Partial depletion of yolk during zebrafish embryogenesis changes the dynamics of methionine cycle and metabolic genes.

BACKGROUND: Limited nutrient availability during development is associated with metabolic diseases in adulthood. The molecular cause for these defects is unclear. Here, we investigate if transcriptional changes caused by developmental malnutrition reveal an early response that can be linked to metabolism and metabolic diseases. RESULTS: We limited nutrient availability by removing yolk from zebrafish (Danio rerio) embryos. We then measured genome expression after 8, 24, 32 h post-fertilization (hpf) by RNA sequencing and 48 hpf by microarray profiling. We assessed the functional impact of deregulated genes by enrichment analysis of gene ontologies, pathways and CpG sites around the transcription start sites. Nutrient depletion during embryogenesis does not affect viability, but induces a bias towards female development. It induces subtle expression changes of metabolic genes: lipid transport, oxidative signaling, and glycolysis are affected during earlier stages, and hormonal signaling at 48 hpf. Co-citation analysis indicates association of deregulated genes to the metabolic syndrome, a known outcome of early-life nutrient depletion. Notably, deregulated methionine cycle genes indicate altered methyl donor availability. We find that the regulation of deregulated genes may be less dependent on methyl donor availability. CONCLUSIONS: The systemic response to reduced nutrient availability in zebrafish embryos affects metabolic pathways and can be linked to metabolic diseases. Further exploration of the reported zebrafish model system may elucidate the consequences of reduced nutrient availability during embryogenesis.

Methods for small RNA preparation for digital gene expression profiling by next-generation sequencing.

Digital gene expression (DGE) profiling techniques are playing an eminent role in the detection, localization, and differential expression quantification of many small RNA species, including microRNAs (1-3). Procedures in small RNA library preparation techniques typically include adapter ligation by RNA ligase, followed by reverse transcription and amplification by PCR. This chapter describes three protocols that were successfully applied to generate small RNA sequencing SOLiD(TM) libraries. The Ambion SREK(TM)-adopted protocol can be readily used for multiplexing samples; the modban-based protocol is cost-efficient, but biased toward certain microRNAs; the poly(A)-based protocol is less biased, but less precise because of the A-tail that is introduced. In summary, each of these protocols has its advantages and disadvantages with respect to the ease of including barcodes, costs, and outcome.

ATP-dependent and independent functions of Rad54 in genome maintenance.

Rad54, a member of the SWI/SNF protein family of DNA-dependent ATPases, repairs DNA double-strand breaks (DSBs) through homologous recombination. Here we demonstrate that Rad54 is required for the timely accumulation of the homologous recombination proteins Rad51 and Brca2 at DSBs. Because replication protein A and Nbs1 accumulation is not affected by Rad54 depletion, Rad54 is downstream of DSB resection. Rad54-mediated Rad51 accumulation does not require Rad54's ATPase activity. Thus, our experiments demonstrate that SWI/SNF proteins may have functions independent of their ATPase activity. However, quantitative real-time analysis of Rad54 focus formation indicates that Rad54's ATPase activity is required for the disassociation of Rad54 from DNA and Rad54 turnover at DSBs. Although the non-DNA-bound fraction of Rad54 reversibly interacts with a focus, independent of its ATPase status, the DNA-bound fraction is immobilized in the absence of ATP hydrolysis by Rad54. Finally, we show that ATP hydrolysis by Rad54 is required for the redistribution of DSB repair sites within the nucleus.

Small RNA expression and strain specificity in the rat.

BACKGROUND: Digital gene expression (DGE) profiling has become an established tool to study RNA expression. Here, we provide an in-depth analysis of small RNA DGE profiles from two different rat strains (BN-Lx and SHR) from six different rat tissues (spleen, liver, brain, testis, heart, kidney). We describe the expression patterns of known and novel micro (mi)RNAs and piwi-interacting (pi)RNAs. RESULTS: We confirmed the expression of 588 known miRNAs (54 in antisense orientation) and identified 56 miRNAs homologous to known human or mouse miRNAs, as well as 45 new rat miRNAs. Furthermore, we confirmed specific A to I editing in brain for mir-376a/b/c and identified mir-377 as a novel editing target. In accordance with earlier findings, we observed a highly tissue-specific expression pattern for all tissues analyzed. The brain was found to express the highest number of tissue-specific miRNAs, followed by testis. Notably, our experiments also revealed robust strain-specific differential miRNA expression in the liver that is caused by genetic variation between the strains. Finally, we identified two types of germline-specific piRNAs in testis, mapping either to transposons or in strand-specific clusters. CONCLUSIONS: Taken together, the small RNA compendium described here advances the annotation of small RNAs in the rat genome. Strain and tissue-specific expression patterns furthermore provide a strong basis for studying the role of small RNAs in regulatory networks as well as biological process like physiology and neurobiology that are extensively studied in this model system.

Repertoire and evolution of miRNA genes in four divergent nematode species.

miRNAs are approximately 22-nt RNA molecules that play important roles in post-transcriptional regulation. We have performed small RNA sequencing in the nematodes Caenorhabditis elegans, C. briggsae, C. remanei, and Pristionchus pacificus, which have diverged up to 400 million years ago, to establish the repertoire and evolutionary dynamics of miRNAs in these species. In addition to previously known miRNA genes from C. elegans and C. briggsae we demonstrate expression of many of their homologs in C. remanei and P. pacificus, and identified in total more than 100 novel expressed miRNA genes, the majority of which belong to P. pacificus. Interestingly, more than half of all identified miRNA genes are conserved at the seed level in all four nematode species, whereas only a few miRNAs appear to be species specific. In our compendium of miRNAs we observed evidence for known mechanisms of miRNA evolution including antisense transcription and arm switching, as well as miRNA family expansion through gene duplication. In addition, we identified a novel mode of miRNA evolution, termed "hairpin shifting," in which an alternative hairpin is formed with up- or downstream sequences, leading to shifting of the hairpin and creation of novel miRNA* species. Finally, we identified 21U-RNAs in all four nematodes, including P. pacificus, where the upstream 21U-RNA motif is more diverged. The identification and systematic analysis of small RNA repertoire in four nematode species described here provides a valuable resource for understanding the evolutionary dynamics of miRNA-mediated gene regulation.