MicroRNAs are important negative regulators of protein-coding gene expression and have been studied intensively over the past years. Several measurement platforms have been developed to determine relative miRNA abundance in biological samples using different technologies such as small RNA sequencing, reverse transcription-quantitative PCR (RT-qPCR) and (microarray) hybridization. In this study, we systematically compared 12 commercially available platforms for analysis of microRNA expression. We measured an identical set of 20 standardized positive and negative control samples, including human universal reference RNA, human brain RNA and titrations thereof, human serum samples and synthetic spikes from microRNA family members with varying homology. We developed robust quality metrics to objectively assess platform performance in terms of reproducibility, sensitivity, accuracy, specificity and concordance of differential expression. The results indicate that each method has its strengths and weaknesses, which help to guide informed selection of a quantitative microRNA gene expression platform for particular study goals.

MicroRNAs (miRNAs) are small non-coding RNAs that associate with Argonaute proteins to regulate gene expression at the post-transcriptional level in the cytoplasm. However, recent studies have reported that some miRNAs localize to and function in other cellular compartments. Mitochondria harbour their own genetic system that may be a potential site for miRNA mediated post-transcriptional regulation. We aimed at investigating whether nuclear-encoded miRNAs can localize to and function in human mitochondria. To enable identification of mitochondrial-enriched miRNAs, we profiled the mitochondrial and cytosolic RNA fractions from the same HeLa cells by miRNA microarray analysis. Mitochondria were purified using a combination of cell fractionation and immunoisolation, and assessed for the lack of protein and RNA contaminants. We found 57 miRNAs differentially expressed in HeLa mitochondria and cytosol. Of these 57, a signature of 13 nuclear-encoded miRNAs was reproducibly enriched in mitochondrial RNA and validated by RT-PCR for hsa-miR-494, hsa-miR-1275 and hsa-miR-1974. The significance of their mitochondrial localization was investigated by characterizing their genomic context, cross-species conservation and instrinsic features such as their size and thermodynamic parameters. Interestingly, the specificities of mitochondrial versus cytosolic miRNAs were underlined by significantly different structural and thermodynamic parameters. Computational targeting analysis of most mitochondrial miRNAs revealed not only nuclear but also mitochondrial-encoded targets. The functional relevance of miRNAs in mitochondria was supported by the finding of Argonaute 2 localization to mitochondria revealed by immunoblotting and confocal microscopy, and further validated by the co-immunoprecipitation of the mitochondrial transcript COX3. This study provides the first comprehensive view of the localization of RNA interference components to the mitochondria. Our data outline the molecular bases for a novel layer of crosstalk between nucleus and mitochondria through a specific subset of human miRNAs that we termed ‘mitomiRs’.

Sinorhizobium meliloti is an alpha-proteobacterium that alternates between a free-living phase in bulk soil or in the rhizosphere of plants and a symbiotic phase within the host plant cells, where the bacteria ultimately differentiate into nitrogen-fixing organelle-like cells, called bacteroids. As a step toward understanding the physiology of S. meliloti in its free-living and symbiotic forms and the transition between the two, gene expression profiles were determined under two sets of biological conditions: growth under oxic versus microoxic conditions, and in free-living versus symbiotic state. Data acquisition was based on both macro- and microarrays. Transcriptome profiles highlighted a profound modification of gene expression during bacteroid differentiation, with 16% of genes being altered. The data are consistent with an overall slow down of bacteroid metabolism during adaptation to symbiotic life and acquisition of nitrogen fixation capability. A large number of genes of unknown function, including potential regulators, that may play a role in symbiosis were identified. Transcriptome profiling in response to oxygen limitation indicated that up to 5% of the genes were oxygen regulated. However, the microoxic and bacteroid transcriptomes only partially overlap, implying that oxygen contributes to a limited extent to the control of symbiotic gene expression.

The production of the Sinorhizobium meliloti exopolysaccharide, succinoglycan, is required for the formation of infection threads inside root hairs, a critical step during the nodulation of alfalfa (Medicago sativa) by S. meliloti. Two bacterial mutations, exoR95::Tn5 and exoS96::Tn5, resulted in the overproduction of succinoglycan and a reduction in symbiosis. Systematic analyses of the symbiotic phenotypes of the two mutants demonstrated their reduced efficiency of root hair colonization. In addition, both the exoR95 and exoS96 mutations caused a marked reduction in the biosynthesis of flagella and consequent loss of ability of the cells to swarm and swim. Succinoglycan overproduction did not appear to be the cause of the suppression of flagellum biosynthesis. Further analysis indicated that both the exoR95 and exoS96 mutations affected the expression of the flagellum biosynthesis genes. These findings suggest that both the ExoR protein and the ExoS/ChvI two-component regulatory system are involved in the regulation of both succinoglycan and flagellum biosynthesis. These findings provide new avenues of understanding of the physiological changes S. meliloti cells go through during the early stages of symbiosis and of the signal transduction pathways that mediate such changes.Sinorhizobium meliloti and its legume host, alfalfa (Medicago sativa), establish an effective nitrogen-fixing symbiosis through a series of signal exchanges that starts with the exchange of Nod (nodulation) factors and flavonoids, which results in the formation of curled alfalfa root hairs that are colonized by S. meliloti cells (13,20,32). The colonized curled root hairs develop infection threads within the root hairs, which allow S. meliloti cells to invade the developing root nodules (14,16,31). A successful invasion of nodules by S. meliloti will result in the formation of pink nitrogen-fixing nodules. The pink color is due to the presence of leghemoglobin. Nodules that are not occupied by S. meliloti and/or not capable of fixing nitrogen are most often white due to the lack of leghemoglobin (20).The formation of infection threads inside root hairs requires the presence of an S. meliloti exopolysaccharide, succinoglycan (9), in addition to the Nod factor (33). Succinoglycan is a polymer that consists of different numbers of a repeating unit consisting of one galactose and seven glucoses with three modification groups: acetyl, pyruvyl, and succinyl (17, 24). All three modifications must be present in order for the S. meliloti succinoglycan to be active in eliciting infection thread formation (9). Surprisingly, overproduction of succinoglycan appears to reduce efficiency of nodulation (12).Two S. meliloti mutants, exoR95::Tn5 and exoS96::Tn5, were isolated based on their ability to overproduce succinoglycan (12). The exoR gene encodes a protein of 268 amino acids that shares no significant homology with any other protein in currently available databases (23). The exoS gene encodes the membrane-bound sensor of the ExoS/ChvI two-component r...

Proteins directing the biosynthesis of galactoglucan (exopolysaccharide II) in Rhizobium melilotiThe soil bacterium Rhizobium meliloti is capable of fixing molecular nitrogen in a symbiotic interaction with alfalfa plants. Bacterial nitrogen fixation takes place within root nodules resulting from a coordinated bacteria-plant interaction which requires the exchange of signals between both symbiotic partners (11, 29). R. meliloti is able to produce two acidic exopolysaccharides (EPSs), succinoglycan (EPS I) and galactoglucan (EPS II). At least one of these EPSs is required for invasion of Medicago sativa nodules by R. meliloti (3,32,36,61,86).EPS I is composed of repeating units containing one galactose and seven glucose molecules joined by ␤-1,4-, ␤-1,3-, and ␤-1,6-glycosidic linkages (63) and is decorated by acetate, pyruvate, and succinyl groups. EPS II consists of alternating glucose and galactose residues joined by ␣-1,3 and ␤-1,3 linkages (38). It is acetylated and pyruvylated. In the Rm1021 and Rm2011 strain backgrounds, the production of EPS II was observed only at low phosphate concentrations (87) or in the presence of a mutation in either the expR (32) or mucR (44, 86) gene. The corresponding gene products are thought to negatively regulate the expression of genes directing the biosynthesis of EPS II.The exo gene cluster, directing the biosynthesis of EPS I (50), and the exp gene cluster, responsible for the synthesis of EPS II (32), are separated by about 200 kb on megaplasmid 2 (15). Whereas the exo gene cluster was extensively studied and functions were assigned to most of the exo gene products (4-8, 12, 33, 34, 58, 64), little is known about the organization of the exp gene cluster and the functions of exp gene products.A 23-kb DNA region involved in EPS II biosynthesis was cloned and characterized by Tn5 mutagenesis (32). Six exp complementation groups required for EPS II biosynthesis in the expR101 mutant background were identified in this region. Here, we report on the 32-kb sequence of the R. meliloti Rm2011 exp gene cluster comprising 25 genes and on the inferred properties of the encoded exp gene products. MATERIALS AND METHODSBacterial strains and plasmids. Strains and plasmids used in this study are listed in Table 1.Media and growth conditions. Escherichia coli strains were grown in Penassay broth (Difco Laboratories) or in LB medium (65) at 37ЊC. R. meliloti strains were grown in TY medium (10), Vincent minimal medium (79), M9 medium (56), or LB medium (65) at 30ЊC.Antibiotics were supplemented as required at the following concentrations (micrograms per milliliter): for R.