A Toolkit and Robust Pipeline for the Generation of Fosmid-Based Reporter Genes in C. elegans

Engineering fluorescent proteins into large genomic clones, contained within BACs or fosmid vectors, is a tool to visualize and study spatiotemporal gene expression patterns in transgenic animals. Because these reporters cover large genomic regions, they most likely capture all cis-regulatory information and can therefore be expected to recapitulate all aspects of endogenous gene expression. Inserting tags at the target gene locus contained within genomic clones by homologous recombination (“recombineering”) represents the most straightforward method to generate these reporters. In this methodology paper, we describe a simple and robust pipeline for recombineering of fosmids, which we apply to generate reporter constructs in the nematode C. elegans, whose genome is almost entirely covered in an available fosmid library. We have generated a toolkit that allows for insertion of fluorescent proteins (GFP, YFP, CFP, VENUS, mCherry) and affinity tags at specific target sites within fosmid clones in a virtually seamless manner. Our new pipeline is less complex and, in our hands, works more robustly than previously described recombineering strategies to generate reporter fusions for C. elegans expression studies. Furthermore, our toolkit provides a novel recombineering cassette which inserts a SL2-spliced intercistronic region between the gene of interest and the fluorescent protein, thus creating a reporter controlled by all 5′ and 3′ cis-acting regulatory elements of the examined gene without the direct translational fusion between the two. With this configuration, the onset of expression and tissue specificity of secreted, sub-cellular compartmentalized or short-lived gene products can be easily detected. We describe other applications of fosmid recombineering as well. The simplicity, speed and robustness of the recombineering pipeline described here should prompt the routine use of this strategy for expression studies in C. elegans

A Genome-Wide RNAi Screen for Factors Involved in Neuronal Specification in Caenorhabditis elegans

One of the central goals of developmental neurobiology is to describe and understand the multi-tiered molecular events that control the progression of a fertilized egg to a terminally differentiated neuron. In the nematode Caenorhabditis elegans, the progression from egg to terminally differentiated neuron has been visually traced by lineage analysis. For example, the two gustatory neurons ASEL and ASER, a bilaterally symmetric neuron pair that is functionally lateralized, are generated from a fertilized egg through an invariant sequence of 11 cellular cleavages that occur stereotypically along specific cleavage planes. Molecular events that occur along this developmental pathway are only superficially understood. We take here an unbiased, genome-wide approach to identify genes that may act at any stage to ensure the correct differentiation of ASEL. Screening a genome-wide RNAi library that knocks-down 18,179 genes (94% of the genome), we identified 245 genes that affect the development of the ASEL neuron, such that the neuron is either not generated, its fate is converted to that of another cell, or cells from other lineage branches now adopt ASEL fate. We analyze in detail two factors that we identify from this screen: (1) the proneural gene hlh-14, which we find to be bilaterally expressed in the ASEL/R lineages despite their asymmetric lineage origins and which we find is required to generate neurons from several lineage branches including the ASE neurons, and (2) the COMPASS histone methyltransferase complex, which we find to be a critical embryonic inducer of ASEL/R asymmetry, acting upstream of the previously identified miRNA lsy-6. Our study represents the first comprehensive, genome-wide analysis of a single neuronal cell fate decision. The results of this analysis provide a starting point for future studies that will eventually lead to a more complete understanding of how individual neuronal cell types are generated from a single-cell embryo

Neuron-type specific regulation of a 3′UTR through redundant and combinatorially acting cis-regulatory elements

3′ Untranslated region (UTR)-dependent post-transcriptional regulation has emerged as a critical mechanism of controlling gene expression in various physiological contexts, including cellular differentiation events. Here, we examine the regulation of the 3′UTR of the die-1 transcription factor in a single neuron of the nematode C. elegans. This 3′UTR shows the intriguing feature of being differentially regulated across the animal's left/right axis. In the left gustatory neuron, ASEL, in which DIE-1 protein is normally expressed in adult animals, the 3′UTR confers no regulatory information, while in the right gustatory neuron, ASER, where DIE-1 is normally not expressed, this 3′UTR confers negative regulatory information. Here, we systematically analyze the cis-regulatory architecture of the die-1 3′UTR using a transgenic, in vivo assay system. Through extensive mutagenesis and sequence insertions into heterologous 3′UTR contexts, we describe three 25-base-pair (bp) sequence elements that are both required and sufficient to mediate the ASER-specific down-regulation of the die-1 3′UTR. These three 25-bp sequence elements operate in both a redundant and combinatorial manner. Moreover, there are not only redundant elements within the die-1 3′UTR regulating its left/right asymmetric activity but asymmetric 3′UTR regulation is itself redundant with other regulatory mechanisms to achieve asymmetric DIE-1 protein expression and function in ASEL versus ASER. The features of 3′UTR regulation we describe here may apply to some of the vast number of genes in animal genomes whose expression is predicted to be regulated through their 3′UTR

Olfaction regulates organismal proteostasis and longevity via microRNA-dependent signalling

The maintenance of proteostasis is crucial for any organism to survive and reproduce in an ever-changing environment, but its efficiency declines with age(1). Post-transcriptional regulators such as micrRNAs (miRNAs) control protein translation of target mRNAs, with major consequences for development, physiology and longevity(2,3). Here we show that food odour stimulates organismal proteostasis and promotes longevity in Caenorhabditis elegans through miR-71-mediated inhibition of tir-1 mRNA stability in olfactory AWC neurons. Screening a collection of miRNAs that control ageing(3), we found that the miRNA miR-71 regulates lifespan and promotes ubiquitin-dependent protein turnover, particularly in the intestine. We show that miR-71 directly inhibits the Toll-receptor-domain protein TIR-1 in AWC olfactory neurons and that disruption of miR-71-tir-1 or loss of AWC olfactory neurons eliminates the influence of food source on proteostasis. miR-71-mediated regulation of TIR-1 controls chemotactic behaviour and is regulated by odour. Thus, odour perception influences cell-type-specific miRNA-target interaction, thereby regulating organismal proteostasis and longevity. We anticipate that the proposed mechanism of food perception will stimulate further research on neuroendocrine brain-to-gut communication and may open the possibility for therapeutic interventions to improve proteostasis and organismal health via the sense of smell, with potential implications for obesity, diabetes and ageing

Mime-seq 2.0: a method to sequence microRNAs from specific mouse cell types

<p>These files contain the raw sequencing data (demultiplexed, unaligned BAM files or FASTQ files) collected as part of following manuscript: "Mime-seq 2.0: a method to sequence microRNAs from specific mouse cell types.". Included are oxidized and unoxidized small RNA sequencing libraries collected from RKO cells (human cell line, timecourse for methyltransferase expression), M. musculus B cells (splenic, CD43 depleted; methyltransferase under Cd79a-Cre driver), M. Musculus Plasma cells (sorted, methyltransferase under Bhlha15-Cre driver). </p><p> </p><p>B cell experiment (<strong>Cd79aCre</strong>) samples:</p><ul><li>unox_HenT6B/HenT6B</li><li>ox_HenT6B/HenT6B</li><li>unox_HenT6B/HenT6B Cd79a-Cre</li><li>ox_HenT6B/HenT6B Cd79a-Cre</li><li>unox_HenT6B/+ Cd79a-Cre</li><li>ox_HenT6B/+ Cd79a-Cre</li></ul><p>Plasma cell titration experiment (<strong>Bhlha15Cre</strong>) samples:</p><ul><li>unox_100</li><li>ox_100</li><li>unox_1</li><li>ox_1</li><li>unox_01</li><li>ox_01</li><li>unox_001</li><li>ox_001</li></ul><p>RKO timecourse (<strong>RKO</strong>) samples:</p><ul><li>unox_0h</li><li>ox_0h</li><li>unox_12h</li><li>ox_12h</li><li>unox_48h</li><li>ox_48h</li><li>unox1_wt</li><li>ox1_wt</li><li>unox2_wt</li><li>ox2_wt</li></ul&gt

Paternal methotrexate exposure affects sperm small RNA content and causes craniofacial defects in the offspring

Folate is an essential vitamin for vertebrate embryo development. Methotrexate (MTX) is a folate antagonist that is widely prescribed for autoimmune diseases, blood and solid organ malignancies, and dermatologic diseases. Although it is highly contraindicated for pregnant women, because it is associated with an increased risk of multiple birth defects, the effect of paternal MTX exposure on their offspring has been largely unexplored. Here, we found MTX treatment of adult medaka male fish (Oryzias latipes) causes cranial cartilage defects in their offspring. Small non-coding RNA (sncRNAs) sequencing in the sperm of MTX treated males identify differential expression of a subset of tRNAs, with higher abundance for specific 5′ tRNA halves. Sperm RNA methylation analysis on MTX treated males shows that m5C is the most abundant and differential modification found in RNAs ranging in size from 50 to 90 nucleotides, predominantly tRNAs, and that it correlates with greater testicular Dnmt2 methyltransferase expression. Injection of sperm small RNA fractions from MTX-treated males into normal fertilized eggs generated cranial cartilage defects in the offspring. Overall, our data suggest that paternal MTX exposure alters sperm sncRNAs expression and modifications that may contribute to developmental defects in their offspring.Fil: Alata Jimenez, Nagif. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Castellano, Mauricio. Instituto Pasteur de Montevideo; Uruguay. Universidad de la Republica; UruguayFil: Santillan, Emilio M.. University Johns Hopkins; Estados UnidosFil: Boulias, Konstantinos. Harvard Medical School; Estados Unidos. Boston Children’s Hospital; Estados UnidosFil: Boan, Agustín Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Arias Padilla, Luisa Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Fernandino, Juan Ignacio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Greer, Eric L.. Harvard Medical School; Estados Unidos. Boston Children’s Hospital; Estados UnidosFil: Tosar, Juan P.. Instituto Pasteur de Montevideo; Uruguay. Universidad de la Republica; UruguayFil: Cochella, Luisa. University Johns Hopkins; Estados UnidosFil: Strobl Mazzulla, Pablo H.. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentin