Identification of long non-protein coding RNAs in chicken skeletal muscle using next generation sequencing

AbstractVertebrate genomes encode thousands of non-coding RNAs including short non-coding RNAs (such as microRNAs) and long non-coding RNAs (lncRNAs). Chicken (Gallus gallus) is an important model organism for developmental biology, and the recently assembled genome sequences for chicken will facilitate the understanding of the functional roles of non-coding RNA genes during development. The present study concerns the first systematic identification of lncRNAs using RNA-Seq to sample the transcriptome during chicken muscle development. A computational approach was used to identify 281 new intergenic lncRNAs in the chicken genome. Novel lncRNAs in general are less conserved than protein-coding genes and slightly more conserved than random non-coding sequences. The present study has provided an initial chicken lncRNA catalog and greatly increased the number of chicken ncRNAs in the non-protein coding RNA database. Furthermore, the computational pipeline presented in the current work will be useful for characterizing lncRNAs obtained from deep sequencing data

Comparative analysis of genome tiling array data reveals many novel primate-specific functional RNAs in human

BACKGROUND: Widespread transcription activities in the human genome were recently observed in high-resolution tiling array experiments, which revealed many novel transcripts that are outside of the boundaries of known protein or RNA genes. Termed as "TARs" (Transcriptionally Active Regions), these novel transcribed regions represent "dark matter" in the genome, and their origin and functionality need to be explained. Many of these transcripts are thought to code for novel proteins or non-protein-coding RNAs. We have applied an integrated bioinformatics approach to investigate the properties of these TARs, including cross-species conservation, and the ability to form stable secondary structures. The goal of this study is to identify a list of potential candidate sequences that are likely to code for functional non-protein-coding RNAs. We are particularly interested in the discovery of those functional RNA candidates that are primate-specific, i.e. those that do not have homologs in the mouse or dog genomes but in rhesus. RESULTS: Using sequence conservation and the probability of forming stable secondary structures, we have identified ~300 possible candidates for primate-specific noncoding RNAs. We are currently in the process of sequencing the orthologous regions of these candidate sequences in several other primate species. We will then be able to apply a "phylogenetic shadowing" approach to analyze the functionality of these ncRNA candidates. CONCLUSION: The existence of potential primate-specific functional transcripts has demonstrated the limitation of previous genome comparison studies, which put too much emphasis on conservation between human and rodents. It also argues for the necessity of sequencing additional primate species to gain a better and more comprehensive understanding of the human genome

Secondary structure prediction for aligned RNA sequences.

Most functional RNA molecules have characteristic secondary structures that are highly conserved in evolution. Here we present a method for computing the consensus structure of a set aligned RNA sequences taking into account both thermodynamic stability and sequence covariation. Comparison with phylogenetic structures of rRNAs shows that a reliability of prediction of more than 80% is achieved for only five related sequences. As an application we show that the Early Noduline mRNA contains significant secondary structure that is supported by sequence covariation

Mycobacterial RNA isolation optimized for non-coding RNA: high fidelity isolation of 5S rRNA from Mycobacterium bovis BCG reveals novel post-transcriptional processing and a complete spectrum of modified ribonucleosides

A major challenge in the study of mycobacterial RNA biology is the lack of a comprehensive RNA isolation method that overcomes the unusual cell wall to faithfully yield the full spectrum of non-coding RNA (ncRNA) species. Here, we describe a simple and robust procedure optimized for the isolation of total ncRNA, including 5S, 16S and 23S ribosomal RNA (rRNA) and tRNA, from mycobacteria, using Mycobacterium bovis BCG to illustrate the method. Based on a combination of mechanical disruption and liquid and solid-phase technologies, the method produces all major species of ncRNA in high yield and with high integrity, enabling direct chemical and sequence analysis of the ncRNA species. The reproducibility of the method with BCG was evident in bioanalyzer electrophoretic analysis of isolated RNA, which revealed quantitatively significant differences in the ncRNA profiles of exponentially growing and non-replicating hypoxic bacilli. The method also overcame an historical inconsistency in 5S rRNA isolation, with direct sequencing revealing a novel post-transcriptional processing of 5S rRNA to its functional form and with chemical analysis revealing seven post-transcriptional ribonucleoside modifications in the 5S rRNA. This optimized RNA isolation procedure thus provides a means to more rigorously explore the biology of ncRNA species in mycobacteria.Singapore-MIT Alliance for Research and TechnologyNational Institute of Environmental Health Sciences (ES017010)National Institute of Environmental Health Sciences (ES002109)Singapore-MIT Alliance (Graduate Fellowship)Singapore. National Research Foundatio

Mammalian microRNAs: a small world for fine-tuning gene expression

The basis of eukaryotic complexity is an intricate genetic architecture where parallel systems are involved in tuning gene expression, via RNA-DNA, RNA-RNA, RNA-protein, and DNA-protein interactions. In higher organisms, about 97% of the transcriptional output is represented by noncoding RNA (ncRNA) encompassing not only rRNA, tRNA, introns, 5′ and 3′ untranslated regions, transposable elements, and intergenic regions, but also a large, rapidly emerging family named microRNAs. MicroRNAs are short 20-22-nucleotide RNA molecules that have been shown to regulate the expression of other genes in a variety of eukaryotic systems. MicroRNAs are formed from larger transcripts that fold to produce hairpin structures and serve as substrates for the cytoplasmic Dicer, a member of the RNase III enzyme family. A recent analysis of the genomic location of human microRNA genes suggested that 50% of microRNA genes are located in cancer-associated genomic regions or in fragile sites. This review focuses on the possible implications of microRNAs in post-transcriptional gene regulation in mammalian diseases, with particular focus on cancer. We argue that developing mouse models for deleted and/or overexpressed microRNAs will be of invaluable interest to decipher the regulatory networks where microRNAs are involved

Cloning and expression of Dazl in Rana pipiens

The DAZ gene on the human Y chromosome has been implicated in male fertility. Humans and other animals possess an autosomal version of this gene, referred to as DAZ-like or Dazl, which functions in gametogenesis and fertility. In order to expand our knowledge of Dazl, I cloned Dazl from the northern leopard frog Rana pipiens. To obtain the clone, I screened a R. pipiens ovary cDNA library with a RpDazl-specific PCR-generated radioactive probe. RpDazl, as determined by sequencing, is 3.5kb in length, with an 843 nucleotide open reading frame (ORF) and a 3\u27 untranslated region (UTR) of 2635 nucleotides. At the amino acid level, the ORF of RpDazl is 51.8% identical to the ORF of Xenopus laevis Dazl or XDazl and 53.6% identical to the ORF of human DAZL. RpDazl has a characteristic RNA recognition motif (RRM) that is 73% identical to the RRM in X. laevis, and contains two smaller motifs within the RRM, RNP-1 and RNP-2. These two smaller motifs are almost 100% conserved across all species studied. RpDazl also contains a DAZ repeat, another characteristic domain of Dazl genes. The DAZ repeat of RpDazl is 69% identical to the DAZ repeat of XDazl. Northern blot analysis revealed a transcript length of 3.8kb and 3.3kb in two independent trials. RT-PCR was performed using template cDNA, made from R. pipiens ovarian RNA, and 23 sets of primers that spanned the cloned sequence. These data authenticated continuity over greater than 3.25kb of the cDNA. Combining results of the Northern blot and RT-PCR experiments, I conclude that the cloned RpDazl is full length. Preliminary data were also obtained regarding the maternal localization of the RpDazl RNA using RT-PCR as well as a computer based sequence analysis program called RepFind. RT-PCR analysis revealed that RpDazl is localized to the vegetal one-third of the oocyte. RepFind revealed clusters of CAC repeats, which are known localization elements. The significance of these clusters, however, is lower than that of the clusters present in the 3\u27 UTR of XDazl. Further analysis will be required to show more precise RpDazl localization, specifically germ plasm association

Modeling RNA folding

In recent years it has become evident that functional RNAs in living organisms are not just curious remnants from a primoridal RNA world but an ubiquitous phenomenon complementing protein enzyme based activity. Functional RNAs, just like proteins, depend in many cases upon their well-defined and evolutionarily conserved three-dimensional structure. In contrast to protein folds, however, RNA molecules have a biophysically important coarse-grained representation: their secondary structure. At this level of resolution at least, RNA structures can be efficiently predicted given only the sequence information. As a consequence, computational studies of RNA routinely incorporate structural information explicitly. RNA secondary structure prediction has proven useful in diverse fields ranging from theoretical models of sequence evolution and biopolymer folding, to genome analysis and even the design biotechnologically or pharmaceutically useful molecules