The human salivary RNA transcriptome revealed by massively parallel sequencing.

BACKGROUND: Evaluation of the salivary transcriptome is an emerging diagnostic technology with discriminatory power for disease detection. This study explored massively parallel sequencing for providing nucleotide-level sequence information for each RNA in saliva. METHODS: Transcriptome profiling with the SOLiD™ system was applied to RNA isolated from unstimulated cell-free saliva (CFS) and whole saliva (WS) from healthy human volunteers. Sequenced reads were aligned to human genome build 18 and the Human Oral Microbiome Database (HOMD). RESULTS: Massively parallel sequencing enabled the acquisition of complete sequence information for each nucleotide position of the human salivary transcriptome through alignment to multiple sequence databases. Approximately 20%-25% of the sequenced reads from CFS aligned to the human genome, and approximately 30% of the sequenced reads aligned to the HOMD. We detected the expression of >4000 coding and noncoding genes in CFS and WS. Gene expression at different genome loci showed that the structural integrity of the transcripts for the annotated genes was preserved in saliva. CONCLUSIONS: A single measurement provided RNA sequence information of gene transcript abundance for both coding and noncoding RNAs and identified sequences from >400 different microbial species within a single sample. Contrary to previous data suggesting that salivary RNA is highly fragmented, in our study the structural integrity of RNA was preserved. The high degree of sequence alignment to annotated exons and introns for each of the respective reference genomes, with sequence coverage spanning the full length of the genes, provides strong evidence that the salivary transcriptome is a complex RNA network.

MicroRNAs and their isomiRs function cooperatively to target common biological pathways.

BACKGROUND: Variants of microRNAs (miRNAs), called isomiRs, are commonly reported in deep-sequencing studies; however, the functional significance of these variants remains controversial. Observational studies show that isomiR patterns are non-random, hinting that these molecules could be regulated and therefore functional, although no conclusive biological role has been demonstrated for these molecules. RESULTS: To assess the biological relevance of isomiRs, we have performed ultra-deep miRNA-seq on ten adult human tissues, and created an analysis pipeline called miRNA-MATE to align, annotate, and analyze miRNAs and their isomiRs. We find that isomiRs share sequence and expression characteristics with canonical miRNAs, and are generally strongly correlated with canonical miRNA expression. A large proportion of isomiRs potentially derive from AGO2 cleavage independent of Dicer. We isolated polyribosome-associated mRNA, captured the mRNA-bound miRNAs, and found that isomiRs and canonical miRNAs are equally associated with translational machinery. Finally, we transfected cells with biotinylated RNA duplexes encoding isomiRs or their canonical counterparts and directly assayed their mRNA targets. These studies allow us to experimentally determine genome-wide mRNA targets, and these experiments showed substantial overlap in functional mRNA networks suppressed by both canonical miRNAs and their isomiRs. CONCLUSIONS: Together, these results find isomiRs to be biologically relevant and functionally cooperative partners of canonical miRNAs that act coordinately to target pathways of functionally related genes. This work exposes the complexity of the miRNA-transcriptome, and helps explain a major miRNA paradox: how specific regulation of biological processes can occur when the specificity of miRNA targeting is mediated by only 6 to 11 nucleotides.

Perturbation of DNA repair gene expression due to interspecies hybridization.

The effect of interspecies hybridization on gene regulation was examined using real-time polymerase chain reaction (RT-PCR) to measure the expression of five base-excision repair genes in brain, eye, gill, liver, and tailfin tissues from Xiphophorus parental species and F(1) hybrids. Relative mRNA levels of uracil N-glycosylase (Ung), Apurinic/apyrimidinic endonuclease (Ape1), polymerase-beta (Polb), flap endonuclease (Fen1), and DNA ligase (Lig1) were measured in three parental Xiphophorus species (X. maculatus Jp 163 B, X. helleri Sarabia, and X. andersi andC) and in two interspecies F(1) hybrids, the Sp-helleri hybrid (X. maculatus Jp 163 BxX. helleri Sarabia) and the Sp-andersi hybrid (X. maculatus Jp 163 BxX. andersi) to identify genes that undergo changes in expression levels upon interspecies hybridization. Significant differences in gene expression were observed between parental animals and their respective F(1) hybrids in both interspecies crosses. Generally, marked increases in DNA repair gene mRNA levels were observed across all tissues in F(1) hybrid animals from the Sp-helleri cross compared to either X. maculatus or X. helleri parents. In contrast, the Sp-andersi F(1) hybrid animals generally exhibited decreased base-excision repair gene expression, although this trend was more specific to individual tissues than observed for Sp-helleri hybrids.

Multiple tissue gene expression analyses in Japanese medaka (Oryzias latipes) exposed to hypoxia.

Due in part to human population growth watersheds and coastal estuaries have been receiving increasing run-off of nutrients and genotoxins. As a consequence, the occurrences of nutrient-driven hypoxia in coastal waters appear to be increasing. Thus, understanding the molecular genetic response to hypoxia by model aquatic organisms is of interest both from environmental and physiological viewpoints. The major objectives of this study are to determine genome-wide gene expression profiles and to better understand how hypoxia influences global gene expression in medaka (Oryzias latipes), a well utilized aquatic model species. Herein we detail our development of a microarray containing 8046 medaka unigenes and describe our experimental results for measuring gene expression changes in the brain, gill, and liver of hypoxia exposed fish. Using conservative selection criteria, we determined that 501 genes in the brain, 442 in the gill, and 715 in the liver were differentially expressed in medaka exposed to hypoxia. These differentially expressed genes fell into a number of biological gene ontology groups related to general metabolism, catabolism, RNA and protein metabolism, etc. Two biological pathways, ubiquitin-proteasome and phosphatidylinositol signaling, were significantly dysregulated in medaka upon hypoxia exposure. Comparative genomics between medaka and human identified several human orthologies associated with known diseases.

Production of F1 interspecies hybrid offspring with cryopreserved sperm from a live-bearing fish, the swordtail Xiphophorus helleri.

Despite study of sperm cryopreservation in more than 200 fish species, production of broods from cryopreserved sperm in live-bearing fish has not been demonstrated. This has not been due to a lack of effort, but instead is a result of the unique morphology, biology, and biochemistry of reproduction in viviparous fishes. For example, sperm of Xiphophorus helleri have a cylindrical nucleus, can swim for days after being activated, have glycolytic capabilities, and can reside in the female reproduction tract for months before fertilization. These traits are not found in fishes with external fertilization. The long-standing research use of the genus Xiphophorus has led to development of over 60 pedigreed lines among the 26 species maintained around the world. These species and lines serve as contemporary models in medical research, although they must be maintained as live populations. Previous attempts at establishing sperm cryopreservation protocols for Xiphophorus have not produced live young. To address this we have been studying the parameters surrounding cryobiology of Xiphophorus sperm and applying this information to an improved understanding of internal fertilization and reproduction. Here we report the first successful fertilization and offspring production by cryopreserved sperm in any live-bearing fish. This claim is supported by our use of artificial insemination between two species that yield distinct hybrid offspring to verify paternity via cryopreserved sperm. We provide a practical approach for preservation of valuable genetic resources from live-bearing fish species, a group that is rapidly being lost due to destruction of native habitats.

Cloning of JunA and JunB and comparison of mRNA expression levels in two Xiphophorus melanoma models.

The cloning and mRNA expression analysis of Xiphophorus maculatus JunA and JunB proto-oncogenes (designated X-JunA and X-JunB, respectively) is described. In mammals, JunA and JunB proteins make up the activator protein-1 (AP-1) transcription factor with related Fos proteins. The deduced amino acid sequences of X-JunA and X-JunB exhibit moderate degrees of similarity when compared to their human homologues, while the regions considered functionally critical, namely, the transactivation domains, DNA-binding domain, and the leucine zipper, are highly conserved. X-JunA and X-JunB mRNA expression levels in six X. maculatus Jp 163 A tissues were assayed by real-time RT-PCR. In addition, X-JunA and X-JunB mRNA levels are compared in skin and tumor tissues derived from two distinct Xiphophorus backcross hybrid tumor models, one of which develops melanoma spontaneously, whereas the other requires induction via UVB exposure for melanoma development. X-JunB mRNA expression was higher than X-JunA expression in tissues from X. maculatus parental animals. X-JunB was also more highly expressed than X-JunA in both spontaneous and induced melanoma tissue and nonmelanotic skin tissue. However, X-JunA mRNA levels were significantly higher in the spontaneous melanomas compared to melanomas induced by UVB exposure. The authors speculate that these findings may indicate that JunA regulation is affected by regulatory differences between the two melanoma model systems.

DNA polymerase beta mRNA and protein expression in Xiphophorus fish.

Herein we report Xiphophorus DNA polymerase beta (XiphPolbeta) mRNA and protein expression levels in brain, liver, gill, and testes tissues from Xiphophorus maculatus, Xiphophorus helleri, and Xiphophorus couchianus parental line fish and two different tumor-bearing Xiphophorus interspecies hybrids. Polymerase beta protein levels in the Xiphophorus tissues were measured by Western blot, and mRNA was measured with a quantitative real time RT-PCR method which employed cRNA construction to produce accurate calibration curves. We found significant differences in both mRNA and protein levels between the tumor-bearing hybrid animals and the three parental species. However, there were no significant differences in either mRNA levels or protein expression observed between the parental species. Thus, interspecies hybridization results in dysregulation of Polbeta expression and this may manifest a modulation in DNA repair capability and susceptibility to latent tumorigenesis.

Gene structure, purification and characterization of DNA polymerase beta from Xiphophorus maculatus.

Cloning of the Xiphophorus maculatus Polbeta gene and overexpression of the recombinant Polbeta protein has been performed. The organization of the XiphPolbeta introns and exons, including intron-exon boundaries, have been assigned and were found to be similar to that for human Polbeta with identical exon sizes except for exon XII coding for an additional two amino acid residues in Xiphophorus. The cDNA sequence encoding the 337-amino acid X. maculatus DNA polymerase beta (Polbeta) protein was subcloned into the Escherichia coli expression plasmid pET. Induction of transformed E. coli cells resulted in the high-level expression of soluble recombinant Polbeta, which catalyzed DNA synthesis on template-primer substrates. The steady-state Michaelis constants (Km) and catalytic efficiencies (kcat/Km) of the recombinant XiphPolbeta for nucleotide insertion opposite single-nucleotide gap DNA substrates were measured and compared with previously published values for recombinant human Polbeta. Steady-state in vitro Km and kcat/Km values for correct nucleotide insertion by XiphPolbeta and human Polbeta were similar, although the recombinant Xiphophorus protein exhibited 2.5-7-fold higher catalytic efficiencies for dGTP and dCTP insertion versus human Polbeta. In contrast, the recombinant XiphPolbeta displayed significantly lower fidelities than human Polbeta for dNTP insertion opposite a single-nucleotide gap at 37 degrees C.

Characterization and purification of flap endonuclease-1 (xiFEN-1) from Xiphophorus maculatus.

The cloning, gene structure, and expression of flap endonuclease-1 (xiFEN1) from Xiphophorus maculates are presented. The xiFEN1 gene structure was found to include 8 exons and 7 introns. The Xiphophorus FEN1 cDNA sequence contained an open reading frame that encoded a 380 amino acid protein with a predicted mass of 43 kDa. The intact FEN1 cDNA was subcloned into a bacterial expression vector (pET101-xiFEN1ct) and recombinant xiFEN1 enzyme purified from E. colicell extracts. The pET101-xiFEN1ct translation product was a 3' fusion protein with a ~3 kDa vector-encoded carboxy terminal extension designed to facilitate protein recognition and purification. The xiFEN1 fusion protein was purified and its amino acid sequence verified by Western blot analysis and tryptic peptide mass fingerprinting. The purified recombinant protein was assessed for enzyme specificity using several different oligonucleotide substrates having select flap overhangs. Also reported are Michaelis steady state kinetic values of enzymatic activity for the xiFEN1 directly compared with human FEN1 activity. xiFEN1 displayed a five-fold greater Km and six-fold lower catalytic efficiency (kcat/Km) than observed for the hFEN1.