Francisco E. Nicolás

Two classes of small antisense RNAs in fungal RNA silencing triggered by non‐integrative transgenes

Transformation of Mucor circinelloides with self‐replicative plasmids containing a wild‐type copy of the carotenogenic gene carB causes silencing of the carB function in 3% of transformants. Genomic analyses revealed a relationship between silenced phenotype and number of copies of plasmids. This phenotype results from a reduction of the steady‐state levels of carB mRNA, a reduction that is not due to differences in the level of transcription, indicating that silencing is post‐transcriptional. Small sense and antisense RNAs have been found to be associated with gene silencing in M.circinelloides. Two size classes of small antisense RNAs, differentially accumulated during the vegetative growth of silenced transformants, have been detected: a long 25‐nucleotide RNA and a short 21‐nucleotide RNA. Secondary sense and antisense RNAs corresponding to sequences of the endogenous gene downstream of the initial triggering molecule have also been detected, revealing the existence of spreading of RNA targeting in fungi. These findings, together with the self‐replicative nature of the triggering molecules, make M.circinelloides a suitable organism for investigating some unresolved questions in RNA silencing.

Experimental identification of microRNA-140 targets by silencing and overexpressing miR-140

MicroRNAs (miRNAs) are short noncoding RNA molecules regulating the expression of mRNAs. Target identification of miRNAs is computationally difficult due to the relatively low homology between miRNAs and their targets. We present here an experimental approach to target identification where the cartilage-specific miR-140 was overexpressed and silenced in cells it is normally expressed in separate experiments. Expression of mRNAs was profiled in both experiments and the intersection of mRNAs repressed by miR-140 overexpression and derepressed by silencing of miR-140 was identified. The intersection contained only 49 genes, although both treatments affected the accumulation of hundreds of mRNAs. These 49 genes showed a very strong enrichment for the miR-140 seed sequence implying that the approach is efficient and specific. Twenty-one of these 49 genes were predicted to be direct targets based on the presence of the seed sequence. Interestingly, none of these were predicted by the published target prediction methods we used. One of the potential target mRNAs, Cxcl12, was experimentally validated by Northern blot analysis and a luciferase reporter assay.

Analyzing mRNA expression identifies Smad3 as a microRNA-140 target regulated only at protein level

mRNA profiling is routinely used to identify microRNA targets, however, this high-throughput technology is not suitable for identifying targets regulated only at protein level. Here, we have developed and validated a novel methodology based on computational analysis of promoter sequences combined with mRNA microarray experiments to reveal transcription factors that are direct microRNA targets at the protein level. Using this approach we identified Smad3, a key transcription factor in the TGFbeta signaling pathway, as a direct miR-140 target. We showed that miR-140 suppressed the TGFbeta pathway through repression of Smad3 and that TGFbeta suppressed the accumulation of miR-140 forming a double negative feedback loop. Our findings establish a valid strategy for the discovery of microRNA targets regulated only at protein level, and we propose that additional targets could be identified by re-analysis of existing microarray datasets.

Ghrelin, Sleep Reduction and Evening Preference: Relationships to CLOCK 3111 T/C SNP and Weight Loss

Background Circadian Locomotor Output Cycles Kaput (CLOCK), an essential element of the positive regulatory arm in the human biological clock, is involved in metabolic regulation. The aim was to investigate the behavioral (sleep duration, eating patterns and chronobiological characteristics) and hormonal (plasma ghrelin and leptin concentrations) factors which could explain the previously reported association between the CLOCK 3111T/C SNP and weight loss. Methodology/Principal Findings We recruited 1495 overweight/obese subjects (BMI: 25–40 kg/m2) of 20–65 y. who attended outpatient obesity clinics in Murcia, in southeastern Spain. We detected an association between the CLOCK 3111T/C SNP and weight loss, which was particularly evident after 12–14 weeks of treatment (P = 0.038). Specifically, carriers of the minor C allele were more resistant to weight loss than TT individuals (Mean±SEM) (8.71±0.59 kg vs 10.4±0.57 kg) C and TT respectively. In addition, our data show that minor C allele carriers had: 1. shorter sleep duration Mean ± SEM (7.0±0.05 vs 7.3±0.05) C and TT respectively (P = 0.039), 2. higher plasma ghrelin concentrations Mean ± SEM (pg/ml) (1108±49 vs 976±47)(P = 0.034); 3. delayed breakfast time; 4. evening preference and 5. less compliance with a Mediterranean Diet pattern, as compared with TT homozygotes. Conclusions/Significance Sleep reduction, changes in ghrelin values, alterations of eating behaviors and evening preference that characterized CLOCK 3111C carriers could be affecting weight loss. Our results support the hypothesis that the influence of the CLOCK gene may extend to a broad range of variables linked with human behaviors.

Cortisol secretary pattern and glucocorticoid feedback sensitivity in women from a Mediterranean area: relationship with anthropometric characteristics, dietary intake and plasma fatty acid profile

Background  Chronic stress is associated with a dysfunctional hypothalamic‐pituitary‐adrenal (HPA) axis consisting on disturbances on the cortisol response and lipid metabolism.

A negative regulator of light-inducible carotenogenesis in Mucor circinelloides

Abstract. Mucor circinelloides responds to blue light by activating carotene biosynthesis. Wild-type strains grown in darkness contain minimal amounts of β-carotene because of the low levels of transcription of the structural genes for carotenogenesis. When exposed to a light pulse, the level of transcription of these genes increases strongly, leading to the formation of high concentrations of β-carotene. The crgA gene is involved in the regulation of light-induced carotenoid biosynthesis. This gene, originally identified as a 3′-truncated ORF which causes carotene over-accumulation in the dark, encodes a protein with a cysteine-rich, zinc-binding, RING-finger motif, as found in diverse groups of regulatory proteins. The expression of the crgA gene is activated by a light pulse, with a time course similar to that of the structural genes for carotenogenesis. To understand the regulatory role of the crgA gene in carotenogenesis, we have used a genetic approach based on the construction of crgA null mutants by gene replacement. Lack of the crgA function provokes the over-accumulation of carotenoids both in the dark and the light. Introduction of the wild-type crgA allele into these mutants restores the wild-type phenotype for carotenogenesis. The high levels of carotenoid accumulation shown by the null crgA mutants are correlated with an increase in the expression of carotenogenic structural genes. These results strongly indicate that crgA acts as a negative regulator of light-inducible carotenogenesis in M. circinelloides.

Endogenous short RNAs generated by Dicer 2 and RNA-dependent RNA polymerase 1 regulate mRNAs in the basal fungus Mucor circinelloides

Endogenous short RNAs (esRNAs) play diverse roles in eukaryotes and usually are produced from double-stranded RNA (dsRNA) by Dicer. esRNAs are grouped into different classes based on biogenesis and function but not all classes are present in all three eukaryotic kingdoms. The esRNA register of fungi is poorly described compared to other eukaryotes and it is not clear what esRNA classes are present in this kingdom and whether they regulate the expression of protein coding genes. However, evidence that some dicer mutant fungi display altered phenotypes suggests that esRNAs play an important role in fungi. Here, we show that the basal fungus Mucor circinelloides produces new classes of esRNAs that map to exons and regulate the expression of many protein coding genes. The largest class of these exonic-siRNAs (ex-siRNAs) are generated by RNA-dependent RNA Polymerase 1 (RdRP1) and dicer-like 2 (DCL2) and target the mRNAs of protein coding genes from which they were produced. Our results expand the range of esRNAs in eukaryotes and reveal a new role for esRNAs in fungi.

Daily profile in two circadian markers “melatonin and cortisol” and associations with metabolic syndrome components☆

OBJECTIVE The aim of the present work was to investigate associations in circadian markers, melatonin (MT) and cortisol, with metabolic syndrome (MetS) parameters, and with leptin, adiponectin and ghrelin plasma values. METHODS The study was conducted in 70 women (mean age: 41±10years) that were classified without MetS (n=30) and with MetS (n=40). Blood collection, plasma separation and processing, and biochemical analyses for plasma lipids were performed. For measuring salivary melatonin, participants collected two samples. The first simple was obtained before lunch (at 14:00 p.m.) and the second sample was taken at night (3:00 a.m.). On a random working day, participants delivered repeated salivary cortisol samples. The first sample was obtained in the morning (09:00 a.m.), then before lunch at (14:00 p.m.), and finally just before bedtime (23:00 p.m.). RESULTS Significant differences were found between the MT measurements taken at night in women without and with MetS. With respect to cortisol, significant differences were found in the different times cortisol levels toward a more flattened pattern among MetS women. Both parameters were positive correlated between them. Of note MT and cortisol night/morning ratios were associated with MetS score and metabolic syndrome components. CONCLUSION The findings indicate that diminished daily amplitude in MT and cortisol circadian patterns was associated with metabolic disturbances in blood pressure, glucose and plasma lipids regulation, ghrelin and adipocyte-secreted hormones such as leptin and adiponectin.

Biogenesis of Y RNA-derived small RNAs is independent of the microRNA pathway

Y RNAs are approximately 100 nucleotide long conserved cytoplasmic non‐coding RNAs, which produce smaller RNA fragments during apoptosis. Here we show that these smaller RNA molecules are also produced in non‐stressed cells and in a range of human cancerous and non‐cancerous cell types. Recent reports have speculated that the cleavage products of Y RNAs enter the microRNA pathway. We tested this hypothesis and found that Y5 and Y3 RNA fragments are Dicer independent, they are in different complexes than microRNAs and that they are not co‐immunoprecipitated with Ago2. Therefore we conclude that Y RNA fragments do not enter the microRNA pathway.

A single dicer gene is required for efficient gene silencing associated with two classes of small antisense RNAs in Mucor circinelloides.

ABSTRACT RNA silencing in the zygomycete Mucor circinelloides exhibits uncommon features, such as induction by self-replicative sense transgenes and the accumulation of two size classes of antisense small interfering RNAs (siRNAs). To investigate whether this silencing phenomenon follows the rules of a canonical RNA-silencing mechanism, we used hairpin RNA (hpRNA)-producing constructs as silencing triggers and analyzed the efficiency and stability of silencing in different genetic backgrounds. We show here that the dsRNA-induced silencing mechanism is also associated with the accumulation of two sizes of antisense siRNAs and that this mechanism is not mediated by the previously known dcl-1 (dicer-like) gene, which implies the existence of an additional dicer gene. An M. circinelloides dcl-2 gene was cloned and characterized, and the corresponding null mutant was generated by gene replacement. This mutant is severely impaired in the silencing mechanism induced by self-replicative sense or inverted-repeat transgenes, providing the first genetic evidence of a canonical silencing mechanism in this class of fungus and pointing to a role for dcl-2 in the mechanism. Moreover, a functional dcl-2 gene is required for the normal accumulation of the two sizes of antisense RNAs, as deduced from the analysis of dcl-2− transformants containing hpRNA-expressing plasmids. In addition to its critical role in transgene-induced silencing, the dcl-2 gene seems to play a role in the control of vegetative development, since the dcl-2 null mutants showed a significant decrease in their production of asexual spores.