA-Seq Datasets.

Long non-coding RNAs (lncRNAs) are emerging as important regulatory molecules in developmental, physiological, and pathological processes. However, the precise mechanism and functions of most of lncRNAs remain largely unknown. Recent advances in high-throughput sequencing of immunoprecipitated RNAs after cross-linking (CLIP-Seq) provide powerful ways to identify biologically relevant protein–lncRNA interactions. In this study, by analyzing millions of RNA-binding protein (RBP) binding sites from 117 CLIP-Seq datasets generated by 50 independent studies, we identified 22,735 RBP–lncRNA regulatory relationships. We found that one single lncRNA will generally be bound and regulated by one or multiple RBPs, the combination of which may coordinately regulate gene expression. We also revealed the expression correlation of these interaction networks by mining expression profiles of over 6000 normal and tumor samples from 14 cancer types. Our combined analysis of CLIP-Seq data and genome-wide association studies data discovered hundreds of disease-related single nucleotide polymorphisms resided in the RBP binding sites of lncRNAs. Finally, we developed interactive web implementations to provide visualization, analysis, and downloading of the aforementioned large-scale datasets. Our study represented an important step in identification and analysis of RBP–lncRNA interactions and showed that these interactions may play crucial roles in cancer and genetic diseases.

Drastic expression change of transposon-derived piRNA-like RNAs and microRNAs in early stages of chicken embryos implies a role in gastrulation

Recent studies have shown that endogenous small RNAs regulate a variety of biological processes during vertebrate development; however, little is known about the role of small RNAs in regulating developmental signaling pathways during early embryogenesis. In this study, we applied Illumina sequencing to characterize an unexpected endogenous small RNA catalog and demonstrated a dramatic transition from transposon-derived piRNA-like small RNAs (pilRNAs) to microRNAs (miRNAs) in pre- and post-gastrula chicken embryos. The comprehensive expression profile of chicken miRNAs at the pre- and post-gastrula stages revealed that most known and new miRNAs were dynamically regulated during development. In addition to embryonic stem cell-related miRNAs, Gene Ontology (GO) analysis showed that miRNAs enriched in early stage chicken embryos targeted multiple signal transduction pathways associated with the reproductive process and embryogenesis, including Wnt and TGF-β, which specifies the neural fate of blastodermal cells. Intriguingly, a large cohort of pilRNAs primarily derived from the active and most abundant transposable elements (TEs) were enriched in chicken stage X blastoderms. Within stage X blastoderms, pilRNAs were specifically localized to the primordial germ cells (PGCs), indicating their post-zygotic origin. Together, these findings imply a role for small RNAs in gastrulation in early stage chicken embryos.

ChIPBase v2.0: decoding transcriptional regulatory networks of non-coding RNAs and protein-coding genes from ChIP-seq data

The abnormal transcriptional regulation of non-coding RNAs (ncRNAs) and protein-coding genes (PCGs) is contributed to various biological processes and linked with human diseases, but the underlying mechanisms remain elusive. In this study, we developed ChIPBase v2.0 (http://rna.sysu.edu.cn/chipbase/) to explore the transcriptional regulatory networks of ncRNAs and PCGs. ChIPBase v2.0 has been expanded with ∼10 200 curated ChIP-seq datasets, which represent about 20 times expansion when comparing to the previous released version. We identified thousands of binding motif matrices and their binding sites from ChIP-seq data of DNA-binding proteins and predicted millions of transcriptional regulatory relationships between transcription factors (TFs) and genes. We constructed ‘Regulator’ module to predict hundreds of TFs and histone modifications that were involved in or affected transcription of ncRNAs and PCGs. Moreover, we built a web-based tool, Co-Expression, to explore the co-expression patterns between DNA-binding proteins and various types of genes by integrating the gene expression profiles of ∼10 000 tumor samples and ∼9100 normal tissues and cell lines. ChIPBase also provides a ChIP-Function tool and a genome browser to predict functions of diverse genes and visualize various ChIP-seq data. This study will greatly expand our understanding of the transcriptional regulations of ncRNAs and PCGs.

tRF2Cancer: A web server to detect tRNA-derived small RNA fragments (tRFs) and their expression in multiple cancers

tRNA-derived small RNA fragments (tRFs) are one class of small non-coding RNAs derived from transfer RNAs (tRNAs). tRFs play important roles in cellular processes and are involved in multiple cancers. High-throughput small RNA (sRNA) sequencing experiments can detect all the cellular expressed sRNAs, including tRFs. However, distinguishing genuine tRFs from RNA fragments generated by random degradation remains a major challenge. In this study, we developed an integrated web-based computing system, tRF2Cancer, to accurately identify tRFs from sRNA deep-sequencing data and evaluate their expression in multiple cancers. The binomial test was introduced to evaluate whether reads from a small RNA-seq data set represent tRFs or degraded fragments. A classification method was then used to annotate the types of tRFs based on their sites of origin in pre-tRNA or mature tRNA. We applied the pipeline to analyze 10 991 data sets from 32 types of cancers and identified thousands of expressed tRFs. A tool called ‘tRFinCancer’ was developed to facilitate the users to inspect the expression of tRFs across different types of cancers. Another tool called ‘tRFBrowser’ shows both the sites of origin and the distribution of chemical modification sites in tRFs on their source tRNA. The tRF2Cancer web server is available at http://rna.sysu.edu.cn/tRFfinder/.

Both endo-siRNAs and tRNA-derived small RNAs are involved in the differentiation of primitive eukaryote Giardia lamblia

Significance Small RNAs (sRNAs) are most important regulators in eukaryotes. Although different kinds of sRNAs have been extensively studied in higher eukaryotes, their role remains largely unknown in protozoa. We have systematically investigated in the full genome the sRNAs of Giardia lamblia, the most primitive eukaryote known. Surprisingly, we have found that two major types of sRNAs (i.e., endogenous siRNAs and tRNA-derived sRNAs) are largely encoded in the genome of G. lamblia, whereas canonical microRNAs could not be identified in this parasite. Additional studies showed that both sRNAs might be involved in the differentiation regulation of G. lamblia. This study indicates that these two kinds of eukaryotic sRNAs emerged in the early evolution of eukaryotes. Small RNAs (sRNAs), including microRNAs and endogenous siRNAs (endo-siRNAs), regulate most important biologic processes in eukaryotes, such as cell division and differentiation. Although sRNAs have been extensively studied in various eukaryotes, the role of sRNAs in the early emergence of eukaryotes is unclear. To address these questions, we deep sequenced the sRNA transcriptome of four different stages in the differentiation of Giardia lamblia, one of the most primitive eukaryotes. We identified a large number of endo-siRNAs in this fascinating parasitic protozoan and found that they were produced from live telomeric retrotransposons and three genomic regions (i.e., endo-siRNA generating regions [eSGRs]). eSGR-derived endo-siRNAs were proven to target mRNAs in trans. Gradual up-regulation of endo-siRNAs in the differentiation of Giardia suggested that they might be involved in the regulation of this process. This hypothesis was supported by the impairment of the differentiation ability of Giardia when GLDICER, essential for the biogenesis of endo-siRNAs, was knocked down. Endo-siRNAs are not the only sRNA regulators in Giardia differentiation, because a great number of tRNAs-derived sRNAs showed more dramatic expression changes than endo-siRNAs in this process. We totally identified five novel kinds of tRNAs-derived sRNAs and found that the biogenesis in four of them might be correlated with that of stress-induced tRNA-derived RNA (sitRNA), which was discovered in our previous studies. Our studies reveal an unexpected complex panorama of sRNA in G. lamblia and shed light on the origin and functional evolution of eukaryotic sRNAs.

Comparative transcriptome analysis of small noncoding RNAs in different stages of Trypanosoma brucei

Trypanosoma brucei, a pathogen of human and domestic animals, is an early evolved parasitic protozoan with a complex life cycle. Most genes of this parasite are post-transcriptionally regulated. However, the mechanisms and the molecules involved remain largely unknown. We have deep-sequenced the small RNAs of two life stages of this parasite--the bloodstream form and the procyclic form. Our results show that the small RNAs of T. brucei could derive from multiple sources, including NATs (natural antisense transcripts), tRNAs, and rRNAs. Most of these small RNAs in the two stages were found to share uniform characteristics. However, our results demonstrate that their variety and expression show significant differences between different stages, indicating possible functional differentiation. Dicer-knockdown evidence further proved that some of the small interfering RNAs (siRNAs) could regulate the expression of genes. Based on the genome-wide analysis of the small RNAs in the two stages of T. brucei, our results not only provide evidence to study their differentiation but also shed light on questions regarding the origins and evolution of small RNA-based mechanisms in early eukaryotes.

Cancer in the parasitic protozoans Trypanosoma brucei and Toxoplasma gondii

Cancer is a general name for more than 100 malignant diseases. It is postulated that all cancers start from a single abnormal cell that grows out of control. Untreated cancers can cause serious consequences and deaths. Great progress has been made in cancer research that has significantly improved our knowledge and understanding of the nature and mechanisms of the disease, but the origins of cancer are far from being well understood due to the limitations of suitable model systems and to the complexities of the disease. In view of the fact that cancers are found in various species of vertebrates and other metazoa, here, we suggest that cancer also occurs in parasitic protozoans such as Trypanosoma brucei, a blood parasite, and Toxoplasma gondii, an obligate intracellular pathogen. Without treatment, these protozoan cancers may cause severe disease and death in mammals, including humans. The simpler genomes of these single-cell organisms, in combination with their complex life cycles and fascinating life cycle differentiation processes, may help us to better understand the origins of cancers and, in particular, leukemias.