Long RNA molecules are at the core of gene regulation across all kingdoms of life, while also serving as genomes in RNA viruses. Few studies have addressed the basic physical properties of long single-stranded RNAs. Long RNAs with nonrepeating sequences usually adopt highly ramified secondary structures and are better described as branched polymers. To test whether a branched polymer model can estimate the overall sizes of large RNAs, we employed fluorescence correlation spectroscopy to examine the hydrodynamic radii of a broad spectrum of biologically important RNAs, ranging from viral genomes to long noncoding regulatory RNAs. The relative sizes of long RNAs measured at low ionic strength correspond well to those predicted by two theoretical approaches that treat the effective branching associated with secondary structure formation—one employing the Kramers theorem for calculating radii of gyration, and the other featuring the metric of maximum ladder distance. Upon addition of multivalent cations, most RNAs are found to be compacted as compared with their original, low ionic-strength sizes. These results suggest that sizes of long RNA molecules are determined by the branching pattern of their secondary structures. We also experimentally validate the proposed computational approaches for estimating hydrodynamic radii of single-stranded RNAs, which use generic RNA structure prediction tools and thus can be universally applied to a wide range of long RNAs. Highly structured RNAs are described by a collapsed polymer chain model with ν close to 0.33, also applicable to other compact biopolymers such as globular proteins  and single-stranded (ss) homopolymeric nucleic acids, both of which behave as linear polymers. Coarse-grained properties of long dsRNAs are well described by semiflexible polymer models such as the wormlike chain , which oproteins , 27.In contrast, due to extensive intramolecular basepairing arising from Watson-Crick complementarity of nucleotides separated by long distances along the chain contour, long ssRNAs fold into effectively branched structures with short duplex regions emanating from single-stranded loops  Fig. 1)Fig. 1). Rh) of a wide range of biologically relevant long RNA molecules at low nanomolar concentration using fluorescence correlation spectroscopy (FCS). The sizes compare well with those predicted by two ensemble averaging methods that take into account the sequence-dependent effective branching of long RNAs. Furthermore, this correlation holds even in the presence of polyvalent cations that enhance tertiary interactions and result in measurable compaction of RNAs, suggesting that these polymer theory-based methods can successfully predict sizes of long RNA molecules under a variety of conditions. Both methods are based on generic RNA structure prediction algorithms and, accordingly, would be widely applicable to other long RNAs with known sequences.Here we examine the sizes , as described in Borodavka et al. .MS2 phage RNA as well as the 3′ and 5′-end fragments of MS2 phage RNAs were transcribed as described in Borodavka et al. . The tema et al. . The XenAmp construct (XhoI restriction site to the 3′-end. Further PCR product purification and cloning into pSMART HCAmp vector were performed the same way as described above for the other DNA templates. Templates for transcription of 16S rRNA and 23S rRNA (16SrRNA_ pSMART_HCAmp and 23SrRNA_ pSMART_HCAmp) were produced by cloning the corresponding genes using genomic DNA extracted from E. coli BL21 cells. The primer pairs 16S_F1/16S_R1 and 23S_F1/23S_R1 (CP001665.1) and region 228583-231490  of the BL21 DE3 E. coli genome. The resulting PCR products corresponding to the 16S and 23R rRNA-coding regions were used as templates for a second round of PCR amplification using 5′-phosphorylated primers 16S_F2/16S_R2 and 23S_F2/23S_R2 (DraI (16S rRNA DNA) and HindIII (23S rRNA DNA) restriction sites at their respective 3′-ends. Further PCR product purification via agarose gel electrophoresis and subsequent cloning into a pSMART HCAmp vector were performed as described above for other DNA templates. The resulting DNA constructs for in vitro transcription of 16S and 23S rRNAs are 16SrRNA_pSMART HCAmp and 23SrRNA_pSMART HCAmp  were used for transformation with the ligated products, the resulting transformants were PCR-screened, and the positive clones were verified by DNA sequencing. The resulting construct HOTAIR_pSMART HCAmp , following the manufacturer’s protocol. Primers NRON_F and NRON_R  were useAmp  was usedSeveral DNA constructs for in vitro transcription were generously provided upon request by various research groups. The DNA template for production of the STNV-C genomic RNA was a gift from Dr. Robert Coutts . DNA conμL of sterile nuclease-free water. MS2-derived RNAs were 3′-end labeled while all others were 5′-end amine-modified RNAs produced by incorporation of amino-GMP and fluorescently labeled as described in Borodavka et al.  following the manufacturer’s protocol. RNAs were purified using RNeasy mini kit  following the manufacturer’s protocol, except for the fluorescently labeled RNAs. In those samples the RNA-loaded column was washed four times with 80% (v/v) ethanol before elution with 30 a et al. . All RNAμW. The immersion oil objective  was used together with low autofluorescence immersion oil . The focus position was adjusted to 20 μm from the coverslip inner surface and precisely maintained by a piezoelectric feedback loop . The photon count was recorded and analyzed by an ALV-5000 multiple tau digital correlator  used in a single channel mode. Multiple runs of up to 100 autocorrelation functions with acquisition scan time of 30 s each were recorded for each of the samples using ALV-correlator software . Calibration of the confocal volume was performed by measuring the diffusion time of AF488-SDP dye (1 nM in RNA measurement buffer) before each data set collection. FCS data were analyzed by nonlinear least-squares fitting with a single-component diffusion model autocorrelation function corrected for the triplet state  power was set to 65 et state  using MAN-morpholino)-propanesulfonic acid (MOPS), 10 mM KOH buffer, pH 7.0 with 1 mM dithiothreitol at 25°C. RNA condensation experiments were performed in the presence of divalent  and trivalent  cations, added to the 0.5–2 nM RNA samples before FCS measurements.RNA measurements were performed with 0.5–2 nM RNA in RNase-free 20 mM 3-(A). To calculate the size of the resulting branched polymer (B), two approaches can be used. The first method makes use of the Kramers theorem  (eff) proportional to the MLD. Treating the resulting linear polymer as an ideal chain then givesb corresponds to the average length of a duplex (≈5 bp) , we use the Boltzmann-averaged MLD (denoted 〈MLD〉), derived from an ensemble of RNA structures generated by prediction algorithms implemented in RNAfold  , 28, 47 bp units . Because RNAfold . Earlierstimated , 42.MLD〉 values were computed from the 100 lowest-energy secondary structures (Rg were estimated using (see Eq. 3) the relationship Rg ∼ (〈MLD〉)1/2. Each tree graph representation was derived from a dot-bracket representation of the secondary structure . The Rg was calculated from the tree graph by treating the vertices as perfectly flexible joints and the edges as rigid phantom bonds , and using the Kramers theorem (Rg of a branched polymer (tree graph) was calculated byL bonds the product of L1(j) and L-L1(j), the numbers of vertices on either side of the jth bond (see B). The square root of Eq. 4 yields the radius of gyration of the tree graph  for long RNAs, ranging from 600 to >9000 nucleotides in length.Due to their large sizes and high conformational flexibility, little is known about the structural organization and physical properties of long RNAs. Some of them, such as viral positive-sense ssRNA genomes, adopt compact conformations as part of their function and facilitate packaging into the confined space of icosahedral viral capsids . Likewis2+) and trivalent  cations. The latter conditions promote formation of tertiary structures  and low ionic strength , and measure sizes of RNA molecules by FCS. In contrast to other ensemble solution techniques , the dilute conditions minimize aggregation due to intermolecular basepairing, which has previously been shown to result in an overestimation of sizes . Furtherructures , 53.MLD〉 values and measured hydrodynamic radii for a range of long RNA molecules examined by FCS. Due to the low RNA concentrations and ionic strength conditions used here , aggregation and tertiary structure formation are unlikely, so that the effects of branching due to secondary structure can be accentuated and be probed directly under close-to-isolated molecule (infinite-dilution) conditions. We note that while the measured Rh broadly increases with the length, the rise significantly deviates from the monotonic behavior expected for the simple scaling laws . This result suggests that linear polymer scaling laws (Eq. 1) are not appropriate to describe long ssRNA, which is an effectively branched polymer. Instead, essential coarse-grained features of their sequences need to be taken into account.s Eq. 1, A. This Rg values based either on the MLD  or the Kramers theorem (C). This result is consistent with most RNA molecules adopting branched structures in which the MLD largely determines the overall size (C) with that of MS2 phage genomic RNA (D). The experimentally determined secondary structure pattern of s11 is significantly less branched than that of the typical prediction for MS2. Furthermore, this is reflected in the relatively large MLD and hydrodynamic size of s11, comparable to that of Ef2 mRNA, which is three times the length. This demonstrates that the relatively simple MLD description can capture the essence of coarse-grained RNA structure, and yields quantitative predictions based on the RNA sequence alone.To account for sequence variations, basepairing, and the resulting branching, we estimate branching patterns using the output of secondary structure algorithms (RNAfold) and ensemble average over the low energy structures. The measured hydrodynamic radii are in good agreement with the theoretical estimates of the  the MLD B, Eq. 3 theorem C. This all size , 42. Thiall size C with tomic RNA D. The e2+, spermidine3+, spermine4+) and/or association with RNA-binding proteins (2+) or trivalent cations , the measured Rh decreases for most RNAs, consistent with compaction driven by electrostatic screening and neutralization. Rh before and after the addition of multivalent cations. The Rh values cluster along the line with the slope between 0.7 and 0.8, indicating that on average the RNAs undergo a 20–25% size compaction compared to their original Rh. As a consequence, the proportionality between Rh and predicted size holds for most of the RNAs even after addition of polyvalent cations. However, there are few RNAs that either fail to further compact (RV s11 No. 1 and Ef2 mRNA No. 6 in Rh change is insignificant at confidence level 90%) or the compaction is more prominent in comparison with other RNAs examined .Further compaction of RNA molecules and the formation of tertiary structure require di- and polyvalent cations , yielding a Rg ∼ 9.3 value that agrees better with the experimental Rh . On the other hand, when secondary structure determination (or prediction) is ambiguous, experimental size measurements by FCS can be used for selecting those structures with MLDs compatible with the experimentally determined hydrodynamic radii. In addition to the MLD prediction limitations, HCV RNA is compacted twofold in the presence of multivalent ions, i.e., to a much higher degree than other RNAs examined here (20–25% reduction), highlighting the importance of repulsive electrostatic interactions at low salt and formation of tertiary contacts stabilized by multivalent cations, not accounted for in our approach.The quantitative relation between the experimental STMV RNA , 55. SucV RNA B;  17, 28), 28Rh anB and C, No. 7, Rh ∼ 17.8 nm). This discrepancy likely reflects the presence of distinct domains within 16S rRNA that make the protein-free 16S rRNA relatively large (measured Rg ∼11.4 nm). Only upon binding of multiple ribosomal proteins does it undergoes gradual compaction to its fully folded functional state (Rg ∼ 7 nm)  , 57.Ef2 mRNA is an example of overestimated size , No. 6 aMLD〉 and the experimental hydrodynamic radius, we generated a scrambled s11 RNA sequence, as described in MLD〉 and is reflected in a concomitant decrease (significant at 99% confidence level) of experimentally measured Rh (A and B).To further test the observed correlation between 〈sured Rh , A and BA) are comparable in length to lncRNAs (Nos. 8 and 11) and the protein-free ribosomal RNA (No. 12), they appear to be smaller in size (see 2–4 kb region in A). However, the Ef2 mRNA transcript (No. 6) is yet even more compact than the comparatively short viral RNAs , suggesting that although there might be evolutionary pressure on genomes of ssRNA viruses to fold into more compact structures (C) to minimize the formation of nonspecific intersegment RNA-RNA contacts, while enabling formation of specific interactions facilitated by the viral RNA chaperones  can be estimated using coarse-grained theoretical predictions, even in the presence of multivalent ions stabilizing tertiary contacts. Because the theoretical approaches used here treat exclusively the branching patterns associated with the RNA secondary structures, our results provide experimental evidence that the overall sizes of long RNAs are determined predominantly by their secondary structure branching patterns . The eff2+  via formA.B., S.W.S., P.G.S., W.M.G., A.B.-S., and R.T. designed research; A.B. and S.W.S. performed research; A.B.-S. and R.T. contributed analytic tools; and A.B., S.W.S., P.G.S., W.M.G., A.B.-S., and R.T. analyzed data and wrote the article.


Spermatozoa of virtually all species can spontaneously take up exogenous DNA or RNA molecules and internalize them into nuclei. In this article I review evidence for a key role of a reverse transcriptase (RT) activity, encoded by LINE-1 retrotransposons, in the fate of the internalized nucleic acid molecules and their implication in transgenerational inheritance. LINE-1-derived RT, present in sperm heads, can reverse-transcribe the internalized molecules in cDNA copies: exogenous RNA is reverse-transcribed in a one-step reaction, whereas DNA is first transcribed into RNA and subsequently reverse-transcribed. Both RNA and cDNA molecules can be delivered from sperm cells to oocytes at fertilization, further propagated throughout embryogenesis and inherited in a non-Mendelian fashion in tissues of adult animals. The reverse-transcribed sequences are extrachromosomal, low-abundance, and mosaic distributed in tissues of adult individuals, where they are variably expressed. These “retrogenes” are transcriptionally competent and induce novel phenotypic traits in animals. Growing evidence indicate that cancer tissues produce DNA- and RNA-containing exosomes. We recently found that these exosomes are released in the bloodstream and eventually taken up into epididymal spermatozoa, consistent with the emerging view that a transgenerational flow of extrachromosomal RNA connects soma to germline and, further, to next generation embryos. Spermatozoa play a crucial bridging role in this process: they act as collectors of somatic information and as delivering vectors to the next generation. On the whole, this phenomenon is compatible with a Lamarckian-type view and closely resembles Darwinian pangenesis. It is a well-established notion that mature spermatozoa have the spontaneous ability to take up exogenous DNA molecules and to internalize them in their nuclei  nor the mechanism of action of embryonic RT are yet fully clarified, emerging data suggest that RT controls the biogenesis of miRNAs, a class of RNA that is globally, yet transiently, suppressed in early embryogenesis , concomiThe link between LINE-1-encoded RT and miRNA biogenesis has been investigated in some depth in cancer cells. In striking analogy with early embryos, RT is also highly expressed in most cancer types from very early stages (reviewed by In recapitulating the aspects discussed so far a framework is beginning to emerge: (1) RNA-containing nanovesicles are released from somatic tissues into the bloodstream; (2) Epididymal spermatozoa take up nanovesicles and internalize them in their nuclei; (3) The internalized RNA molecules are processed and their copy number is amplified, via the RT/DNA-dependent RNA polymerase interplay; (4) Somatic RNAs, or their cDNA copies, are delivered from sperm to embryos at fertilization.The first three steps continuously renew the RNA storage in sperm heads. The last one, i.e., the delivery of processed somatic RNA to oocytes, can recur at each round of fertilization. Through this process sperm RNA is transmitted from one generation to the next, which can contribute to the embryo fitness and in principle expand the adaptation of the newborn to diverse environmental conditions. It is reasonable to assume that a large proportion of the males living in a same ecological niche and exposed to the same stimuli, produce sperm RNA cargos of similar composition; under constant environmental conditions, these RNA cargo would be continuously delivered to the progeny via fertilization throughout generations. It is not unreasonable to hypothesize that, in the long run , the sperm RNAs promote the “assimilation” of new trait(s), to use Waddington’s concept . In otheThe first (epigenetic) step builds on the established regulatory functions of miRNAs and tsRNAs, which can modulate the expression of relevant genes. It is reasonable to hypothesize that RNAs delivered by sperm cells at fertilization also exert these regulatory functions and remodulate the gene expression profile in early embryos. Consequently, new genetic circuits  become functionally active, or/and pre-existing ones are rewired.Canalized circuits would then reach their final state through targeted retrotransposition events (genetic step). New retrotranspositions can provide additional layers of control, by inserting protein-binding sites , as well as enhancers, promoters, insulators, etc. in new sites within the genome.Figure 1.Thus, a hybrid epigenetic/genetic process drives the remodeling of the embryonic chromatin architecture, as schematized in Figure 1) constitute minimal contributions toward the epigenetic activation of novel canalization(s) by “deepening the canal” , while non-active pathways become shallower . When the cumulative effects of the sperm-delivered RNA overcome the buffering capacity of the embryos, the novel canalized genomic circuit  has the potential to redirect the embryonic ontogenesis and generate phenotypic novelties.The process is seen as progressive in nature, based on the assumption that the “quanta” of sperm-derived regulatory RNAs delivered to the embryo at fertilization  contribute to establish novel regulatory circuits in at least three ways: (i) they provide new protein-binding and regulatory sites; (ii) they contribute new miRNA-coding sequences that expand the overall diversity in the RNA population, and (iii) they stabilize the newly remodeled landscape by fixing the chromatin architecture.Targeted retrotransposition events constitute the genetic component of the model, contributing to the functional reshaping of the embryo regulatory circuits. Targeted insertions . In analogy with Lamarckism, this hypothesis is based on the assumption that extrachromosomal transgenerational inheritance can affect ontogenesis and generate evolutionarily significant, stably acquired variations. Darwinian pangenesis  is the oCS has conceived the model and wrote the manuscript.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Pseudomonas aeruginosa. Our results indicated different rRNA removal efficiency for the three procedures, with the Ribo-Zero kit yielding the highest degree of rRNA depletion, which translated into enhanced enrichment of non-rRNA transcripts and increased depth of RNA-seq coverage. The results indicated that, in addition to improving RNA-seq sensitivity, efficient rRNA removal enhanced detection of low abundance transcripts via qPCR. Finally, we demonstrate that the Ribo-Zero kit also exhibited the highest efficiency when P. aeruginosa/Staphylococcus aureus co-culture RNA samples were tested.Global transcriptomic analysis via RNA-seq is often hampered by the high abundance of ribosomal (r)RNA in bacterial cells. To remove rRNA and enrich coding sequences, subtractive hybridization procedures have become the approach of choice prior to RNA-seq, with their efficiency varying in a manner dependent on sample type and composition. Yet, despite an increasing number of RNA-seq studies, comparative evaluation of bacterial rRNA depletion methods has remained limited. Moreover, no such study has utilized RNA derived from bacterial biofilms, which have potentially higher rRNA:mRNA ratios and higher rRNA carryover during RNA-seq analysis. Presently, we evaluated the efficiency of three subtractive hybridization-based kits in depleting rRNA from samples derived from biofilm, as well as planktonic cells of the opportunistic human pathogen  It is increasingly preferred over microarrays for its greater dynamic range, independence from probes, and alternative applications including discovery of new transcripts, mapping of transcription start sites, and sequencing of novel small RNAs. Despite reduced size, prokaryotic transcriptomes are nonetheless complex and provide unique analysis challenges. Bacterial transcriptomes contain protein-coding RNAs, transfer (t)RNAs, transfer messenger (tm)RNA, small regulatory (s)RNAs, and ribosomal (r)RNAs. The rRNA accounts for more than 85% of the prokaryotic cellular RNA content2569Studies of regulatory networks in bacteria often utilize analyses of gene expression. High-throughput The subtractive hybridization procedure, available as several commercial kits, has become the most common choice for rRNA depletion prior to prokaryotic RNA-seq analyses. Subtractive hybridization kits, such as the Ambion MICROBExpress™ Bacterial mRNA Enrichment Kit, which until recently has been considered to be one of the best and most widely used choices, rely on oligonucleotide probes to capture 16S and 23S rRNA. Such kits have been used on single-species cultures, multi-species communities, and environmental samples. The efficiency of these methods, however, has varied in a manner dependent on bacterial species and sample composition, with significant carryover of rRNA often observed following RNA-seq data analysis, with up to 50% of reads corresponding to rRNA111211Pseudomonas aeruginosa, an opportunistic human pathogen associated with infections of surgical sites, urinary tract, chronic and burn wounds, and lungs of cystic fibrosis patients, and one of the most frequent colonizers of medical devices. The aims of such studies have included identifying antisense and other small regulatory RNAs, determining the targets of regulatory proteins, assessing antibiotic resistance fitness costs, and analyzing the process of biofilm formation that represents a distinct mode of growth enabling the bacterium to colonize and persist within the infected host1415161718P. aeruginosa rRNA depletion by various methods, however, has not been evaluated. This is especially important considering that previous reports have demonstrated low yields of non-rRNA reads (5–30%) in cDNA libraries following depletion, with the problem being exacerbated in biofilm samples13Pseudomonas aeruginosa” or “biofilms” in combination with the respective kit names reveal similar numbers of projects utilizing the two procedures. Therefore, the present work was assessed the efficiency of three commercially available subtractive hybridization-based rRNA depletion kits: Illumina Ribo-Zero rRNA Removal Kit (Bacteria), Ambion MICROBExpress™ Bacterial mRNA Enrichment Kit and the Life Technologies RiboMinus Transcriptome Isolation Kit, Bacteria.An increasing number of studies have utilized RNA-seq to elucidate the regulatory processes behind growth and pathogenesis of P. aeruginosa biofilm RNA samples, the Ribo-Zero kit outperformed the other two kits, reducing rRNA to less than 1% of all RNA-seq reads, and substantially improved the detection of low abundance transcripts during both RNA-seq and qPCR analyses. The Ribo-Zero kit also exhibited superior rRNA depletion efficiency when P. aeruginosa planktonic and P. aeruginosa/Staphylococcus aureus co-cultures samples were tested.The MICROBExpress procedure employs capture oligonucleotides for 16S and 23S rRNA, which subsequently hybridize to oligonucleotides on derivatized magnetic beads to remove the rRNA. The RiboMinus procedure entails hybridization of 16S and 23S rRNA to rRNA sequence-specific 5′-biotin labeled oligonucleotide probes, which are then removed from the sample together with the bound rRNA using streptavidin-coated magnetic beads. Similarly, the Ribo-Zero kit relies on biotinylated rRNA capture probes, which, following hybridization to the target rRNA molecules, are captured by magnetic beads. In contrast to MICROBExpress and RiboMinus, the RiboZero kit also targets 5S rRNA. We provide evidence that, when tested using unmodified manufacturers’ protocols for the removal of rRNA from P. aeruginosa biofilm samples, we have subjected 4 μg of DNAse-treated RNA isolated form 3-day-old PAO1 biofilms to treatment with the Illumina Ribo-Zero rRNA Removal Kit (Bacteria), Ambion MICROBExpress™ Bacterial mRNA Enrichment Kit and the Life Technologies RiboMinus Transcriptome Isolation Kit, Bacteria. In order to ensure valid comparison of the rRNA depletion methods, the procedures were performed on aliquots of the same input RNA sample, with the comparisons repeated using biological triplicates. Following rRNA depletion and ethanol/acetate precipitation, the resulting RNA was subjected to quantitative and qualitative analysis to determine yield and RNA species size distribution. Qubit fluorimetric analysis revealed the highest yield for the MICROBExpress-treated sample, with approximately 400 ng or 10% of the input RNA recovered  corresponds to the amplification cycle when the product fluorescence (i.e. abundance) exceeds the background level, with lower Cq values corresponding to higher amounts of template, we accordingly expected the MICROBExpress sample to exhibit significantly higher Cq values than the input RNA, but lower Cq readings than the RiboMinus and Ribo-Zero samples, with Ribo-Zero demonstrating the highest Cq values. In agreement with this, the Cq values for 16S and 23S rRNA exceeded 20 for the Ribo-Zero samples, while products were detectable following 10–12 qPCR cycles in the MICROBExpress and RiboMinus samples PCR. Following reverse transcriptase (RT)-PCR using the input untreated RNA and the three rRNA-depleted samples, the resulting cDNA was subjected to qPCR analysis using primers for zer data , we anti samples . Surprisof 10–12 . Consideof 10–12 ,C.q values and lower copy numbers for 5S rRNA in the Ribo-Zero, but not the RiboMinus or the MICROBExpress samples relative to the input RNA  compared to those detected in the MICROBExpress and RiboMinus samples  and RiboMinus (58.3 ± 1.8%) samples than for the Ribo-Zero sample (2.9 ± 0.7%) . As four samples . While t samples . The RNA samples . TogetheP. aeruginosa genome. Concurrently with lowering the rRNA reads number, the Ribo-Zero kit significantly increased the number of reads mapping to non-rRNA features including protein-coding transcripts, non-coding RNAs (ncRNAs), and tRNAs relative to the MICROBExpress and RiboMinus samples  SsrA, a bacterial RNA molecule with dual tRNA-like and mRNA-like properties, which was found to be the most abundant mRNA species in all three samples tested. Relative SsrA levels increased from 7–8% in the MICROBExpress or RiboMinus samples to close to 40% of all counts in the Ribo-Zero sample ,E. DespiR2 > 0.995), suggesting a roughly five-fold enrichment of non-rRNA reads in the Ribo-Zero sample  than the other two procedures  when all transcripts were considered , but was still below that observed for the Ribo-Zero replicates (R2 = 0.99921).Moreover, comparison of the RNA-seq counts of biological duplicates for each of the rRNA depletion kits revealed higher correlation of cDNA libraries produced following treatment with the Ribo-Zero kit  in flasks at 220 rpm. Biofilms were grown in 20-fold diluted LB medium using a continuous flow tube reactor system  with an inner surface area of 25 cm2 at a flow rate of 0.1 ml/min288 cells according to the manufacturer’s protocol, with the following modifications. For biofilm experiments, P. aeruginosa cells were treated with 400 μg/mL lysozyme in TE for 5 min at room temperature prior to RNeasy RNA isolation. For planktonic and co-culture experiments, cells were treated with 400 μg/mL lysozyme and 100 μg/mL lysostaphin in TE for 30 min at 37 °C prior to RNeasy RNA isolation. The RNeasy elution step was also modified to use 75 uL of 1X TE (10 mM Tris-HCl 1 mM EDTA) buffer to ensure downstream stability of the RNA during the RNA-seq library preparation. The RNA was subsequently subjected to DNase treatment  according to the manufacturer’s protocol. RNA quantity and quality were assessed as described below.Samples of biofilm cells were collected as described above into 3 mL of RNAprotect Bacteria Reagent (Qiagen). Following 10-minute room temperature incubation, mRNA isolation  was carried out using approximately 3 × 10Where indicated, RNA or DNA was quantified using Qubit RNA HS Assay Kit and Qubit dsDNA HS Assay Kit (Life Technologies), respectively, on the Qubit 2.0 Fluorometer (Life Technologies). In addition, where indicated, fragment size distribution of RNA or DNA samples was assessed on the Agilent 2100 BioAnalyzer using the Agilent RNA 6000 Pico Kit or Agilent High Sensitivity DNA Kit (Agilent Technologies), respectively. The BioAnalyzer was also used to assess the quality of DNAse-treated total RNA, with only RNA samples exhibiting an RNA integrity number (RIN) >9 selected for downstream processingA total of 4 ug RNA was subjected to rRNA depletion using the Illumina Ribo-Zero rRNA Removal Kit (Bacteria), Ambion MICROBExpress™ Bacterial mRNA Enrichment Kit and the Life Technologies RiboMinus Transcriptome Isolation Kit, Bacteria, according to the manufacturers’ protocols. RNA quantity and quality post-depletion was assessed using the Agilent 2100 Bioanalyzer instrument as described above. In order to ensure valid comparison of the rRNA depletion methods, the procedures were performed on aliquots of the same input RNA sample, with the comparisons repeated using biological triplicates.http://www.ncbi.nlm.nih.gov/Traces/sra/sra.cgi) with accession number SRP072237 under BioProject PRJNA316121.Whole transcriptome RNA-Seq was carried out using the ION Torrent Personal Genome System (Life Technologies). Following rRNA depletion and BioAnalyzer quality assessment performed as discussed above, the cDNA libraries was constructed using the Ion Total RNA-Seq kit v2 (Life technologies) according to the manufacturer’s protocol. Following BioAnalyzer size distribution assessment and Qubit quantitation, the cDNA libraries were used to prepare sequencing templates using the Ion PGM Template OT2 200 kit on the Ion OneTouch™2 instrument (Life Technologies), followed by Ion Sphere™ Quality Control Kit assessment and template-positive Ion Sphere™ Particle (ISP) enrichment using the Ion OneTouch™ ES instrument. Sequencing was carried out using Ion Sequencing 200 Kit v2 and the Ion 318 Chip on the Ion Torrent PGM sequencing platform (Life Technologies). For each rRNA depletion method, RNA-sequencing was performed in duplicate using distinct biological replicates. The raw sequencing data are available in the NCBI short read archive (http://www.bioinformatics.bbsrc.ac.uk/projects/fastqc/]) was used to generate statistics and evaluate the quality of the generated RNA-seq reads. Bowtie 2P. aeruginosa PAO1 reference genomeFastQC  with oligonucleotides listed in mreB, brlR, and PA0701 transcript copy numbers versus Cq values, as calculated using the CFX Manager Software (BioRad), qPCR was also performed using serial 10-fold dilutions of P. aeruginosa genomic DNA. Subsequently, the standard curves were used for calculations of template copy numbers in the indicated samples. Melting curve analyses were employed to verify specific single product amplification. The qRT-PCR analysis was performed using biological triplicates.cDNA synthesis was performed using the iScript™ cDNA Synthesis Kit (BioRad), with 10 ng or 1 μg of total RNA or 10 ng of rRNA-depleted RNA  used as the template. Subsequently, qPCR for indicated transcripts was performed using the BioRad CFX Connect Real-Time PCR Detection System and SsoAdvanced™ SYBRHow to cite this article: Petrova, O. E. et al. Comparative evaluation of rRNA depletion procedures for the improved analysis of bacterial biofilm and mixed pathogen culture transcriptomes. Sci. Rep.7, 41114; doi: 10.1038/srep41114 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The last few years has witnessed the emergence of a novel class of long non-coding RNA known as circular RNA (circRNA). These molecules are characterised by their circularity formed through the back splicing of 3’ and 5’ ends of transcript segments produced by one or more of its exons. CircRNAs function as transcriptional modulators, microRNA regulators, as well as template for translation. In our current study, we profiled circRNA expression in post-mortem brain samples from Schizophrenia (SZ) and control subjects using next-generation sequencing technology to discover the association of these novel RNA molecules with the pathogenesis of SZ.Total RNA from cerebral cortex (BA46) of 17 SZ patients and 18 healthy controls were subjected to ribosomal RNA depletion and then RNase R treatment to further deplete linear RNA and enrich for exonuclease resistant circRNA transcripts. Sequencing libraries were constructed using Illumina TruSeq RNA Library Prep Kit (LT) (150 cycles) and sequenced by an Illumina NexSeq500. Sequencing data was analysed by the CIRCexplorer2 pipeline to identify circRNA transcripts. To validate the sequencing findings, real-time PCR was performed using outward primers sets designed to specifically amplify circular transcripts.We discovered a large number of distinct circRNAs , many of which were highly expressed throughout the cohort. Surprisingly, a large proportion (52%) of the identified circRNAs sequences were novel or not previously reported. Differential expression analysis suggested that there was substantial alteration in circRNA expression in SZ. More than two thirds of these molecules displayed decreased expression, whereas the remainder were upregulated. Functional annotation of the host genes was significantly enrichment for terms-related to neurobiology and neurocognitive impairment including clusters such as neurogenesis, differentiation and synapse. Many of these circRNAs were also predicted to interact with miRNAs, supporting a potential miRNA sponging function for these circRNA.RNA sequencing in the human postmortem DLPFC revealed dysregulation of circRNA expression in schizophrenia. This alteration was characterized by a substantial decrease in circRNA expression in the disorder. Bioinformatic predictions of circRNA interaction suggest they function as miRNA regulators and may have a broader role in etiology or pathophysiology of the disorder.


N1-methyladenosine (m1A), which is typically found at position 58 in the tRNA T-loop of eukaryotes, functions to stabilize tRNA tertiary structure 5C) in tRNA can stabilize the secondary structure and maintain the anticodon stem-loop conformation Ever since the first RNA nucleoside modification was characterized in 1957 N6-methyladenosine (m6A) was revealed to be also present in mRNA several decades ago 6A modification in mRNA has been shown to play roles in gene expression regulation through multiple pathways, including affecting the stability, translation, splicing, and secondary structure of RNA molecules 6A, several other modifications, including N6, 2′-O-dimethyladenosine (m6Am), inosine (I), m5C, Ψ, m1A, and 5-hydroxylmethylcytidine (hm5C), have also been revealed and mapped in mRNA and long ncRNA (lncRNA) 1A-associated translational regulation Interestingly, Studies in the field of epitranscriptomics have revealed the functional importance of RNA modifications, yet many questions remain to be answered. While recent progresses in epitranscriptomics have been summarized in many excellent reviews 5C and m1A for example, most of the modified sites can only be detected with resolutions of wider than 50 nucleotides 1A in position 1322 of 28S rRNA and position 58 of tRNAMet in humans, are modified with high stoichiometry 6A-level and isoform-characterization sequencing (m6A-LAIC-seq). m6A-LAIC-seq is capable of detecting m6A in a quantitative manner e.g., clinical samples). Besides the widely-used NGS-based approaches, the rapidly-evolving third-generation sequencing (TGS) will, hopefully, provide a better approach for transcriptome-wide modification mapping. The single-molecule technologies, including PacBio’s single molecule real-time (SMRT) sequencing and Oxford Nanopore’s nanopore sequencing, have the potentials to directly detect modifications in the original nucleotide molecule Interest in the functional studies of RNA modifications has been revived in recent years. The majority of these studies are highly dependent on the existing transcriptome-wide mapping methods with the utilization of next-generation sequencing (NGS) technologies 6A, Ψ, and m1A, would affect the Watson–Crick base-pairing, hence influencing the local RNA structure Most of the transcriptome-wide modification studies generate a large amount of sequencing data, bioinformatic tools are therefore greatly needed to aid the data analyses. Specifically, RIP-based transcriptome-wide mapping technologies are commonly used in epitranscriptomic studies. However, most of the currently available peak calling algorithms, such as zero-inflated negative binomial algorithm (ZINBA) 6A modification, including regulation on mRNA stability, translation efficiency, and exon inclusion, relied on manipulating the methyltransferases METTL3/METTL14/WTAP 6A were found to be mediated by its readers, mainly the YT521-B homology (YTH) domain-containing proteins 6A, identification of their writer, eraser, and reader proteins would be very important and helpful in exploring their functions, as well as revealing the underlying mechanisms. The initial discovery of m6A readers relies on the RNA pull-down assay using synthesized oligonucleotides containing m6A in vitro incubation of an unmodified RNA oligonucleotide with different fractions from cell extract. Such strategy proved successful in discovering METTL3 , one component of m6A writer de novo through bioinformatic prediction based on the conservation of protein domains involved, followed by the experimental validation using assays like knock-down or overexpression 5C, whose writer could target both mRNA and tRNA, it could be difficult to distinguish the effect caused by the modifications in mRNA or in tRNA. Under such circumstances, assays like photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) or individual-nucleotide resolution CLIP (iCLIP) could be very useful to identify the global targets of a writer.For modification studies, it is important to identify the enzymes responsible for the installation and removal of a modification , as well as the modification binding proteins (“reader”). By manipulating the expression of the writer or eraser (if any) for a modification, we can observe the global change of the modification and further investigate the downstream biological effects. For example, many functional studies of mA comprehensive database for curating and sharing epitranscriptomic data should be established. The National Institutes of Health (NIH) has built the “Roadmap Epigenomics Mapping Consortium” to integrate the public resources for epigenomics studies 6A is the first RNA modification being profiled transcriptome-wide with its writer, eraser, and reader identified, the functional roles of this methylation have received extensive attention in the field. Hopefully, besides m6A, other modifications in mRNA could also share the limelight. In fact, potential functions of a certain type of modification could be hinted by its distribution pattern. Take m1A for example, a prominent feature of highly-enriched distribution pattern in 5′UTRs and start codon has been revealed in both humans and mice 1A may be involved in translational regulation. Besides, the intrinsic property of a modification would also provide us some clues for generating new hypotheses. Modifications occurring in the Watson–Crick edge, such as m5C and m1A, would affect the normal base pairing, hence altering the local RNA structure or causing recoding of the translated peptides 6A has been suggested to connect with cell differentiation and cancer formation Since m6A, m1A, and Ψ, have been identified in MALAT1MALAT1. Along with the development of transcriptome-wide mapping technologies in the foreseeable future , integral studies of the RNA modifications could come into play.The study of epitranscriptome could evolve toward an integral manner that comprehensively considers the effect of multiple types of modifications in the transcriptome. Most of the current studies in epitranscriptome are focused on a single particular modification. However, various modifications in RNA may interplay with each other, forming “networks” to modulate the physiological pathways. For instance, multiple types of modifications, including m6A have brought an initial period of prosperity in the emerging field of epitranscriptomics; in the meantime, several other modifications have also been identified and mapped in mRNAs and lncRNAs. We envision that with more endeavor being made in this field, researchers will get a better understanding of RNA modifications and their functions in a more comprehensive way.Collectively, the functional studies of mThe authors declare no competing financial interests.


Oxytricha trifallax maintains two genomes: a germline genome that is active only during sexual conjugation and a transcriptionally active, somatic genome that derives from the germline via extensive sequence reduction and rearrangement. Previously, we found that long noncoding (lnc) RNA “templates”—telomere-containing, RNA-cached copies of mature chromosomes—provide the information to program the rearrangement process. Here we used a modified RNA-seq approach to conduct the first genome-wide search for endogenous, telomere-to-telomere RNA transcripts. We find that during development, Oxytricha produces long noncoding RNA copies for over 10,000 of its 16,000 somatic chromosomes, consistent with a model in which Oxytricha transmits an RNA-cached copy of its somatic genome to the sexual progeny. Both the primary sequence and expression profile of a somatic chromosome influence the temporal distribution and abundance of individual template RNAs. This suggests that Oxytricha may undergo multiple rounds of DNA rearrangement during development. These observations implicate a complex set of thousands of long RNA molecules in the wiring and maintenance of a highly elaborate somatic genome architecture.The ciliate  Oxytricha trifallax (Oxytricha produces complete RNA copies of thousands of its somatic chromosomes during nuclear differentiation and development.Long noncoding RNAs (lncRNAs), defined as transcripts >200 nt with no protein coding function, were once thought to represent primarily nonfunctional “junk” transcription. However, the discovery of ∼10,000 lncRNA loci in the human genome  and evidrifallax . Here, wOxytricha is a microbial eukaryote with two kinds of nuclei per cell. The smaller micronucleus (MIC) contains the germline, which provides haploid gametic nuclei for sexual conjugation. The larger macronucleus (MAC) contains the somatic genome, which is the source of gene transcription during asexual growth and reproduction. While the micronuclear genome consists of long diploid chromosomes, the MAC genome contains over 16,000 different chromosomes, most of which bear only a single gene and are a median length of 2515 bp (mean 3.2 kb) at a typical copy number of ∼1900n  to mark segments of the MIC genome for retention rather than elimination program chromosomal rearrangements . Oxytricelopment ; RT-PCR elopment . Injectielopment . Point melopment  and, in erations . This suerations .Oxytricha trifallax and the first such survey of telomere-to-telomere RNA transcripts in any eukaryote. We detect the presence of over 10,000 different lncRNAs corresponding to complete MAC chromosomes. These findings support the template RNA-guided model of DNA rearrangement and underscore the importance of long RNAs in the programming and maintenance of Oxytricha’s genome architecture.Here we report the first global survey of template RNAs in Oxytricha strains are combined but before mating begins; 6 h post-mixing, shortly after the first putative template RNAs were detected in Oxytricha MAC genome containing high-confidence chromosomes short enough to be amplified by the PCR step in the sequencing pipeline. Ultimately, we recovered RNA-seq read pairs corresponding to 10,507 different chromosomes, representing more than 2/3 of all completely assembled chromosomes in the somatic genome. This includes RNA-seq reads detected from 3230 chromosomes as early as the time of cell mixing (0 h), much earlier than expected. This set is enriched in chromosomes with a high level of expression during development, and transcription might occasionally begin at or within either telomere, where RNA polymerase localizes  .To investigate why template RNAs were absent for a subset of  motifs) . OverallWe propose that these motifs disrupt transcription of the template RNA and prevent RNA polymerase II  from reaFurthermore, experiments using RT-PCR and gene-specific primers recovered sense, antisense, or both strands corresponding to lncRNA transcripts with a telomere sequence at one end for five (out of five) MAC chromosomes whose template RNAs were absent from our RNA-seq survey  than those whose templates were only detected later in development. Chromosomes whose gene products are required early might assemble first, especially if the cell relies on zygotic transcription from the developing macronucleus while the parental nucleus degrades. On the other hand, chromosomes that bear genes whose products are not required until vegetative growth, i.e., until nuclear differentiation is complete, are less critical and can be rearranged later during development.This variation in individual template RNA levels across development suggests that chromosome rearrangement may occur in waves, rather than all at once. Chromosomes whose templates appear in early development (at zero or 6 h post-mixing of mating types) have significantly higher RNA-seq expression levels during genome rearrangement , although they have fewer scrambled pointers, which require reordering during rearrangement, relative to chromosomes with templates found at all time points surveyed . Those chromosomes whose templates are absent from our RNA-seq data set have a greater number of MDSs than those that are present  as well as more scrambled pointers . Conversely, chromosomes with only a single MDS, which require no rearrangement or IES elimination but do require telomere addition, appear at significantly more time points than those with more than one MDS . Overall, chromosomes that require the greatest number of MDS joining or rearrangement events have the most transiently available template RNAs.In addition, some features of a chromosome's rearrangement map appear to correlate with when and how long its RNA templates are present. Chromosomes whose templates appear in only a single time point have a significantly greater number of pointers (the short direct repeats in the germline where somatic segments recombine) than those present at all time points  to descramble and therefore require longer-lived templates, it is intriguing that such complex chromosomes are produced instead by some of the most transient lncRNAs in our data set. Overall, the heterogeneity of the lncRNA population during development suggests that different somatic chromosomes may differ in their rearrangement pathways, and that the entire population of molecules does not differentiate in lockstep. Such a strategy may reflect the cell's need to properly assemble over 16,000 chromosomes to build its somatic genome. In the future, it would be fruitful to extend the types of single-locus studies of DNA rearrangement pathways in Stylonychia lemnae are also implicated in telomere sequence regulation  using a TRIzol extraction kit (Invitrogen). We DNase-treated (Turbo DNAse) 10 µg RNA, precipitated it in phenol–chloroform, and resuspended it in 24 µL nuclease-free water . Reverse transcription reactions were performed with Superscript III enzyme on 3 µg RNA, using a telomeric primer (5′-CCCAAAACCCCAAAACC-3′). In addition, control reactions at each time point without reverse transcriptase showed no qualitative product , but werTouchdown PCR products were sheared to 400 bp using the Covaris MiniTube system and gel purified with the QIAquick Gel Extraction Kit to obtain 350–450 bp fragments. Library preparation was carried out using a standard Illumina protocol, with end repair, A-tailing, and adaptor ligation, followed by a second round of gel purification. We eluted  purified product into 10 µL nuclease-free water and amplified 5 µL by 12 cycles of PCR before gel purification, validation on a Bio-Analyzer, and sequencing on an Illumina GAIIx HiSeq instrument. Raw RNA-seq read files are available on SRA under accession SRP079066.Oxytricha trifallax macronuclear genome , as the incubation time we used for PCR precluded the capture of RNAs corresponding to longer chromosomes. Because template RNAs are expected to cover the entire chromosome without splicing, we used the nonspliced aligner BWA MEM  and downstream  regions were extracted. We also removed noncoding regions <5 nt long from the data set according to the requirements of the motif-finding algorithm. We used FIRE-1.1a  for discWe used the R programming environment  for all http://trifallax.princeton.edu/cms/databases/raw-data/transcriptome/reads/RNA-seq/WUGSC) and piRNA data from We used publicly available mRNA data from


A subset of human box C/D snoRNA species have target GAC sequences that lead to formation of N6‐methyladenine at a key trans Hoogsteen‐sugar A·G base pair, of which half are methylated in vivo. The GAC target is conserved only in those that are methylated. Methylation prevents binding of the 15.5‐kDa protein and the induced folding of the RNA. Thus, the assembly of the box C/D snoRNP could in principle be regulated by RNA methylation at its critical first stage. Crystallography reveals that N6‐methylation of adenine prevents the formation of trans Hoogsteen‐sugar A·G base pairs, explaining why the box C/D RNA cannot adopt its kinked conformation. More generally, our data indicate that sheared A·G base pairs (but not Watson–Crick base pairs) are more susceptible to disruption by N6mA methylation and are therefore possible regulatory sites. The human signal recognition particle RNA and many related Alu retrotransposon RNA species are also methylated at N6 of an adenine that forms a sheared base pair with guanine and mediates a key tertiary interaction.N In archaea and the eukaryotes, the box C/D snoRNP complexes direct the site‐specific 2′‐trans sugar‐Hoogsteen G·A base pairs (sometimes termed sheared base pairs) that position the conserved adenine nucleobases to make key cross‐strand hydrogen bonds that stabilize the conformation .Analysis of the targets for methylation by the METTL3–METTL14 methyl transferase complex in eukaryotes shows the preferred sequence to be DRACH , with GAC as the most common site of methylation 6‐methyladenine sites in vivo. From this, we identified 14 human sequences that were shown to be N6‐methylated at A1n (eight from box D and six from box D’) (Table Using the snoRNABase ’) Table . Most of6‐methylation of A1n in these k‐turn‐forming snoRNA species is functionally significant then we might expect that the ‐1n = C sequence that is required to generate the methylation target should be conserved in other eukaryotes. We therefore examined the conservation of the ‐1n position in other organisms using the snoRNABase and snOPY databases . Outside the conserved C, D, C’, D’ and guide region, only the ‐1n C and its complementary paired base (‐1b = G) are 100% conserved .We have seen that in a subset of human box C/D k‐turn sequences the ‐1n position is a cytosine, creating a GAC methylation target on the non‐bulged strand. The METTL3‐METTL14 methyl transferase complex could therefore convert the A1n of the k‐turn to NArcheoglobus fulgidus L7Ae (Af L7Ae) protein  indicative of a folding of the k‐turns  snoRNA, and one box C’/D’ k‐turn from SNORD62A (U62A) snoRNA Fig . The unmtein Fig A and C. urns Fig B and D. 6‐methyladenine at the 1n position exhibited very different behavior. Methylated versions of both U13 and U62A RNA failed to form discrete retarded complexes observed by gel electrophoresis  compared to the unmethylated RNA. Moreover, the shape of the transition indicates rather greater cooperativity. These results suggest non‐specific binding of multiple protein molecules that fails to fold the methylated RNA into a normal k‐turn conformation.By contrast, the corresponding RNA species with Nesis Fig A and C, esis Fig B and D s6A‐methylated RNA is different, having lower values of ∆H and T∆S and weaker apparent binding affinity, with a consequently different 15.5k protein–RNA ratio. This clearly reflects a significantly different protein binding mode between unmethylated and N6A‐methylated RNA, consistent with the results of the electrophoretic and FRET analysis, and confirming non‐specific binding when the snoRNA is methylated.We have explored the binding of 15.5k protein to SNORD62A (U62A) snoRNA with and without N6 methylation of A1n using isothermal titration calorimetry . Binding6‐methylation of A1n disrupts the proper binding and folding of box C/D and C’/D’ k‐turns by the human 15.5k protein.Evidently, N6‐methyladenine on k‐turn conformation, we have used X‐ray crystallography to investigate the structural effect of this modification on different base pairs involving adenine. Each species was formed by the hybridization of a self‐complementary strand that contains a 5‐bromocytosine nucleotide used to calculate phases for the diffraction in most cases. Crystallographic statistics are presented in In order to understand the structural basis for the effect of N6‐methyladenine paired with uridine (PDB 5LR5). This sequence contains GGACU, which is methylated with the highest frequency in cellular RNA 6mA‐U base pairs are normal Watson–Crick pairs  at 1.65‐Å resolution and the methylated RNA (PDB 5LR4) at 1.72 Å (trans sugar‐Hoogsteen base pairs with two H‐bonds between GN2:AN7 (2.8 Å) and AN6:GN3 . In marked contrast, in the N6‐methyladenine‐containing duplex the guanine and N6‐methyladenine do not form base pairs . In its new position, AN6 donates a H‐bond to the O2′ of the opposing G (2.8 Å) and the methyl group is free from steric clash.We studied another duplex of sequence identical to that of the previous one, except that the G‐C base pairs flanking the central G·A pairs were replaced by G·U pairs Fig D and E. t 1.72 Å . The unm RNA PDB LR3 at 1. RNA PDB LR3 at 1.6‐methylation of adenine in this context is the complete disruption of the trans sugar‐Hoogsteen base pair with guanine and is therefore incompatible with the k‐turn conformation. This can be seen if a methyl group is added to the N6 position of A1n in a standard k‐turn, whereupon the methyl can be seen to clash with the ribose of G1b  to be methylated in vivo. Importantly, the ‐1n = C is strongly conserved for those that are methylated, whereas this is not true for those that are not methylated.The great majority of known k‐turn‐forming sequences have a C‐G base pair in the ‐1b, ‐1n position, so creating a GAG sequence on the unbulged strand that is not a target for N6‐methylation of A1n in human box C/D and C’/D’ k‐turns known to undergo methylation in vivo prevent the binding of the 15.5k protein and the concomitant folding of the k‐turn into the kinked conformation. This is the first stage of the assembly of the box C/D snoRNP, without which formation of an active methylation complex cannot proceed further 6mA methylation of the guide RNA species. It has been shown that binding 15.5k protein stabilizes box C/D snoRNA We have shown here that NIs this restricted solely to box C/D snoRNA? 15.5k additionally binds the U4/U6.U5 tri‐snRNP in spliceosome assembly 6mA methylation at G·A base pairs in other systems. In fact one of the box C/D k‐turns we identified as methylated  was made using Jalview  http://weblogo.threeplusone.com.A full alignment of 262 of human box C/D sequences taken from snoRNABase http://snoopy.med.miyazaki-u.ac.jp) were checked with the Microsoft Word search function and visual alignment to identify box C/D RNAs with 2n = G, 1n = A, and ‐1n = C sequence.Human box C/D sequences extracted from snoRNABase Box C/D sequences extracted from snoRNABase http://mirlab.sysu.edu.cn/rmbase/index.php in order to discover their A1n methylation status.Candidate box C/D snoRNAs were used to search RMBase t‐BDMS phosphoramidite chemistry et altert‐butyldimethyl‐silyl (t‐BDMS) protection t‐BDMS‐protected N6‐methyl‐A‐CE phosphoramidite was obtained from Glen Research. Fluorescein (Link Technologies) and Cy3  were attached to the 5′ termini of the oligonucleotides as phosphoramidites in the final cycle of synthesis as required. Unmodified RNA was deprotected in a 25% ethanol/ammonia solution at room temperature for 3 h and evaporated to dryness. Oligonucleotides containing N6‐methyladenine were further deprotected for 2 h at 65°C. Oligonucleotides containing 5‐bromocytidine (ChemGenes) were deprotected for 36 h at 20°C. All oligoribonucleotides were redissolved in 115 μl of anhydrous DMSO, 60 μl triethylamine (Aldrich), and 75 μl triethylamine trihydrofluoride (Aldrich) to remove t‐BDMS groups, and agitated at 65°C in the dark for 2.5 h. After cooling on ice for 10 min, 250 μl RNA quenching buffer (Glen Research) was added to stop the reaction, and the oligonucleotides were desalted using NAP‐10 columns .RNA oligonucleotides were synthesized using RNA for crystallization was purified by gel electrophoresis in polyacrylamide under denaturing conditions in the presence of 7 M urea. The full‐length RNA product was visualized by UV shadowing. The band was excised and electroeluted using an Elutrap Electroelution System  into 45 mM Tris‐borate (pH 8.5), 5 mM EDTA buffer for 8 h at 200 V at 4°C. The RNA was precipitated with ethanol, washed once with 70% ethanol, and suspended in water.Fluorophore‐labeled oligoribonucleotides were purified by gel electrophoresis under denaturing conditions (as described below) and subjected to further purification by reversed‐phase HPLC on a C18 column , using an acetonitrile gradient with an aqueous phase of 100 mM triethylammonium acetate (pH 7.0). Duplex species used for FRET experiments were prepared by mixing equimolar quantities of the appropriate oligoribonucleotides and annealing them in 90 mM Tris‐borate (pH 8.5), 10 mM EDTA, 25 mM NaCl, by slow cooling from 90°C to 4°C. They were purified by electrophoresis in 12% polyacrylamide under nondenaturing conditions and recovered by electroelution, followed by ethanol precipitation.Archeoglobus fulgidus L7Ae protein was expressed and purified as previously published Escherichia coli BL21 (DE3) pLysS induced with 0.2 mM isopropyl β‐D‐1‐thiogalactopyranoside at 22°C. Harvested cells were resuspended in PBS buffer and lysed by sonication. Insoluble protein was removed by ultracentrifugation at 45,000 g for 60 min at 4°C. The solution was then applied to Glutathione Sepharose 4B resin  equilibrated in buffer PBS. The GST tag was cleaved by incubation with 40 U/mg thrombin in 20 mM Tris–HCl (pH 8.0), 50 mM NaCl for 8 h at 25°C. In order to remove contaminating RNA the protein was applied to a heparin column  in 20 mM Tris–HCl (pH 8.0) with a 50–2,000 mM NaCl gradient; 15.5k protein eluted at 500 mM NaCl. The purified protein was concentrated to 10 mg/ml.Human 15.5k protein was expressed as an N‐terminal GST fusion in 6‐A1n‐substituted variant (from position −2 to 4). The sequences used were (written 5′–3′):Fluorescence resonance energy transfer efficiency was measured from duplex RNA terminally 5′‐labeled with fluorescein and Cy3. RNA duplexes contained a central box C/D sequence or NU13top: F –CCAGUCAGUGAGCGUGAUGCAUGUCAGGU62Atop: F –CCAGUCAGUGGGAGAUGAAGCAUGUCAGGU48top: F –CCAGUCAGUGGUGAUGAUGCAUGUCAGGlower strand: Cy3‐CCUGACAUGCUGACCCACUGACUGGNote that the lower strand is the same in each case.EFRET) were measured using the acceptor normalization method Absorption spectra were measured in 90 mM Tris‐borate (pH 8.3) in a 5‐mm path‐length cuvette in 2 μl volumes using a NanoDrop 2000c spectrophotometer (Thermo Scientific). Spectra were deconvoluted using a corresponding RNA species labeled only with Cy3, and fluorophore absorption ratios calculated using a MATLAB program. Fluorescence spectra were recorded in 90 mM Tris‐borate (pH 8.3) at 4°C using an SLM‐Aminco 8100 fluorimeter. Spectra were corrected for lamp fluctuations and instrumental variations, and polarization artifacts were avoided by setting excitation and emission polarizers crossed at 54.7°. Values of FRET efficiency  was incubated in 45 mM Tris‐borate (pH 8.3) with various concentrations of proteins (15.5k or AfL7Ae) in a final volume of 10 μl at 7°C for 60 min. An equal volume of loading buffer containing 10% glycerol and 45 mM Tris‐borate (pH 8.3) was added to the reactions, which were then electrophoresed in 10% native polyacrylamide gels in 45 mM Tris‐borate (pH 8.3) at 20 V/cm at 7°C for 1 h. Fluorescent RNA was visualized using a Typhoon 9500 fluorimager .2 to a final concentration of 20 μM. 15.5k was prepared in the same binding buffer with a concentration of 200 μM. Solutions were degassed for 2–5 min before loading. The sample cell was filled with 200 μl of RNA. 15.5k protein was injected into the RNA solution, using 0.4 μl for the first injection and 2 μl for the subsequent 19 injections using a computer‐controlled 40‐μl microsyringe with an injection interval of 120 s. Titration of protein into the binding buffer or titration of the binding buffer into the RNA solution produced negligible heat evolution. Integrated heat data were analyzed using a one‐set‐of‐sites model in MicroCal Origin following the manufacturer's instructions. The first data point was excluded in the analysis. The binding parameters ΔH , K (M−1) and n (moles of 15.5k bound per RNA) were fitted. The binding free energy ΔG and reaction entropy ΔS were calculated using the relationships ΔG = −RT ln K  and ΔG = ΔH − TΔS. The dissociation constant Kd was calculated as 1/K.Titrations were performed at 298 K using an ITC‐200 microcalorimeter (GE). RNA solutions were prepared by diluting concentrated stocks into the binding buffer containing 40 mM HEPES (pH 7.5), 100 mM KCl, 10 mM MgCl2 was added to a final concentration of 10 mM. The complete conditions used for crystallization of each species are given in The sequences are shown in Fig 1/2 and density map as described previously All data were collected on beamline I02 of Diamond Light Source , and processed by XIA2 version 0.4.0.0 2. Models were adjusted manually using Coot All the structures’ initial phases were acquired from the SAD data by locating the bromine atoms with Autosol in the PHENIX suite, except for 5LR3 that was determined by soaking with CuClCoordinates of the crystal structures described in this work are deposited with the PDB with accession numbers 5LR5, 5LQO, 5LQT, 5LR3, and 5LR4.LH performed bioinformatic analysis and crystallography, SA carried out RNA synthesis, JW performed bioinformatic analysis, and LH and DMJL planned experiments, analyzed data and wrote the paper.The authors declare that they have no conflict of interest.AppendixClick here for additional data file.Review Process FileClick here for additional data file.


Caenorhabditis elegans. Cholinergic motor neuron classes of the ventral nerve cord can be subdivided into subclasses along the anterior-posterior (A-P) axis based on synaptic connectivity patterns and molecular features. The conserved COE-type terminal selector UNC-3 not only controls the expression of traits shared by all members of a neuron class, but is also required for subclass-specific traits expressed along the A-P axis. UNC-3, which is not regionally restricted, requires region-specific cofactors in the form of Hox proteins to co-activate subclass-specific effector genes in post-mitotic motor neurons. This intersectional gene regulatory principle for neuronal subclass diversification may be conserved from nematodes to mice.A core principle of nervous system organization is the diversification of neuron classes into subclasses that share large sets of features but differ in select traits. We describe here a molecular mechanism necessary for motor neurons to acquire subclass-specific traits in the nematode DOI:http://dx.doi.org/10.7554/eLife.25751.001 Caenorhabditis elegans nervous system , retrovesicular ganglion, and preanal ganglion. As shown in To be able to study the problem of how neuronal subclass diversity is generated, we use the nervous system of the nematode  classes . Synapti classes . One parC. elegans wiring diagram reveals a correlation of connectivity differences with MN cell body position along the A-P axis and, more specifically, with the location of individual class members in the retrovesicular ganglion, VNC and preanal ganglion. For example, although all nine members of the DA neuron class share multiple connectivity features, such as input from the command (pre-motor) interneurons AVA and AVD and output to dorsal body wall muscle, the DA neurons located in the preanal ganglion (DA8 and DA9) display several synaptic connectivity differences when compared to the more anteriorly located DA neurons (DA1-DA7) which are located in the VNC and retrovesicular ganglion (www.wormwiring.org). Subclass diversity based on synaptic connectivity is not only evident in the DA class, but it is a recurring theme for all cholinergic MN classes , but not in the retrovesicular ganglion (VA1) or preanal ganglion  to specify the correct pattern of presynaptic input from AVA pre-motor neurons (unc-129 is selectively expressed in the DA neurons of the retrovesicular ganglion and VNC (DA1-7) and not in DA8 and DA9 neurons in the preanal ganglion  cooperates with a region-specific transcriptional code (Hox) to distinguish MN class members from one another, and thereby generate subclass diversity.Here, we provide insights into the molecular logic for how this subclass diversity is genetically encoded along the A-P axis. We describe that the terminal selector-type transcription factor UNC-3, which controls the identity of all distinct MN classes , 2011, iwww.wormbase.org) or that we identified in surveys of expression patterns of specific gene families. All of the genes examined are continuously expressed in cholinergic MNs throughout the C. elegans lifespan. Confirming the notion that all neurons of a given class share a number of molecular features, we observed that several of these MN-expressed genes  are expressed in all individual members of a given class. We call these ‘MN class-specific’ markers  molecularly subdivide each MN class , Table 1glr-4, which codes for an ionotropic glutamate receptor (GluR) subunit, is only expressed in DA9  by all MN classes, as well as class-specific features, that are shared by each member of an individual MN class  shows that the COE motif is essential for this expression . We conclude that UNC-3 is required for subclass diversity in DA, VA and AS neurons by controlling directly the expression of subclass-specific genes, as evidenced by the overrepresentation of COE motifs in the cis-regulatory region of these genes , suggespression . Similar in VA12 . Within se genes .unc-3 is expressed in all nine neurons of the DA class and all twelve neurons of the VA class  and found that three out of the six C. elegans Hox genes are differentially expressed in subsets of MNs that belong to the same class  in DA8 and/or DA9 neurons. However, lin-39/Dfd/Hox4-Hox5 and mab-5/Antp/Hox6-Hox8 are expressed in more anteriorly located DA neurons , while lin-39 and mab-5 are expressed in the more anteriorly located VA neurons along the VNC  in distinct anatomical regions along the A-P axis  led us to investigate whether region-specific regulatory factors cooperate with UNC-3 to control the expression of MN subclass-specific features. Hox genes were previously shown to be expressed in spatially distinct domains of the ans body . We pursme class . Specifi markers . We foun neurons . Within  the VNC . Within  the VNC . Neuronsnd mab-5 . We concen class . – the Clin-39 and mab-5  and egl-5 (expressed in DA9) are, like unc-3, also required for the expression of MN subclass-specific genes. Given the early patterning defects that result from Hox gene removal in other organisms  are controlled by EGL-5, although the effects are milder compared to unc-3 null mutants  and not in other VA neurons along the VNC, and we find that the VA12-expressed genes mig-13 and flp-18 are under the control of egl-5 , which are direct UNC-3 target genes in all MNs  or triple  mutants, we would observe stronger effects compared to single mutants for unc-3 and egl-5 or double lin-39 mab-5 mutants. Since animals carrying the unc-3 null allele (e151) displayed severe effects (100% penetrance) in terms of MN subclass-specific gene expression  and del-1 (VA1-VA11 marker) are milder, thereby enabling combined mutant analysis. We find that unc-3(fp8); lin-39 mab-5 triple mutants display a more severe effect on unc-129 and del-1 expression when compared to lin-39 mab-5 double or unc-3(fp8) single mutants (unc-129 and del-1 expression in unc-3(fp8); lin-39 mab-5 triple mutants together with the absence of cross-regulatory interactions between UNC-3 and Hox lead us to conclude that these regulatory factors act synergistically to generate DA and VA subclass diversity by controlling the expression of subclass-specific genes. We summarize these findings into a genetic model shown in If UNC-3 and Hox proteins act synergistically, we would predict that by generating double  in minimal cis-regulatory elements (243 bp for unc-129 and 488 bp for del-1) that drive reporter gene expression in DA1-7/DB and VA1-11/VB MNs, respectively (unc-129 element (243 bp LIN-39 SITES 6 & 7 MUT) and observed a dramatic loss of reporter gene expression in the DA1-7 and DB neurons  resulted in a reduction of reporter gene expression in VA1-11 and VB neurons  cells are transformed to those of their anterior homologues, suggesting homeotic transformation  in VA12 of egl-5 mutants. We found that 100% of egl-5 mutants display del-1 de-repression in VA12, while in 84.2% of egl-5 mutants unc-129 is de-repressed in DA9  we used to drive egl-5 expression is also expressed in DA9, we observed partial rescue of unc-129 de-repression in DA9 of egl-5 null animals, indicating that EGL-5 acts cell autonomously  where it runs anteriorly to form en passant neuromuscular synapses with dorsal body wall muscle . Since our expression analysis revealed that the Antennapedia-type Hox gene mab-5 is expressed in DA8 and not in DA9  animals  in cfi-1 mutants . The expt in DA9 . cfi-1 eby unc-3  and we i DA1-DA8 . The los mutants  and, con mutants . cfi-1 r mutants . There i mutants .bnc-1. This Zn finger transcription factor is expressed in VA/VB MNs, is a direct target of UNC-3, and antagonizes the activity of UNC-3 on a number of effector genes in VA/VB  are expressed in subsets of neurons of the DA, VA, and AS classes, and thereby differentially intersect with unc-3, which is expressed in all DA, VA, and AS neurons. These three Hox genes act synergistically and cooperate with UNC-3 in an intersectional manner to control the expression of MN subclass-specific genes along the A-P axis  involves class-specific direct repression of effector genes  is mainly generated through positive regulatory inputs provided by UNC-3 and Hox proteins to subclass-specific effector genes  as Hox downstream targets and (ii) by demonstrating that distinct Hox genes intersect with a broadly-acting, terminal selector-type transcription factor and act together at the cis-regulatory level of effector genes to generate MN subclass diversity. These genes encode proteins critical for MN function throughout life, such as ion channels and neurotransmitter receptors. Given the maintained expression of Hox genes in adult C. elegans MNs and mouse CNS  of the synaptic input that the DA9 MN receives from AVG interneurons. Apart from adding a temporal dimension to the function of egl-5/Abd-B/Hox9-Hox13, these findings also demonstrate that Hox genes are required for synaptic input received by MNs, extending previous reports on the requirement of Hox genes for MN to muscle connectivity , EGL-5 also represses the expression of lin-39/Scr/Dfd/Hox4-Hox5, a mid-body Hox gene required for the expression of subclass-specific genes in more anterior DA and VA neurons  signaling from a neighboring cell to the P12 neuroblast (which produces the VA12 neuron) is necessary for the initiation of egl-5 expression in P12  MNs along the A-P axis of the mouse spinal cord  as a food source as previously described (unc-3 (e151) X, unc-3 (n3435) X, unc-3 (fp8) X, lin-39(n1760), mab-5(e1239), egl-5(n945), him-5(e1490), egl-5 (tm4746), egl-5(u202), ceh-20(ok541). bnc-1 (ot845 [bnc-1::+mNG+AID]) CRISPR allele, unc-3 (ot839 [UNC-3::+GFP]) CRISPR allele.Worms were grown at 20°C or 25°C on nematode growth media (NGM) plates seeded with bacteria , pEVL194 (acr-2::NLG-1::GFP11), inx-18p::wCherry; pRF4), kasEx1 [Punc-4prom::egl-5 + myo-2::gfp], kasEx2 [Punc-4prom::egl-5 + myo-2::gfp], wyIs226 [mig-13prom::mCherry::rab-3]wdIs3 X or wdIs6 II , mizEx57 [glr-4prom::egl-5], otEx6502[cfi-1fosmid::gfp], juIs14[acr-2prom::gfp], kasEx3-5 [Pdel-1488bp prom::NLS-YFP], kasEx6-8 [Pdel-1488bp prom LIN-39 sites 5 & 6 MUT::YFP], kasEx9-11 [Pdel-1488bp prom LIN-39 site 4 MUT::YFP], otEx5733-5 [Pdel-11827bp prom::GFP], otEx5739-41 [Pdel-11827bp prom COE3 MUT::GFP], kasEx12[cfi-1prom::tagRFP].mxIs28 While the reviewers find the conclusions of this study compelling, we were not completely convinced regarding the novelty. The role of unc-3 in MN specification has been previously demonstrated by the authors. The role of Hox genes in neuronal subclass specification, including MN specification, has been extensively studied in several other model systems. The strength in this study lies in the identification of an extensive repertoire of subclass-specific MN effector genes, and the demonstration that these genes, as well as neuronal morphology and connectivity, are controlled by a COE-Hox code. In addition, the enhancer analysis, combined with ModEncode ChIP data, provides a much more elaborate molecular resolution for MN subclass specification than that described in other systems. Addressing the following concerns, in particular points 1 and 2 below, could help strengthen the manuscript.Essential revisions:1) In a recent study from the Hobert lab, the group identified seven transcription factors acting together with Unc-3 to control MN specification (PMID 28056346). It would be interesting to see if any of these regulators  are regulated by the Hox homeotic genes studied in the current study, and vice versa. This would only need to be analyzed in the relevant cells i.e., egl-5 vs. cfi-1 in DA9, etc.cfi-1/Arid3a and bnc-1/Bnc1/2, two out of the seven repressors for which we have generated reliable reagents to monitor and quantify their expression. In wild-type animals, cfi-1 is expressed in several cholinergic  and GABAergic  MN classes of the C. elegans ventral nerve cord, while bnc-1 is selectively expressed in all 12 VA neurons, most VB neurons (not in VB1) and 2 of 3 SAB neurons (SABVL/R) . In brief, we found that HOX genes indeed regulate the expression of these subtype-specific repressors, explained in detail in a new section on p.16/17 and in a new We pursued this point by evaluating the expression of 2) A recent study in Drosophila reveals that the Hox homeotic gene Ubx acts in both MNs and their target muscles to control the formation of neuromuscular connections (PMID 27913640). This touches upon the long-standing idea that TFs that are expressed both in neurons and the target tissues could coordinate axon-target pathfinding via activating the same surface molecules in both cells. On that note, can they rule out a role for egl-5 in the target muscle as an underlying reason for the synapse location defects observed in these mutants ?egl-5 is also expressed in posterior body wall muscle and found this to be the case. We therefore performed an additional rescue experiment by providing EGL-5 in the muscle (using the myo-3 promoter) of egl-5 mutants and observed partial rescue of the DA9 synapse location phenotype. However, the muscle-specific rescue was less convincing than the neuronal DA9-specific rescue and adding the muscle-expressed construct to the neuronal expressed construct did not obviously improve rescue. Due to the inconclusive nature of these results, we would therefore prefer to refrain from raising these points in the manuscript.Following this suggestion, we assessed whether 3) There are a few places where the authors imply that certain conceptual findings are novel, but have been well-characterized in the literature at a neuron-specific level. The transformation of posterior neuronal identities to an anterior fate, and in some cases the derepression of Hox genes, have been shown in flies, worms, and mice. These include the transformation of peptidergic neurons in fly Hox mutants (PMID:20485487), and the derepression of Hox1 genes in Hox3 mouse mutants (PMID:12954718), and the derepression of Hox genes in Hoxc9 mutants (PMID:20826310). The authors should rephrase statements that imply novelty in cases where it has already been demonstrated at a neuronal subtype-specific level.We do not disagree with what the reviewers say, but want to emphasize that what makes our paper novel is the cellular and molecular resolution with which we characterize Hox gene function and its collaboration with UNC-3. Meaning, we show how HOX genes interact with terminal selectors at the cis-regulatory level, with the resolution of single targets and single cells.As per the reviewers’ suggestion, we have modified the text in Results to acknowledge that our findings are consistent with previous studies in worms, flies and mice. As suggested, we cite these papers PMID:20485487, PMID:12954718, PMID: 20826310, PMID: 26539892 in two occasions in the Results section:egl-5 represses the mid-body Hox gene lin-39 in DA9 and VA12 neurons.“In flies, worms and mice, Hox genes specifying posterior structures or neurons often repress the expression and activity of more anterior Hox genes . Consistent with these reports, our analysis demonstrates, with single neuron resolution, that the posterior Hox gene “To summarize, our findings suggest homeotic transformations on a single neuron level and are consistent with previous Hox studies describing the transformation of posterior neuronal identities to an anterior fate .”Moreover, we have added a new paragraph in Discussion that discusses the conserved aspect of our findings regarding posterior Hox gene activity and neuronal diversity.4) The reviewers do not believe the data showing expression of Hox proteins and Ebf2 in median motor column neurons in mice adds much to the paper. Hox genes are expressed by most motor neuron subtypes, and there is no current evidence that they are important for the development of this motor neuron subtype. The authors should remove this section from the main Results, perhaps mention it in the Discussion, and show it as a supplement.possible in vertebrates is a notion that we would like to display in the main text. Specifically, we believe that the expression of Ebf2 particularly in the median motor column (MMC) is an important finding that highlights the potential evolutionary implications of our C. elegans results. The C. elegans motor neurons in the ventral nerve cord and the two flanking ganglia  could perhaps be envisioned as an “ancient MMC” since these motor neurons innervate body wall muscles. There is indeed no current evidence in the mouse literature suggesting that Hox genes are important for the development/diversity of body wall muscle-innervating motor neurons. However, it is conceivable that this lack of evidence is most likely due to the lack of molecular markers that can subdivide MMC motor neurons into MMC subclasses along the A-P axis. On the other hand, the plethora of C. elegans motor neuron markers allowed us to reveal that Hox genes are required for the development and diversity of body wall muscle-innervating motor neurons.We respectfully disagree with the respect to eliminate this data. As detailed above, we think one key novelty of our finding is integration of UNC-3/EBF and HOX gene function. To show that such integrated function may also be 5) One limitation of this analysis is the attempt to generalize the finding of Unc-3 controlling some motor neuron subtype genes to a general modus operandi. It is not clear whether Unc-3 motifs are overrepresented in all subtype specific promoters or a few were selected because of Unc-3 sites and ENCODE data.unc-3 dependent as revealed by our mutant analysis. To help the reader, we have clarified this point in the Results  and have also added one extra column in unc-129 – functional Hox (LIN-39) binding sites are also present in the cis-regulatory region of the subclass-specific gene del-1. This new data are now shown in UNC-3 motifs are indeed overrepresented in the subclass-specific gene promoters. Eleven out of the 12 subclass-specific gene promoters contain at least one UNC-3 (COE) motif. In addition, 9 out of 10 of the subclass-specific genes tested for UNC-3 dependency are indeed 6) Terminology: we understand the challenging complexity of the system, but the terminology still seems unnecessarily confusing. For example, in Inspired by other examples in biology – inter-cellular vs. intra-cellular, inter-chromosomal vs. intra-chromosomal, etc. –, we attempted to use the terms inter-class vs. intra-class to refer to neuronal diversity. However, we recognize that the terminology can be confusing especially between intra-class and sub-class. As suggested, we have replaced throughout the manuscript and figures the term “intraclass” with “subclass” for clarity.We have also modified the sentence "However, the manner by which UNC-3 activity is restricted to individual subclasses is fundamentally distinct from the manner by which UNC-3 activity is restricted to distinct MN classes." to "However, the manner by which UNC-3 activity is restricted to individual subclasses (members of a given class) is fundamentally distinct from the manner by which UNC-3 activity is restricted to distinct MN classes."