MiR-103a-3p targets the 5\u27 UTR of GPRC5A in pancreatic cells.

MicroRNAs (miRNAs) are short noncoding RNAs that regulate the expression of their targets in a sequence-dependent manner. For protein-coding transcripts, miRNAs regulate expression levels through binding sites in either the 3\u27 untranslated region (3\u27 UTR) or the amino acid coding sequence (CDS) of the targeted messenger RNA (mRNA). Currently, for the 5\u27 untranslated region (5\u27 UTR) of mRNAs, very few naturally occurring examples exist whereby the targeting miRNA down-regulates the expression of the corresponding mRNA in a seed-dependent manner. Here we describe and characterize two miR-103a-3p target sites in the 5\u27 UTR of GPRC5A, a gene that acts as a tumor suppressor in some cancer contexts and as an ongocene in other cancer contexts. In particular, we show that the interaction of miR-103a-3p with each of these two 5\u27 UTR targets reduces the expression levels of both GPRC5A mRNA and GPRC5A protein in one normal epithelial and two pancreatic cancer cell lines. By ectopically expressing sponges that contain instances of the wild-type 5\u27 UTR targets we also show that we can reduce miR-103a-3p levels and increase GPRC5A mRNA and protein levels. These findings provide some first knowledge on the post-transcriptional regulation of this tumor suppressor/oncogene and present additional evidence for the participation of 5\u27 UTRs in miRNA driven post-transcriptional regulatory control

MINTmap: fast and exhaustive profiling of nuclear and mitochondrial tRNA fragments from short RNA-seq data.

Transfer RNA fragments (tRFs) are an established class of constitutive regulatory molecules that arise from precursor and mature tRNAs. RNA deep sequencing (RNA-seq) has greatly facilitated the study of tRFs. However, the repeat nature of the tRNA templates and the idiosyncrasies of tRNA sequences necessitate the development and use of methodologies that differ markedly from those used to analyze RNA-seq data when studying microRNAs (miRNAs) or messenger RNAs (mRNAs). Here we present MINTmap (for MItochondrial and Nuclear TRF mapping), a method and a software package that was developed specifically for the quick, deterministic and exhaustive identification of tRFs in short RNA-seq datasets. In addition to identifying them, MINTmap is able to unambiguously calculate and report both raw and normalized abundances for the discovered tRFs. Furthermore, to ensure specificity, MINTmap identifies the subset of discovered tRFs that could be originating outside of tRNA space and flags them as candidate false positives. Our comparative analysis shows that MINTmap exhibits superior sensitivity and specificity to other available methods while also being exceptionally fast. The MINTmap codes are available through https://github.com/TJU-CMC-Org/MINTmap/ under an open source GNU GPL v3.0 license

Pattern-based phylogenetic distance estimation and tree reconstruction

We have developed an alignment-free method that calculates phylogenetic distances using a maximum likelihood approach for a model of sequence change on patterns that are discovered in unaligned sequences. To evaluate the phylogenetic accuracy of our method, and to conduct a comprehensive comparison of existing alignment-free methods (freely available as Python package decaf+py at http://www.bioinformatics.org.au), we have created a dataset of reference trees covering a wide range of phylogenetic distances. Amino acid sequences were evolved along the trees and input to the tested methods; from their calculated distances we infered trees whose topologies we compared to the reference trees. We find our pattern-based method statistically superior to all other tested alignment-free methods on this dataset. We also demonstrate the general advantage of alignment-free methods over an approach based on automated alignments when sequences violate the assumption of collinearity. Similarly, we compare methods on empirical data from an existing alignment benchmark set that we used to derive reference distances and trees. Our pattern-based approach yields distances that show a linear relationship to reference distances over a substantially longer range than other alignment-free methods. The pattern-based approach outperforms alignment-free methods and its phylogenetic accuracy is statistically indistinguishable from alignment-based distances.Comment: 21 pages, 3 figures, 2 table

The emerging roles of GPRC5A in diseases.

The \u27Retinoic Acid-Inducible G-protein-coupled receptors\u27 or RAIG are a group comprising the four orphan receptors GPRC5A, GPRC5B, GPRC5C and GPRC5D. As the name implies, their expression is induced by retinoic acid but beyond that very little is known about their function. In recent years, one member, GPRC5A, has been receiving increasing attention as it was shown to play important roles in human cancers. As a matter of fact, dysregulation of GPRC5A has been associated with several cancers including lung cancer, breast cancer, colorectal cancer, and pancreatic cancer. Here we review the current state of knowledge about the heterogeneity and evolution of GPRC5A, its regulation, its molecular functions, and its involvement in human disease

A new computational method for the detection of horizontal gene transfer events

In recent years, the increase in the amounts of available genomic data has made it easier to appreciate the extent by which organisms increase their genetic diversity through horizontally transferred genetic material. Such transfers have the potential to give rise to extremely dynamic genomes where a significant proportion of their coding DNA has been contributed by external sources. Because of the impact of these horizontal transfers on the ecological and pathogenic character of the recipient organisms, methods are continuously sought that are able to computationally determine which of the genes of a given genome are products of transfer events. In this paper, we introduce and discuss a novel computational method for identifying horizontal transfers that relies on a gene's nucleotide composition and obviates the need for knowledge of codon boundaries. In addition to being applicable to individual genes, the method can be easily extended to the case of clusters of horizontally transferred genes. With the help of an extensive and carefully designed set of experiments on 123 archaeal and bacterial genomes, we demonstrate that the new method exhibits significant improvement in sensitivity when compared to previously published approaches. In fact, it achieves an average relative improvement across genomes of between 11 and 41% compared to the Codon Adaptation Index method in distinguishing native from foreign genes. Our method's horizontal gene transfer predictions for 123 microbial genomes are available online at

A sensitive, support-vector-machine method for the detection of horizontal gene transfers in viral, archaeal and bacterial genomes

In earlier work, we introduced and discussed a generalized computational framework for identifying horizontal transfers. This framework relied on a gene's nucleotide composition, obviated the need for knowledge of codon boundaries and database searches, and was shown to perform very well across a wide range of archaeal and bacterial genomes when compared with previously published approaches, such as Codon Adaptation Index and C + G content. Nonetheless, two considerations remained outstanding: we wanted to further increase the sensitivity of detecting horizontal transfers and also to be able to apply the method to increasingly smaller genomes. In the discussion that follows, we present such a method, Wn-SVM, and show that it exhibits a very significant improvement in sensitivity compared with earlier approaches. Wn-SVM uses a one-class support-vector machine and can learn using rather small training sets. This property makes Wn-SVM particularly suitable for studying small-size genomes, similar to those of viruses, as well as the typically larger archaeal and bacterial genomes. We show experimentally that the new method results in a superior performance across a wide range of organisms and that it improves even upon our own earlier method by an average of 10% across all examined genomes. As a small-genome case study, we analyze the genome of the human cytomegalovirus and demonstrate that Wn-SVM correctly identifies regions that are known to be conserved and prototypical of all beta-herpesvirinae, regions that are known to have been acquired horizontally from the human host and, finally, regions that had not up to now been suspected to be horizontally transferred. Atypical region predictions for many eukaryotic viruses, including the α-, β- and γ-herpesvirinae, and 123 archaeal and bacterial genomes, have been made available online at

IsomiR Expression Profiles in Human Lymphoblastoid Cell Lines Exhibit Population and Gender Dependencies.

For many years it was believed that each mature microRNA (miRNA) existed as a single entity with fixed endpoints and a \u27static\u27 and unchangeable primary sequence. However, recent evidence suggests that mature miRNAs are more \u27dynamic\u27 and that each miRNA precursor arm gives rise to multiple isoforms, the isomiRs. Here we report on our identification of numerous and abundant isomiRs in the lymphoblastoid cell lines (LCLs) of 452 men and women from five different population groups. Unexpectedly, we find that these isomiRs exhibit an expression profile that is population-dependent and gender-dependent. This is important as it indicates that the LCLs of each gender/population combination have their own unique collection of mature miRNA transcripts. Moreover, each identified isomiR has its own characteristic abundance that remains consistent across biological replicates indicating that these are not degradation products. The primary sequences of identified isomiRs differ from the known miRBase miRNA either at their 5´-endpoint (leads to a different \u27seed\u27 sequence and suggests a different targetome), their 3´-endpoint, or both simultaneously. Our analysis of Argonaute PAR-CLIP data from LCLs supports the association of many of these newly identified isomiRs with the Argonaute silencing complex and thus their functional roles through participation in the RNA interference pathway

On the expanding roles of tRNA fragments in modulating cell behavior

The fragments that derive from transfer RNAs (tRNAs) are an emerging category of regulatory RNAs. Known as tRFs, these fragments were reported for the first time only a decade ago, making them a relatively recent addition to the ever-expanding pantheon of non-coding RNAs. tRFs are short, 16-35 nucleotides (nts) in length, and produced through cleavage of mature and precursor tRNAs at various positions. Both cleavage positions and relative tRF abundance depend strongly on context, including the tissue type, tissue state, and disease, as well as the sex, population of origin, and race/ethnicity of an individual. These dependencies increase the urgency to understand the regulatory roles of tRFs. Such efforts are gaining momentum, and comprise experimental and computational approaches. System-level studies across many tissues and thousands of samples have produced strong evidence that tRFs have important and multi-faceted roles. Here, we review the relevant literature on tRF biology in higher organisms, single cell eukaryotes, and prokaryotes

YAMAT-seq: an efficient method for high-throughput sequencing of mature transfer RNAs.

Besides translation, transfer RNAs (tRNAs) play many non-canonical roles in various biological pathways and exhibit highly variable expression profiles. To unravel the emerging complexities of tRNA biology and molecular mechanisms underlying them, an efficient tRNA sequencing method is required. However, the rigid structure of tRNA has been presenting a challenge to the development of such methods. We report the development of Y-shaped Adapter-ligated MAture TRNA sequencing (YAMAT-seq), an efficient and convenient method for high-throughput sequencing of mature tRNAs. YAMAT-seq circumvents the issue of inefficient adapter ligation, a characteristic of conventional RNA sequencing methods for mature tRNAs, by employing the efficient and specific ligation of Y-shaped adapter to mature tRNAs using T4 RNA Ligase 2. Subsequent cDNA amplification and next-generation sequencing successfully yield numerous mature tRNA sequences. YAMAT-seq has high specificity for mature tRNAs and high sensitivity to detect most isoacceptors from minute amount of total RNA. Moreover, YAMAT-seq shows quantitative capability to estimate expression levels of mature tRNAs, and has high reproducibility and broad applicability for various cell lines. YAMAT-seq thus provides high-throughput technique for identifying tRNA profiles and their regulations in various transcriptomes, which could play important regulatory roles in translation and other biological processes

The biogenesis pathway of tRNA-derived piRNAs in Bombyx germ cells.

Transfer RNAs (tRNAs) function in translational machinery and further serves as a source of short non-coding RNAs (ncRNAs). tRNA-derived ncRNAs show differential expression profiles and play roles in many biological processes beyond translation. Molecular mechanisms that shape and regulate their expression profiles are largely unknown. Here, we report the mechanism of biogenesis for tRNA-derived Piwi-interacting RNAs (td-piRNAs) expressed in Bombyx BmN4 cells. In the cells, two cytoplasmic tRNA species, tRNAAspGUC and tRNAHisGUG, served as major sources for td-piRNAs, which were derived from the 5\u27-part of the respective tRNAs. cP-RNA-seq identified the two tRNAs as major substrates for the 5\u27-tRNA halves as well, suggesting a previously uncharacterized link between 5\u27-tRNA halves and td-piRNAs. An increase in levels of the 5\u27-tRNA halves, induced by BmNSun2 knockdown, enhanced the td-piRNA expression levels without quantitative change in mature tRNAs, indicating that 5\u27-tRNA halves, not mature tRNAs, are the direct precursors for td-piRNAs. For the generation of tRNAHisGUG-derived piRNAs, BmThg1l-mediated nucleotide addition to -1 position of tRNAHisGUG was required, revealing an important function of BmThg1l in piRNA biogenesis. Our study advances the understanding of biogenesis mechanisms and the genesis of specific expression profiles for tRNA-derived ncRNAs