5′HS5 of the Human β-globin Locus Control Region Is Dispensable for the Formation of the β-globin Active Chromatin Hub

Hypersensitive site 5 (5′HS5) of the β-globin Locus Control Region functions as a developmental stage-specific border in erythroid cells. Here, we have analyzed the role of 5′HS5 in the three dimensional organization of the β-gene locus using the Chromatin Conformation Capture (3C) technique. The results show that when 5′HS5 is deleted from the locus, both remote and internal regulatory elements are still able to interact with each other in a three-dimensional configuration termed the Active Chromatin Hub. Thus, the absence of 5′HS5 does not have an appreciable effect on the three dimensional organization of the β-globin locus. This rules out models in which 5′HS5 nucleates interactions with remote and/or internal regulatory elements. We also determined the binding of CTCF, the only defined insulator protein in mammalian cells, to 5′HS5 by using chromatin immunoprecipitation (ChIP) assays. We detect low levels of CTCF binding to 5′HS5 in primitive erythroid cells, in which it functions as a border element. Surprisingly, we also observe binding levels of CTCF to 5′HS5 in definitive erythroid cells. Thus, binding of CTCF to 5′HS5 per se does not render it a functional border element. This is consistent with the previous data suggesting that CTCF has dual functionality.     

Genome-wide analysis of single non-templated nucleotides in plant endogenous siRNAs and miRNAs

Plant small RNAs are subject to various modifications. Previous reports revealed widespread 3′ modifications (truncations and non-templated tailing) of plant miRNAs when the 2′-O-methyltransferase HEN1 is absent. However, non-templated nucleotides in plant heterochromatic siRNAs have not been deeply studied, especially in wild-type plants. We systematically studied non-templated nucleotide patterns in plant small RNAs by analyzing small RNA sequencing libraries from Arabidopsis, tomato, Medicago, rice, maize and Physcomitrella. Elevated rates of non-templated nucleotides were observed at the 3′ ends of both miRNAs and endogenous siRNAs from wild-type specimens of all species. ‘Off-sized’ small RNAs, such as 25 and 23 nt siRNAs arising from loci dominated by 24 nt siRNAs, often had very high rates of 3′-non-templated nucleotides. The same pattern was observed in all species that we studied. Further analysis of 24 nt siRNA clusters in Arabidopsis revealed distinct patterns of 3′-non-templated nucleotides of 23 nt siRNAs arising from heterochromatic siRNA loci. This pattern of non-templated 3′ nucleotides on 23 nt siRNAs is not affected by loss of known small RNA 3′-end modifying enzymes, and may result from modifications added to longer heterochromatic siRNA precursors.     

Identification and Characterization of aarF, a Locus Required for Production of Ubiquinone in Providencia stuartii and Escherichia coli and for Expression of 2′-N-Acetyltransferase in P. stuartii

Providencia stuartii contains a chromosomal 2′-N-acetyltransferase [AAC(2′)-Ia] involved in the O acetylation of peptidoglycan. The AAC(2′)-Ia enzyme is also capable of acetylating and inactivating certain aminoglycosides and confers high-level resistance to these antibiotics when overexpressed. We report the identification of a locus in P. stuartii, designated aarF, that is required for the expression of AAC(2′)-Ia. Northern (RNA) analysis demonstrated that aac(2′)-Ia mRNA levels were dramatically decreased in a P. stuartii strain carrying an aarF::Cm disruption. The aarF::Cm disruption also resulted in a deficiency in the respiratory cofactor ubiquinone. The aarF locus encoded a protein that had a predicted molecular mass of 62,559 Da and that exhibited extensive amino acid similarity to the products of two adjacent open reading frames of unknown function (YigQ and YigR), located at 86 min on the Escherichia coli chromosome. An E. coli yigR::Kan mutant was also deficient in ubiquinone content. Complementation studies demonstrated that the aarF and the E. coli yigQR loci were functionally equivalent. The aarF or yigQR genes were unable to complement ubiD and ubiE mutations that are also present at 86 min on the E. coli chromosome. This result indicates that aarF (yigQR) represents a novel locus for ubiquinone production and reveals a previously unreported connection between ubiquinone biosynthesis and the regulation of gene expression.     

The Impact of 3′UTR Variants on Differential Expression of Candidate Cancer Susceptibility Genes

Variants in regulatory regions are predicted to play an important role in disease susceptibility of common diseases. Polymorphisms mapping to microRNA (miRNA) binding sites have been shown to disrupt the ability of miRNAs to target genes resulting in differential mRNA and protein expression. Skin tumor susceptibility 5 (Skts5) was identified as a locus conferring susceptibility to chemically-induced skin cancer in NIH/Ola by SPRET/Outbred F1 backcrosses. To determine if polymorphisms between the strains which mapped to putative miRNA binding sites in the 3′ untranslated region (3′UTR) of genes at Skts5 influenced expression, we conducted a systematic evaluation of 3′UTRs of candidate genes across this locus. Nine genes had polymorphisms in their 3′UTRs which fit the linkage data and eight of these contained polymorphisms suspected to interfere with or introduce miRNA binding. 3′UTRs of six genes, Bcap29, Dgkb, Hbp1, Pik3cg, Twistnb, and Tspan13 differentially affected luciferase expression, but did not appear to be differentially regulated by the evaluated miRNAs predicted to bind to only one of the two isoforms. 3′UTRs from four additional genes chosen from the locus that fit less stringent criteria were evaluated. Ifrd1 and Etv1 showed differences and contained polymorphisms predicted to disrupt or create miRNA binding sites but showed no difference in regulation by the miRNAs tested. In summary, multiple 3′UTRs with putative functional variants between susceptible and resistant strains of mice influenced differential expression independent of predicted miRNA binding.     

CvfA Protein and Polynucleotide Phosphorylase Act in an Opposing Manner to Regulate Staphylococcus aureus Virulence

We previously identified CvfA (SA1129) as a Staphylococcus aureus virulence factor using a silkworm infection model. S. aureus cvfA-deleted mutants exhibit decreased expression of the agr locus encoding a positive regulator of hemolysin genes and decreased hemolysin production. CvfA protein hydrolyzes a 2′,3′-cyclic phosphodiester bond at the RNA 3′ terminus, producing RNA with a 3′-phosphate (3′-phosphorylated RNA, RNA with a 3′-phosphate). Here, we report that the cvfA-deleted mutant phenotype (decreased agr expression and hemolysin production) was suppressed by disrupting pnpA-encoding polynucleotide phosphorylase (PNPase) with 3′- to 5′-exonuclease activity. The suppression was blocked by introducing a pnpA-encoding PNPase with exonuclease activity but not by a pnpA-encoding mutant PNPase without exonuclease activity. Therefore, loss of PNPase exonuclease activity suppressed the cvfA-deleted mutant phenotype. Purified PNPase efficiently degraded RNA with 2′,3′-cyclic phosphate at the 3′ terminus (2′,3′-cyclic RNA), but it inefficiently degraded 3′-phosphorylated RNA. These findings indicate that 3′-phosphorylated RNA production from 2′,3′-cyclic RNA by CvfA prevents RNA degradation by PNPase and contributes to the expression of agr and hemolysin genes. We speculate that in the cvfA-deleted mutant, 2′,3′-cyclic RNA is not converted to the 3′-phosphorylated form and is efficiently degraded by PNPase, resulting in the loss of RNA essential for expressing agr and hemolysin genes, whereas in the cvfA/pnpA double-disrupted mutant, 2′,3′-cyclic RNA is not degraded by PNPase, leading to hemolysin production. These findings suggest that CvfA and PNPase competitively regulate RNA degradation essential for S. aureus virulence.     

In Vivo 23Na Nuclear Magnetic Resonance Study of Maintenance of a Sodium Gradient in the Ruminal Bacterium Fibrobacter succinogenes S85

Sodium gradients (ΔpNa) were measured in resting cells of Fibrobacter succinogenes by in vivo ²³Na nuclear magnetic resonance using Tm(DOTP)⁵⁻ [thulium(III) 1,4,7,10-tetraazacyclododecane-N′,N′′,N′′′-tetramethylenephosphonate] as the shift reagent. This bacterium was able to maintain a ΔpNa of −55 to −40 mV for extracellular sodium concentrations ranging from 30 to 200 mM. Depletion of Na⁺ ions during the washing steps led to irreversible damage (modification of glucose metabolism and inability to maintain a sodium gradient).     

Evidence that DNase I hypersensitive site 5 of the human beta-globin locus control region functions as a chromosomal insulator in transgenic mice

We have previously reported that DNase I hypersensitive site 5 (5′HS5) of the human β-globin locus control region functions as a chromatin insulator in stable transfection assays. In this report we show that a 3.2 kb DNA fragment containing the entire 5′HS5 region can protect a position-sensitive ^Aγ-globin gene against position effects in transgenic mice. Bracketing is required for function of 5′HS5 as an insulator. The 5′HS5 insulator operates in adult as well as in embryonic murine erythroid cells. The insulator has no significant stimulatory effects of its own. These results indicate that 5′HS5 can function as a chromatin insulator in vivo.     

Inhibition of translation by IFIT family members is determined by their ability to interact selectively with the 5′-terminal regions of cap0-, cap1- and 5′ppp- mRNAs

Ribosomal recruitment of cellular mRNAs depends on binding of eIF4F to the mRNA’s 5′-terminal ‘cap’. The minimal ‘cap0’ consists of N7-methylguanosine linked to the first nucleotide via a 5′-5′ triphosphate (ppp) bridge. Cap0 is further modified by 2′-O-methylation of the next two riboses, yielding ‘cap1’ (m7GpppNmN) and ‘cap2’ (m7GpppNmNm). However, some viral RNAs lack 2′-O-methylation, whereas others contain only ppp- at their 5′-end. Interferon-induced proteins with tetratricopeptide repeats (IFITs) are highly expressed effectors of innate immunity that inhibit viral replication by incompletely understood mechanisms. Here, we investigated the ability of IFIT family members to interact with cap1-, cap0- and 5′ppp- mRNAs and inhibit their translation. IFIT1 and IFIT1B showed very high affinity to cap-proximal regions of cap0-mRNAs (K_1/2,app ∼9 to 23 nM). The 2′-O-methylation abrogated IFIT1/mRNA interaction, whereas IFIT1B retained the ability to bind cap1-mRNA, albeit with reduced affinity (K_1/2,app ∼450 nM). The 5′-terminal regions of 5′ppp-mRNAs were recognized by IFIT5 (K_1/2,app ∼400 nM). The activity of individual IFITs in inhibiting initiation on a specific mRNA was determined by their ability to interact with its 5′-terminal region: IFIT1 and IFIT1B efficiently outcompeted eIF4F and abrogated initiation on cap0-mRNAs, whereas inhibition on cap1- and 5′ppp- mRNAs by IFIT1B and IFIT5 was weaker and required higher protein concentrations.     

Protospacer Adjacent Motif (PAM)-Distal Sequences Engage CRISPR Cas9 DNA Target Cleavage

The clustered regularly interspaced short palindromic repeat (CRISPR)-associated enzyme Cas9 is an RNA-guided nuclease that has been widely adapted for genome editing in eukaryotic cells. However, the in vivo target specificity of Cas9 is poorly understood and most studies rely on in silico predictions to define the potential off-target editing spectrum. Using chromatin immunoprecipitation followed by sequencing (ChIP-seq), we delineate the genome-wide binding panorama of catalytically inactive Cas9 directed by two different single guide (sg) RNAs targeting the Trp53 locus. Cas9:sgRNA complexes are able to load onto multiple sites with short seed regions adjacent to ^5′NGG^3′ protospacer adjacent motifs (PAM). Yet among 43 ChIP-seq sites harboring seed regions analyzed for mutational status, we find editing only at the intended on-target locus and one off-target site. In vitro analysis of target site recognition revealed that interactions between the 5′ end of the guide and PAM-distal target sequences are necessary to efficiently engage Cas9 nucleolytic activity, providing an explanation for why off-target editing is significantly lower than expected from ChIP-seq data.     

Microbial Transformations of Natural Antitumor Agents: Products of Rotenone and Dihydrorotenone Transformation by Cunninghamella blakesleeana

Various species of Absidia, Aspergillus, Cunninghamella, Trichothecium, Penicillium, and Phanerochaete were found to transform rotenone to one or more metabolites. Two biotransformation products were isolated from a preparative-scale incubation of rotenone with Cunninghamella blakesleeana and identified as 1′,2′-dihydro-1′,2′-dihydroxyrotenone and 3′-hydroxyrotenone (amorphigenin). The catalytic reduction of the isopropylene side chain of rotenone resulted in the formation of 1′,2′-dihydrorotenone. The latter was transformed by C. blakesleeana to 2′-hydroxy-1′,2′-dihydrorotenone.     

Intertwined pathways for Argonaute-mediated microRNA biogenesis in Drosophila

Although Dicer is essential for general microRNA (miRNA) biogenesis, vertebrate mir-451 is Dicer independent. Instead, its short pre-miRNA hairpin is ‘sliced’ by Ago2, then 3′-resected into mature miRNAs. Here, we show that Drosophila cells and animals generate functional small RNAs from mir-451-type precursors. However, their bulk maturation arrests as Ago-cleaved pre-miRNAs, which mostly associate with the RNAi effector AGO2. Routing of pre-mir-451 hairpins to the miRNA effector AGO1 was inhibited by Dicer-1 and its partner Loqs. Loss of these miRNA factors promoted association of pre-mir-451 with AGO1, which sliced them and permitted maturation into ∼23–26 nt products. The difference was due to the 3′ modification of single-stranded species in AGO2 by Hen1 methyltransferase, whose depletion permitted 3′ trimming of Ago-cleaved pre-miRNAs in AGO2. Surprisingly, Nibbler, a 3′–5′ exoribonuclease that trims ‘long’ mature miRNAs in AGO1, antagonized miR-451 processing. We used an in vitro reconstitution assay to identify a soluble, EDTA-sensitive activity that resects sliced pre-miRNAs in AGO1 complexes. Finally, we use deep sequencing to show that depletion of dicer-1 increases the diversity of small RNAs in AGO1, including some candidate mir-451-like loci. Altogether, we document unexpected aspects of miRNA biogenesis and Ago sorting, and provide insights into maturation of Argonaute-cleaved miRNA substrates.     

Characterization of the 2',3' cyclic phosphodiesterase activities of Clostridium thermocellum polynucleotide kinase-phosphatase and bacteriophage lambda phosphatase

Clostridium thermocellum polynucleotide kinase-phosphatase (CthPnkp) catalyzes 5′ and 3′ end-healing reactions that prepare broken RNA termini for sealing by RNA ligase. The central phosphatase domain of CthPnkp belongs to the dinuclear metallophosphoesterase superfamily exemplified by bacteriophage λ phosphatase (λ-Pase). CthPnkp is a Ni²⁺/Mn²⁺-dependent phosphodiesterase-monoesterase, active on nucleotide and non-nucleotide substrates, that can be transformed toward narrower metal and substrate specificities via mutations of the active site. Here we characterize the Mn²⁺-dependent 2′,3′ cyclic nucleotide phosphodiesterase activity of CthPnkp, the reaction most relevant to RNA repair pathways. We find that CthPnkp prefers a 2′,3′ cyclic phosphate to a 3′,5′ cyclic phosphate. A single H189D mutation imposes strict specificity for a 2′,3′ cyclic phosphate, which is cleaved to form a single 2′-NMP product. Analysis of the cyclic phosphodiesterase activities of mutated CthPnkp enzymes illuminates the active site and the structural features that affect substrate affinity and k_cat. We also characterize a previously unrecognized phosphodiesterase activity of λ-Pase, which catalyzes hydrolysis of bis-p-nitrophenyl phosphate. λ-Pase also has cyclic phosphodiesterase activity with nucleoside 2′,3′ cyclic phosphates, which it hydrolyzes to yield a mixture of 2′-NMP and 3′-NMP products. We discuss our results in light of available structural and functional data for other phosphodiesterase members of the binuclear metallophosphoesterase family and draw inferences about how differences in active site composition influence catalytic repertoire.     

Role of the nucleotide excision repair gene ERCC1 in formation of recombination-dependent rearrangements in mammalian cells

Spontaneous recombination between direct repeats at the adenine phosphoribosyltransferase (APRT) locus in ERCC1-deficient cells generates a high frequency of rearrangements that are dependent on the process of homologous recombination, suggesting that rearrangements are formed by misprocessing of recombination intermediates. Given the specificity of the structure-specific Ercc1/Xpf endonuclease, two potential recombination intermediates are substrates for misprocessing in ERCC1^– cells: heteroduplex loops and heteroduplex intermediates with non-homologous 3′ tails. To investigate the roles of each, we constructed repeats that would yield no heteroduplex loops during spontaneous recombination or that would yield two non-homologous 3′ tails after treatment with the rare-cutting endonuclease I-SceI. Our results indicate that misprocessing of heteroduplex loops is not the major source of recombination-dependent rearrangements in ERCC1-deficient cells. Our results also suggest that the Ercc1/Xpf endonuclease is required for efficient removal of non-homologous 3′ tails, like its Rad1/Rad10 counterpart in yeast. Thus, it is likely that misprocessing of non-homologous 3′ tails is the primary source of recombination-dependent rearrangements in mammalian cells. We also find an unexpected effect of ERCC1 deficiency on I-SceI-stimulated rearrangements, which are not dependent on homologous recombination, suggesting that the ERCC1 gene product may play a role in generating the rearrangements that arise after I-SceI-induced double-strand breaks.