Project description:The tooth is a convenient experimental model for the study of the basic mechanisms of organ development, including differentiation, cellular interaction, morphogenesis, and production and mineralization of extracellular matrices. The rodent incisor teeth grow continuously and exhibit all stages of tooth development at any time. This characteristic makes them convenient models for the study of enamel formation, which occurs in several distinct stages along the tooth axis. The distribution and structure of mouse incisor enamel resemble that of the rat. The enamel covers only the labial aspect of the tooth, and can be divided into four layers: a thin inner prism-free layer, inner enamel with prism decussation, i.e. transverse rows of prisms with prisms inclined medially and laterally in alternate rows, outer enamel with parallel prisms inclined incisally and a thin superficial prism-free layer. Recently, we have described how this elaborate organization of the enamel is established in the initial enamel formed on the unerupted and unworn incisal tip of the incisors. The very first enamel formed, i.e. the most incisally situated enamel, is always prism-free, corresponding to the superficial layer in fully established enamel, although thicker. Going in apical direction, i.e. in the direction of initiation of new enamel formation, isolated prisms appear among the crystals continuous with the prism-free zone crystals. These initial prisms are in general inclined incisally, corresponding to the prisms in the outer enamel layer in the fully established enamel. Inner enamel with the characteristic decussation pattern is added somewhat further apically and increases in thickness with increasing enamel thickness. Based on the structure of the enamel that they produce, the ameloblasts producing the initial, thin, prism-free enamel at the incisal tip of the unworn mouse incisor are probably differently configured (e.g. lacking Tomes' processes), probably differently organized (e.g. not organized in transverse rows moving sideways in opposite directions), and probably have a shorter life-span than the more apically situated ameloblasts which produce thicker enamel with the full four-layered configuration of mouse incisor enamel. For this reason it would be of interest to compare the gene expression profile of the incisal tip segment where prism-free enamel is being formed with the profile of the immediately adjacent segment where enamel with all four layers is being formed. As long as the incisor is unworn enamel formation and gene expression in these segments will likely reflect mechanistic differences between these segments as regards enamel biosynthesis. MicroRNAs (miRNA) are a class of non-coding RNAs that regulate gene expression at a post-transcriptional level, and are considered as important regulatory molecules during foetal development. By binding to target mRNA, miRNA induce mRNA decay or translation repression. Recent bioinformatic predictions of miRNA targets in vertebrates indicate that hundreds of miRNAs are responsible for regulating the expression of up to 30% of the human protein-coding genes, and miRNAs have recently also been shown to regulate expression of hundreds of mRNAs in cultured cells. Recently, miRNA expression profiles of the developing murine molar tooth germ and submandibular salivary gland have been established. We here report expression profiling of miRNAs present in the two adjacent segments of the incisor tooth germ on which we also have carried out mRNA expression profiling. The results suggest that also miRNAs are abundantly, and differentially, expressed during development of the incisor tooth germ in mouse. By analogy with the mRNA data also miRNA expression is dynamic, with respect to both the two incisor segments investigated and to the developmental stage of the incisor.

Project description:Long non-coding RNAs (lncRNAs) are generally defined as RNA transcripts longer than 200 nucleotides that are not translated into proteins. Recently, many small open reading frames (smORFs) embedded in lncRNA scripts have been verified to be able to encode functional polypeptides (namely lncRNA-SEPs here). Although collaborative analysis by advanced genomics, bioinformatics and proteomics largely drives SEPs discovery, the poor predictability, diminutive size and low abundance still challenge systematic identification of SEPs from different biological samples. Here, we took advantage of the NONCODE database that deposited with the most complete collection and annotation of lncRNA transcripts from different species to build a database that to maximally collect all putative small ORFs from human and mouse lncRNA transcripts. Two effective and complementary polypeptides enrichment strategies (30 kDa MWCO filter and C8 SPE column) were also integrated to further improve the discovery of novel lncRNA-SEPs. These efforts led to the discovery of 362 lncRNA-SEPs from 8 human cell lines and 238 lncRNA-SEPs from 3 mouse cell lines and 8 mouse tissues. 18 out of these lncRNA-SEPs were verified experimentally by multiple technologies including in vitro expression, immunoblotting and parallel reaction monitoring-based mass spectrometry (PRM-MS) in 293T cells. Further bioinformatic analysis reveals that the physical and chemical properties of these novel lncRNA-SEPs, such as amino acid composition and codon usage, are varied from canonical proteins. Intriguingly, nearly 70% of the identified lncRNA-SEPs were found to be initiated with non-AUG start codons. Collectively, the efficient workflows presented in this study enables us identify 600 novel lncRNA-SPEs from multiple cell lines and tissues, which should represent the largest number of MS-detected lncRNA-encoding SEPs ever reported to date. These novel lncRNA-SEPs not only could provide new clues for the annotation of the noncoding elements in the genome, but also could serve as a valuable resource for the functional characterization of individual lncRNA-SEPs.

Project description:Recent advances in transcriptome sequencing have enabled the discovery of thousands of long non-coding RNAs (lncRNAs) across many species. Though several lncRNAs have been shown to play important roles in diverse biological processes, the functions and mechanisms of most lncRNAs remain unknown. Two significant obstacles lie between transcriptome sequencing and functional characterization of lncRNAs: identifying truly non-coding genes from de novo reconstructed transcriptomes, and prioritizing the hundreds of resulting putative lncRNAs for downstream experimental interrogation. We present slncky, a computational lncRNA discovery tool that produces a high-quality set of lncRNAs from RNA-sequencing data and further uses evolutionary constraint to prioritize lncRNAs that are likely to be functionally important. Our automated filtering pipeline is comparable to manual curation efforts and more sensitive than previously published computational approaches. Furthermore, we develop a sensitive alignment pipeline for aligning lncRNA loci and propose new evolutionary metrics relevant for analyzing sequence and transcript evolution. Our analysis reveals that evolutionary selection acts in several distinct patterns, and uncovers two notable classes of intergenic lncRNAs: one showing strong purifying selection on RNA sequence and another where constraint is restricted to the regulation but not the sequence of the transcript. To study a comprehensive and comparable set of lncRNAs expressed in the pluripotent state, we analyzed RNA-Seq data from pluripotent cells derived from several strains and species, and grown in two physiological conditions. First we derived ânaÃ¯veâ ES cells (ESCs) from each of three different mice strains: 129SvEv, NOD, and Mus musculus castaneus (cast) mouse, a wild mouse subspecies originally from Thailand, as well as naÃ¯ve induced pluripotent stem (iPS) cells from rat and human. Next, to facilitate comparisons across pluripotent cells from different species, we also cultured mouse and rat cells in âprimedâ epiblast stem cell (EpiSC) culture conditions, since human iPS cells in culture are much more similar molecularly and functionally to mouse primed EpiSCs rather than to a ground state naÃ¯ve ESCs. We polyA selected RNA from each cell type and deeply sequenced on HiSeq2500

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Genome regulation by long noncoding RNAs during amelogenesis

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