Jian-You Liao

Deep Sequencing of Human Nuclear and Cytoplasmic Small RNAs Reveals an Unexpectedly Complex Subcellular Distribution of miRNAs and tRNA 3′ Trailers

Background MicroRNAs (miRNAs) are ∼22-nt small non-coding regulatory RNAs that have generally been considered to regulate gene expression at the post-transcriptional level in the cytoplasm. However, recent studies have reported that some miRNAs localize to and function in the nucleus. Methodology/Principal Findings To determine the number of miRNAs localized to the nucleus, we systematically investigated the subcellular distribution of small RNAs (sRNAs) by independent deep sequencing sequenced of the nuclear and cytoplasmic pools of 18- to 30-nucleotide sRNAs from human cells. We identified 339 nuclear and 324 cytoplasmic known miRNAs, 300 of which overlap, suggesting that the majority of miRNAs are imported into the nucleus. With the exception of a few miRNAs evidently enriched in the nuclear pool, such as the mir-29b, the ratio of miRNA abundances in the nuclear fraction versus in the cytoplasmic fraction vary to some extent. Moreover, our results revealed that a large number of tRNA 3′trailers are exported from the nucleus and accumulate in the cytoplasm. These tRNA 3′ trailers accumulate in a variety of cell types, implying that the biogenesis of tRNA 3′ trailers is conserved and that they have a potential functional role in vertebrate cells. Conclusion/Significance Our results provide the first comprehensive view of the subcellular distribution of diverse sRNAs and new insights into the roles of miRNAs and tRNA 3′ trailers in the cell.

Overexpression of microRNA OsmiR397 improves rice yield by increasing grain size and promoting panicle branching

Increasing grain yields is a major focus of crop breeders around the world. Here we report that overexpression of the rice microRNA (miRNA) OsmiR397, which is naturally highly expressed in young panicles and grains, enlarges grain size and promotes panicle branching, leading to an increase in overall grain yield of up to 25% in a field trial. To our knowledge, no previous report has shown a positive regulatory role of miRNA in the control of plant seed size and grain yield. We determined that OsmiR397 increases grain yield by downregulating its target, OsLAC, whose product is a laccase-like protein that we found to be involved in the sensitivity of plants to brassinosteroids. As miR397 is highly conserved across different species, our results suggest that manipulating miR397 may be useful for increasing grain yield not only in rice but also in other cereal crops.

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.

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.

mirExplorer: detecting microRNAs from genome and next generation sequencing data using the AdaBoost method with transition probability matrix and combined features.

microRNAs (miRNAs) represent an abundant group of small regulatory non-coding RNAs in eukaryotes. The emergence of Next-generation sequencing (NGS) technologies has allowed the systematic detection of small RNAs (sRNAs) and de novo sequencing of genomes quickly and with low cost. As a result, there is an increased need to develop fast miRNA prediction tools to annotate miRNAs from various organisms with a high level of accuracy, using the genome sequence or the NGS data. Several miRNA predictors have been proposed to achieve this purpose. However, the accuracy and fitness for multiple species of existing predictors needed to be improved. Here, we present a novel prediction tool called mirExplorer, which is based on an integrated adaptive boosting method and contains two modules. The first module named mirExplorer-genome was designed to de novo predict pre-miRNAs from genome, and the second module named mirExplorer-NGS was used to discover miRNAs from NGS data. A set of novel features of pre-miRNA secondary structure and miRNA biogenesis has been extracted to distinguish real pre-miRNAs from pseudo ones. We used outer-ten-fold cross-validation to verify the mirExplorer-genome computation, which obtained a specificity of 95.03% and a sensitivity of 93.71% on human data. This computation was made on test data from 16 species, and it achieved an overall accuracy of 95.53%. Systematic outer-ten-fold cross-validation of the mirExplorer-NGS model achieved a specificity of 98.3% and a sensitivity of 97.72%. We found that the good performance of the mirExplorer-NGS model was upheld across species from vertebrates to plants in test datasets. The mirExplorer is available as both web server and software package at http://biocenter.sysu.edu.cn/mir/.

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.

Genome-wide evolutionary analysis of the noncoding RNA genes and noncoding DNA of Paramecium tetraurelia

The compact genome of the unicellular eukaryote Paramecium tetraurelia contains noncoding DNA (ncDNA) distributed into >39,000 intergenic sequences and >90,000 introns of 390 base pairs (bp) and 25 bp on average, respectively. Here we analyzed the molecular features of the ncRNA genes, introns, and intergenic sequences of this genome. We mainly used computational programs and comparative genomics possible because the P. tetraurelia genome had formed throughout whole-genome duplications (WGDs). We characterized 417 5S rRNA, snRNA, snoRNA, SRP RNA, and tRNA putative genes, 415 of which map within intergenic sequences, and two, within introns. The evolution of these ncRNA genes appears to have mainly involved purifying selection and gene deletion. We then compared the introns that interrupt the protein-coding gene duplicates arisen from the recent WGD and identified a population of a few thousands of introns having evolved under most stringent constraints (>95% of identity). We also showed that low nucleotide substitution levels characterize the 50 and 80-115 base pairs flanking, respectively, the stop and start codons of the protein-coding genes. Lower substitution levels mark the base pairs flanking the highly transcribed genes, or the start codons of the genes of the sets with a high number of WGD-related sequences. Finally, adjacent to protein-coding genes, we characterized 32 DNA motifs able to encode stable and evolutionary conserved RNA secondary structures and defining putative expression controlling elements. Fourteen DNA motifs with similar properties map distant from protein-coding genes and may encode regulatory ncRNAs.

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.

A Contig-Based Strategy for the Genome-Wide Discovery of MicroRNAs without Complete Genome Resources

MicroRNAs (miRNAs) are important regulators of many cellular processes and exist in a wide range of eukaryotes. High-throughput sequencing is a mainstream method of miRNA identification through which it is possible to obtain the complete small RNA profile of an organism. Currently, most approaches to miRNA identification rely on a reference genome for the prediction of hairpin structures. However, many species of economic and phylogenetic importance are non-model organisms without complete genome sequences, and this limits miRNA discovery. Here, to overcome this limitation, we have developed a contig-based miRNA identification strategy. We applied this method to a triploid species of edible banana (GCTCV-119, Musa spp. AAA group) and identified 180 pre-miRNAs and 314 mature miRNAs, which is three times more than those were predicted by the available dataset-based methods (represented by EST+GSS). Based on the recently published miRNA data set of Musa acuminate, the recall rate and precision of our strategy are estimated to be 70.6% and 92.2%, respectively, significantly better than those of EST+GSS-based strategy (10.2% and 50.0%, respectively). Our novel, efficient and cost-effective strategy facilitates the study of the functional and evolutionary role of miRNAs, as well as miRNA-based molecular breeding, in non-model species of economic or evolutionary interest.

Different Types of Small RNAs in Protozoa

The origin and evolution of small RNA molecules is a long-standing mystery. The protozoa represent the earliest eukaryotes and encompass rich diverse genetic resources. However, there are many contradictions and disputes prevalent in studies on protozoan microRNAs. In this review, we summarize the research on the analysis of small RNA transcriptome data on two representative protozoans, Trypanosoma brucei and Giardia lamblia , as generated by high throughput sequencing. The results show that these protozoans do not have canonical miRNAs. Unexpectedly, there are many types of endogenous small interfering RNAs (endo-siRNAs). In addition, stress induced tRNA derived small RNAs (sitRNAs) were observed in different life cycle stages of these parasites. In total, there are six kinds of sitRNAs in G. lamblia . These small RNAs are then found to be involved in the differentiation processes of primitive eukaryotes. These results showed that systematic analysis of small RNAs in the protozoa revealed a more complex picture than previously thought.