Targeted CRISPR disruption reveals a role for RNase MRP RNA in human preribosomal RNA processing.

MRP RNA is an abundant, essential noncoding RNA whose functions have been proposed in yeast but are incompletely understood in humans. Mutations in the genomic locus for MRP RNA cause pleiotropic human diseases, including cartilage hair hypoplasia (CHH). Here we applied CRISPR-Cas9 genome editing to disrupt the endogenous human MRP RNA locus, thereby attaining what has eluded RNAi and RNase H experiments: elimination of MRP RNA in the majority of cells. The resulting accumulation of ribosomal RNA (rRNA) precursor-analyzed by RNA fluorescent in situ hybridization (FISH), Northern blots, and RNA sequencing-implicates MRP RNA in pre-rRNA processing. Amelioration of pre-rRNA imbalance is achieved through rescue of MRP RNA levels by ectopic expression. Furthermore, affinity-purified MRP ribonucleoprotein (RNP) from HeLa cells cleaves the human pre-rRNA in vitro at at least one site used in cells, while RNP isolated from cells with CRISPR-edited MRP loci loses this activity, and ectopic MRP RNA expression restores cleavage activity. Thus, a role for RNase MRP in human pre-rRNA processing is established. As demonstrated here, targeted CRISPR disruption is a valuable tool for functional studies of essential noncoding RNAs that are resistant to RNAi and RNase H-based degradation.

Inhibition of telomerase RNA decay rescues telomerase deficiency caused by dyskerin or PARN defects.

Mutations in the human telomerase RNA component (hTR), the telomerase ribonucleoprotein component dyskerin (DKC1) and the poly(A) RNase (PARN) can lead to reduced levels of hTR and to dyskeratosis congenita (DC). However, the enzymes and mechanisms responsible for hTR degradation are unknown. We demonstrate that defects in dyskerin binding lead to hTR degradation by PAPD5-mediated oligoadenylation, which promotes 3'-to-5' degradation by EXOSC10, as well as decapping and 5'-to-3' decay by the cytoplasmic DCP2 and XRN1 enzymes. PARN increased hTR levels by deadenylating hTR, thereby limiting its degradation by EXOSC10. Telomerase activity and proper hTR localization in dyskerin- or PARN-deficient cells were rescued by knockdown of DCP2 and/or EXOSC10. Prevention of hTR RNA decay also led to a rescue of localization of DC-associated hTR mutants. These results suggest that inhibition of RNA decay pathways might be a useful therapy for some telomere pathologies.

Metals other than uranium affected microbial community composition in a historical uranium-mining site.

To understand the links between the long-term impact of uranium and other metals on microbial community composition, ground- and surface water-influenced soils varying greatly in uranium and metal concentrations were investigated at the former uranium-mining district in Ronneburg, Germany. A soil-based 16S PhyloChip approach revealed 2358 bacterial and 35 archaeal operational taxonomic units (OTU) within diverse phylogenetic groups with higher OTU numbers than at other uranium-contaminated sites, e.g., at Oak Ridge. Iron- and sulfate-reducing bacteria (FeRB and SRB), which have the potential to attenuate uranium and other metals by the enzymatic and/or abiotic reduction of metal ions, were found at all sites. Although soil concentrations of solid-phase uranium were high, ranging from 5 to 1569 µg·g (dry weight) soil(-1), redundancy analysis (RDA) and forward selection indicated that neither total nor bio-available uranium concentrations contributed significantly to the observed OTU distribution. Instead, microbial community composition appeared to be influenced more by redox potential. Bacterial communities were also influenced by bio-available manganese and total cobalt and cadmium concentrations. Bio-available cadmium impacted FeRB distribution while bio-available manganese and copper as well as solid-phase zinc concentrations in the soil affected SRB composition. Archaeal communities were influenced by the bio-available lead as well as total zinc and cobalt concentrations. These results suggest that (i) microbial richness was not impacted by heavy metals and radionuclides and that (ii) redox potential and secondary metal contaminants had the strongest effect on microbial community composition, as opposed to uranium, the primary source of contamination.

3' terminal diversity of MRP RNA and other human noncoding RNAs revealed by deep sequencing.

BACKGROUND: Post-transcriptional 3' end processing is a key component of RNA regulation. The abundant and essential RNA subunit of RNase MRP has been proposed to function in three distinct cellular compartments and therefore may utilize this mode of regulation. Here we employ 3' RACE coupled with high-throughput sequencing to characterize the 3' terminal sequences of human MRP RNA and other noncoding RNAs that form RNP complexes. RESULTS: The 3' terminal sequence of MRP RNA from HEK293T cells has a distinctive distribution of genomically encoded termini (including an assortment of U residues) with a portion of these selectively tagged by oligo(A) tails. This profile contrasts with the relatively homogenous 3' terminus of an in vitro transcribed MRP RNA control and the differing 3' terminal profiles of U3 snoRNA, RNase P RNA, and telomerase RNA (hTR). CONCLUSIONS: 3' RACE coupled with deep sequencing provides a valuable framework for the functional characterization of 3' terminal sequences of noncoding RNAs.

Cystic fibrosis transmembrane conductance regulator knockout mice exhibit aberrant gastrointestinal microbiota.

The composition of the gastrointestinal microbiome is increasingly recognized as a crucial contributor to immune and metabolic homeostasis-deficiencies in which are characteristic of cystic fibrosis (CF) patients. The murine model (CFTR (-/-) , CF), has, in previous studies, demonstrated characteristic CF gastrointestinal (GI) manifestations including slowed transit and significant upregulation of genes associated with inflammation. To determine if characteristics of the microbiome are associated with these phenotypes we used a phylogenetic microarray to compare small intestine bacterial communities of wild type and congenic CF mice. Loss of functional CFTR is associated with significant decreases in GI bacterial community richness, evenness and diversity and reduced relative abundance of putative protective species such as Acinetobacter lwoffii and a multitude of Lactobacilliales members. CF mice exhibited significant enrichment of Mycobacteria species and Bacteroides fragilis, previously associated with GI infection and immunomodulation. Antibiotic administration to WT and CF animals resulted in convergence of their microbiome composition and significant increases in community diversity in CF mice. These communities were characterized by enrichment of members of the Lactobacillaceae and Bifidobacteriaceae and reduced abundance of Enterobacteriaceae and Clostridiaceae. These data suggest that Enterobacteria and Clostridia species, long associated with small intestinal overgrowth and inflammatory bowel disease, may suppress both ileal bacterial diversity and the particular species which maintain motility and immune homeostasis in this niche. Thus, these data provide the first indications that GI bacterial colonization is strongly impacted by the loss of functional CFTR and opens up avenues for alternative therapeutic approaches to improve CF disease management.

Influence of geogenic factors on microbial communities in metallogenic Australian soils.

Links between microbial community assemblages and geogenic factors were assessed in 187 soil samples collected from four metal-rich provinces across Australia. Field-fresh soils and soils incubated with soluble Au(III) complexes were analysed using three-domain multiplex-terminal restriction fragment length polymorphism, and phylogenetic (PhyloChip) and functional (GeoChip) microarrays. Geogenic factors of soils were determined using lithological-, geomorphological- and soil-mapping combined with analyses of 51 geochemical parameters. Microbial communities differed significantly between landforms, soil horizons, lithologies and also with the occurrence of underlying Au deposits. The strongest responses to these factors, and to amendment with soluble Au(III) complexes, was observed in bacterial communities. PhyloChip analyses revealed a greater abundance and diversity of Alphaproteobacteria (especially Sphingomonas spp.), and Firmicutes (Bacillus spp.) in Au-containing and Au(III)-amended soils. Analyses of potential function (GeoChip) revealed higher abundances of metal-resistance genes in metal-rich soils. For example, genes that hybridised with metal-resistance genes copA, chrA and czcA of a prevalent aurophillic bacterium, Cupriavidus metallidurans CH34, occurred only in auriferous soils. These data help establish key links between geogenic factors and the phylogeny and function within soil microbial communities. In particular, the landform, which is a crucial factor in determining soil geochemistry, strongly affected microbial community structures.

RNase P branches out from RNP to protein: organelle-triggered diversification?

RNase P is the enzyme that removes 5' leader sequences from precursor tRNAs. Remarkably, in most organisms, RNase P is a ribonucleoprotein particle where the RNA component is responsible for catalysis. In this issue of Genes & Development, Gutmann and colleagues (pp. 1022-1027) report the first organism, Arabidopsis thaliana, to employ protein-only RNase P in both its nucleus and organelles. An intriguing possibility is that replacement of RNase P ribonucleoprotein particles (RNPs) by proteins may have been triggered by the acquisition of organelles.

Differences in crop bacterial community structure between hoatzins from different geographical locations.

The hoatzin is the only known folivorous bird with foregut fermentation, and is distributed in Venezuela in rivers of the central savannas to the eastern Orinoco River. Differences in diet are expected to affect the digestive microbiota and we hypothesized that hoatzins from different habitats might have a different crop microbiota. We thus characterized the microbiota of six birds from the Cojedes and Orinoco Rivers using the G2 PhyloChip and, in parallel, we compared plant availability and foraging behavior of the hoatzins from the two locations. Plant composition differed between the 2 locations, which shared 5 out of 18 plant families and 1 plant genus--Coccoloba--that was highly consumed in both locations. The PhyloChip detected ∼1600 phylotypes from 42 phyla. There was a core microbiota with ~50% of the OTUs shared by at least 4 of the 6 individuals, but there were also differences in the crop microbiota of animals from the two regions. There existed a higher relative abundance of Alphaproteobacteria and Actinobacteria in the crops of birds from the Cojedes River and of Clostridia and Deltaproteobacteria in the crops of birds from the Orinoco River. The results showed both a core crop microbiota and also the bacterial taxa responsible for geographical differences among individuals from the two locations with different vegetation, suggesting an effect of both diet and geography in shaping the crop bacterial community of the hoatzin.

Comparative analyses of foregut and hindgut bacterial communities in hoatzins and cows.

Foregut fermentation occurs in mammalian ruminants and in one bird, the South American folivorous hoatzin. This bird has an enlarged crop with a function analogous to the rumen, where foregut microbes degrade the otherwise indigestible plant matter, providing energy to the host from foregut fermentation, in addition to the fermentation that occurs in their hindguts (cecum/colon). As foregut fermentation represents an evolutionary convergence between hoatzins and ruminants, our aim was to compare the community structure of foregut and hindgut bacterial communities in the cow and hoatzin to evaluate the influences of host phylogeny and organ function in shaping the gut microbiome. The approach used was to hybridize amplified bacterial ribosomal RNA genes onto a high-density microarray (PhyloChip). The results show that the microbial communities cluster primarily by functional environment (foreguts cluster separately from hindguts) and then by host. Bacterial community diversity was higher in the cow than in the hoatzin. Overall, compared with hindguts, foreguts have higher proportions of Bacteroidetes and Spirochaetes, and lower proportions of Firmicutes and Proteobacteria. The main host differences in gut bacterial composition include a higher representation of Spirochaetes, Synergistetes and Verrucomicrobia in the cow. Despite the significant differences in host phylogeny, body size, physiology and diet, the function seems to shape the microbial communities involved in fermentation. Regardless of the independent origin of foregut fermentation in birds and mammals, organ function has led to convergence of the microbial community structure in phylogenetically distant hosts.

Soil bacterial communities of a calcium-supplemented and a reference watershed at the Hubbard Brook Experimental Forest (HBEF), New Hampshire, USA.

Soil Ca depletion because of acidic deposition-related soil chemistry changes has led to the decline of forest productivity and carbon sequestration in the northeastern USA. In 1999, acidic watershed (WS) 1 at the Hubbard Brook Experimental Forest (HBEF), NH, USA was amended with Ca silicate to restore soil Ca pools. In 2006, soil samples were collected from the Ca-amended (WS1) and reference watershed (WS3) for comparison of bacterial community composition between the two watersheds. The sites were about 125 m apart and were known to have similar stream chemistry and tree populations before Ca amendment. Ca-amended soil had higher Ca and P, and lower Al and acidity as compared with the reference soils. Analysis of bacterial populations by PhyloChip revealed that the bacterial community structure in the Ca-amended and the reference soils was significantly different and that the differences were more pronounced in the mineral soils. Overall, the relative abundance of 300 taxa was significantly affected. Numbers of detectable taxa in families such as Acidobacteriaceae, Comamonadaceae, and Pseudomonadaceae were lower in the Ca-amended soils, while Flavobacteriaceae and Geobacteraceae were higher. The other functionally important groups, e.g. ammonia-oxidizing Nitrosomonadaceae, had lower numbers of taxa in the Ca-amended organic soil but higher in the mineral soil.

Differential growth responses of soil bacterial taxa to carbon substrates of varying chemical recalcitrance.

Soils are immensely diverse microbial habitats with thousands of co-existing bacterial, archaeal, and fungal species. Across broad spatial scales, factors such as pH and soil moisture appear to determine the diversity and structure of soil bacterial communities. Within any one site however, bacterial taxon diversity is high and factors maintaining this diversity are poorly resolved. Candidate factors include organic substrate availability and chemical recalcitrance, and given that they appear to structure bacterial communities at the phylum level, we examine whether these factors might structure bacterial communities at finer levels of taxonomic resolution. Analyzing 16S rRNA gene composition of nucleotide analog-labeled DNA by PhyloChip microarrays, we compare relative growth rates on organic substrates of increasing chemical recalcitrance of >2,200 bacterial taxa across 43 divisions/phyla. Taxa that increase in relative abundance with labile organic substrates (i.e., glycine, sucrose) are numerous (>500), phylogenetically clustered, and occur predominantly in two phyla (Proteobacteria and Actinobacteria) including orders Actinomycetales, Enterobacteriales, Burkholderiales, Rhodocyclales, Alteromonadales, and Pseudomonadales. Taxa increasing in relative abundance with more chemically recalcitrant substrates (i.e., cellulose, lignin, or tannin-protein) are fewer (168) but more phylogenetically dispersed, occurring across eight phyla and including Clostridiales, Sphingomonadalaes, Desulfovibrionales. Just over 6% of detected taxa, including many Burkholderiales increase in relative abundance with both labile and chemically recalcitrant substrates. Estimates of median rRNA copy number per genome of responding taxa demonstrate that these patterns are broadly consistent with bacterial growth strategies. Taken together, these data suggest that changes in availability of intrinsically labile substrates may result in predictable shifts in soil bacterial composition.

Developmental microbial ecology of the crop of the folivorous hoatzin.

The hoatzin (Opisthocomus hoazin) is a South American strict folivorous bird, with a crop microbial ecosystem that ferments dietary plants. Chicks progressively become independent from the adult-fed regurgitated crop liquids, and we hypothesized that the crop bacterial ecosystem develops through ecological succession mechanisms, as they grow into adults. The aim of this work was to compare the crop bacterial community in hoatzins from three age groups: newly hatched chicks, juveniles and adults by sequencing 16S rRNA genes and using the G2 PhyloChip. Cloning yielded a total of 2123 nearly full-length sequences binned into 294 operational taxonomic units (OTUs) (with <97% homology) belonging to 7 phyla, with 91% of novel OTUs. The microarray identified a diverse bacterial community dominated by Firmicutes and Bacteroidetes, with approximately 1400 taxa grouped in 40 phyla that included those detected by cloning. In comparison with the adult, the hoatzin chick crop had a greater abundance of Flavobacteriaceae, Clostridiaceae and Lachnospiraceae but lacked phyla DSS1, Deferribacteres and Termite group 1, which were mostly present in adults. The overall community structure of the crop of the hoatzin changes with age in a complex manner, probably responding to new niches made available through dietary changes related to the transition from dependent to independent feeding.

Induction of intestinal Th17 cells by segmented filamentous bacteria.

The gastrointestinal tract of mammals is inhabited by hundreds of distinct species of commensal microorganisms that exist in a mutualistic relationship with the host. How commensal microbiota influence the host immune system is poorly understood. We show here that colonization of the small intestine of mice with a single commensal microbe, segmented filamentous bacterium (SFB), is sufficient to induce the appearance of CD4(+) T helper cells that produce IL-17 and IL-22 (Th17 cells) in the lamina propria. SFB adhere tightly to the surface of epithelial cells in the terminal ileum of mice with Th17 cells but are absent from mice that have few Th17 cells. Colonization with SFB was correlated with increased expression of genes associated with inflammation and antimicrobial defenses and resulted in enhanced resistance to the intestinal pathogen Citrobacter rodentium. Thus, manipulation of this commensal-regulated pathway may provide new opportunities for enhancing mucosal immunity and treating autoimmune disease.