Escherichia coli genome for RNA sequences with high affinity to RNA Pol by performing genomic SELEX. This approach led to the identification of RNA polymerase-binding APtamers termed “RAPs”. RAPs can have positive and negative effects on gene expression. A subgroup is able to downregulate transcription via the activity of the termination factor Rho. In this study, we used a similar SELEX setup using yeast genomic DNA as source of RNA sequences and highly purified yeast RNA Pol II as bait and obtained almost 1300 yeast-derived RAPs. Yeast RAPs are found throughout the genome within genes and antisense to genes, they are overrepresented in the non-transcribed strand of yeast telomeres and underrepresented in intergenic regions. Genes harbouring a RAP are more likely to show lower mRNA levels. By determining the endogenous expression levels as well as using a reporter system, we show that RAPs located within coding regions can reduce the transcript level downstream of the RAP. Here we demonstrate that RAPs represent a novel type of regulatory RNA signal in Saccharomyces cerevisiae that act in cis and interfere with the elongating transcription machinery to reduce the transcriptional output.Transcription as the key step in gene expression is a highly regulated process. The speed of transcription elongation depends on the underlying gene sequence and varies on a gene by gene basis. The reason for this sequence dependence is not known in detail. Recently, our group studied the cross talk between the nascent RNA and the transcribing RNA polymerase by screening the  Transcription of mRNA is a highly regulated process influenced by numerous protein factors at each stage of the transcription cycle –7. RNAs trans-acting RNAs have been reported to directly bind to RNA polymerases and modulate its activity: 6S RNA (E. coli), B2 RNA (M. musculus), and Alu RNAs (H. sapiens) all inhibit transcription at the stage of initiation [in vitro selected artificial RNA, the FC aptamer, is able to exhibit inhibition of transcription of yeast RNA Pol II by binding to the active center cleft [putL and putR RNA elements interact with the beta-prime subunit of RNA polymerase thereby inducing transcription antitermination [Only a few itiation –18. Moreer cleft ,20. In pmination ,22.E. coli. A combination of SELEX and Next Generation Sequencing led to the identification of sequences within the nascent transcripts, termed RAPs (RNA polymerase binding APtamers), that show a very high affinity to RNA polymerase. A subgroup of RAPs was identified to exert a negative effect on transcription. These iRAPs (inhibitory RAPs) can downregulate elongation in cis via combined mechanisms mediated by the bacterial termination factor Rho [In a recent publication, our group showed for the first time that nascent RNA is able to directly regulate RNA polymerase activity in ctor Rho .cis in the budding yeast S. cerevisiae. We chose a similar strategy as used for E. coli aiming to identify RNA elements that have a high affinity to RNA Polymerase II (RNA Pol II) using a genomic SELEX screen. This approach allowed us to identify almost 1300 yeast RAPs. We show that RAP activity is not only restricted to prokaryotes but can also affect eukaryotic RNA polymerase II. RAPs in S. cerevisiae represent cis-acting regulatory RNA signals that can interfere with the elongation machinery possibly causing premature intragenic termination of transcription.Here, we addressed the question whether specific sequences in the nascent RNA can interact with RNA polymerase II and modulate transcription in S. cerevisiae, we chose the unbiased approach of Genomic SELEX . We constructed an initial library starting from short (30-400nt) fragments of the yeast genome to screen for RNAs binding with high-affinity to a complete purified yeast RNA Pol II 12 subunit complex. In course of the abovementioned SELEX procedure [th cycle  and very highly abundant transcripts . Importantly, sense RAPs were found to be enriched in genes with low transcript levels   . We furt.3*10−5) .CAA-, CCA-, or AU-rich and vary between 14 (CAA-rich) to 22 nucleotides (CCA and AU-rich) in length  of the andidate  since itr search . To inveP levels  at all aGal1-10prGFPHIS3 reporter construct to determine the minimal RAP size necessary to down-regulate elongation and to specify the sequence requirements by performing mutational analyzes. First, we introduced the identified minimal consensus motif obtained from our search with Homer software  (HIS3 expression levels (CYC8 RAP133 into the reporter. This sequence of 24bp represents that particular region of the identified RAP that shows the highest read count derived from the genomic SELEX sequencing. This sequence had a similar repressive effect (~40% drop) on HIS3 expression as the full length RAP133 of CYC8  . This miCAA to AAC or ACA repeats completely altered the amino acid composition of the encoded proteins but did not alleviate the repressive RAP effect n-3’ . This enrichment was almost two times higher than for the GU-rich strand , known to be transcribed in yeast forming the long non-coding RNA termed TERRA ([C-rich and G-rich telomere ends into the GFPHIS3 reporter system (C-rich strand caused a repressive effect on HIS3 expression (50%) whereas the G-rich sequence did not CA-rich sd TERRA  (TUP1 contains 2 sense and 1 overlapping antisense) 10 show a significant drop in transcript levels downstream of the RAP. The reduction ranged from ~20%  shows an effect on expression levels  . Exponennd SRV2)  were harn levels . RAPs int levels . Howeverrrp6Δ. This data set enables the detection of CUTs which were initially observed in this mutant background. We analyzed the transcriptional landscape of the 10 candidates that showed an effect in our qRT-PCR analysis to ensure that neither antisense transcription, nor products of cryptic and pervasive transcription (such as Cryptic Unstable Transcripts-CUTs and stable unannotated transcripts-SUTs) could interfere with our approach. We used the available data set derived from high resolution tiling microarrays by Xu et al.  includinS. cerevisiae can be responsible for cis-active inhibition of transcription.To confirm that the reduction in transcript levels is specific for RAP-containing genes we analyzed six control genes of varying expression levels for which no RAPs had been detected and did not observe any reduction in transcript levels towards the 3’ end of the genes . These rMOT3, CBK1 and PUF3 under the control of the inducible, highly active GAL1-10 promoter  and one located downstream of the RAP (probe2)  and PUF3 (~100-1000bp). These signals were not detectable when probe2, located downstream of the RAPs, was used  and in the middle of the locus (probe2)  . Both prwas used . We perfe points . The CBKern Blot . These r(probe2) . Both prThe size of the RNAs detected by Northern Blotting indicated that the majority of the short transcripts ended upstream of the RAP sequence. To map their 3’ ends we performed 3’RACE using adapter-labeled RNA see . The PCRMOT3 expression pattern between wild type and rrp6Δmutant. The short transcripts detected by probe1 displayed a similar intensity in both strain backgrounds  and could also not detect any difference between wild type and rrp6Δ strain -cleavage factor (CF) dependent pathway being responsible for mRNAs, and the NNS (Nrd1-Nab31-Sen1) pathway, which is tightly connected to exosome function and necessary for termination of snoRNAs, cryptic transcripts and other RNA species [MOT3, CYC8, CBK1 or SSL2  show a slightly decreased occupancy of RNA Pol II downstream of the RAP  of the largest RNA Pol II subunit Rpb1 at the selected targets analyzed for transcriptional effects in  the RAP . The hosse sites . Howeverene body . These rene body .CYC8, CBK1, SSL2, PUF3, BUD8, SSM4)  . HoweverTo confirm this assumption on a global scale we used two different strategies. In a first approach we removed the first 200nts of every gene to avoid signal distortion caused by RNA Pol II accumulation immediately downstream of the TSS ,48 and S. Gene boIn a second, RAP focused approach the ratios of RNA Pol II occupancy densities upstream (gene start to RAP) and downstream (RAP to gene end) were calculated and tested for significance. As a background control we used randomized positions preserving the relative location of the RAPs within the genes. The difference between expected frequencies from the control and the observed counts of the RAP containing genes was tested for significance by using a chi-squared test . We observed that RAP host genes have a higher tendency for a decrease of RNA Pol II density compared to the randomized control . These oE. coli genome as source of RNAs and bacterial RNA Pol as bait [A key step in evolution is the shift of RNA-driven RNA synthesis to protein-catalysed RNA synthesis. For this to occur RNAs and proteins had to co-evolve accompanied by a continuous cross-talk between the two macromolecules. We hypothesized that during protein-catalysed RNA synthesis the nascent RNA constantly interacts with RNA polymerases leading to elongation kinetics that are dependent on the underlying RNA sequence. In order to deepen our understanding of the cross-talk between RNA polymerases and nascent RNA, we searched for RNA sequences that have the capacity to interact with the transcription machinery. Previously, we identified such RNA sequences (termed RAPs) in a genomic SELEX experiment using the  as bait . For theE. coli, the primary outcome of the SELEX experiment is that the obtained sequences do not manifest a single common motif but are rather diverse. RNA Pol II is a very large protein complex composed of 12 subunits, each of them containing potential interaction sites for RNA and thus, the diversity of the identified sequences is not utterly surprising. Nevertheless, we could identify some motifs such as the CAA-rich motif in a subset of characterized aptamers. The tested RAP-containing genes harbouring this sequence motif all showed a drop of transcript levels downstream of the RAP . Assuming that RAPs with diverse sequences could interfere with transcription we tested mRNA levels flanking the aptamers of 14 RAP-containing genes in their endogenous context. We found that 10 of them had diminished transcript levels downstream of the RAPs. RAPs that were found to be inactive in our approach could still have an impact on transcription that requires detection via different assays. Trans-acting regulatory RNAs such as the B2 or Alu RNAs or the synthetic Fc aptamer that binds yeast RNA Pol II have been previously identified and some RAPs might work in a similar manner [cis-acting RAPs and did not test potential effects in trans. RAPs could also positively affect transcription via a mechanism that is similar to the antiterminating effect of the E. coli phage putR and putL elements [Similar to the approach performed in r manner ,18,49–52elements . Since m1-3ACACC-3’5’-ACACC(n)) (53). The number of Rap1 molecules associated with the telomere is the major determinant of telomere length and Rap1 recruits the silencers Sir2, Sir3 and Sir4 [GU-rich control sequence does not have any effect on elongation. Therefore, we suggest that the observed RAP effect is indeed caused by the telomeric CA-rich transcript.Interestingly, we found that RAPs encoded in subtelomeric and telomeric regions also have a repressive effect on elongation. This indicates that cells might use RAPs for silencing of certain regions such as telomeres, origins of replication or centromeres. Telomere ends are covered with the DNA-binding protein Rap1 with a recognition sequence that is encoded in both strands (and Sir4 . MoreoveCACC-3’) . The numand Sir4 . HoweverCA-rich motif (and the CAA-rich motif of CYC8 and CBK1) we could confirm that the underlying double stranded DNA or encoded protein sequence are not responsible for the RAP effect  which could indeed point to backtracked elongation complexes. However, available high-resolution NET-Seq and CRAC-Seq data sets show that RNA Pol II occupancy levels are slightly but significantly diminished downstream of RAP sequences and we could not observe any RNA Pol II peaks on single loci in these setups. This finding rather points to a RAP-induced intragenic termination event which is corroborated by the fact that TFIIS does not have any impact on the upstream/downstream transcript ratios at RAP host genes. Nevertheless, we cannot completely exclude the possibility that pausing and backtracking might be involved since we only analyzed a small number of RAP-containing genes and pausing events might simply be masked in our genome-wide analysis. Moreover, we have not investigated the impact of post-transcriptional events, such as RNA degradation in the cytoplasm, in this study.Elongation of mRNA is not a homogenous process. Several factors influence the elongation rate of individual genes. In fact, the dynamics of elongating RNA Pol II are highly gene-specific suggesting that the underlying DNA sequence and the regulatory features of a gene are significant factors for elongation efficiency –4,6,48. E. coli where the inhibitory RAP effect is clearly dependent on the activity of the termination factor Rho. Therefore, we speculate that cis-acting RAPs in S. cerevisiae might exert their effects by directly regulating elongating RNA Pol II without the need of additional protein factors.Interestingly, the observed diminished RNA Pol II levels seem not to be mediated by the two major termination pathways in yeast. This is in contrast to the findings in The disruption of elongation at RAP host genes resulted in the generation of multiple short transcript species with undefined 3’ ends identified by Northern Blotting and 3’ RACE. Although some of these transcripts end within or in close proximity to the RAPs, the majority ends upstream which could be explained by degradation events. A recent publication proposed a model where the early elongation complex is always in a state poised for termination leading to short, unstable transcripts that are substrates for the NNS-Exosome pathway similar to CUTs . The majE. coli RAPs and bacterial RNA polymerase [E. coli are able to modulate gene expression under stress conditions and strongly reduce transcription interference from antisense transcripts [In conclusion, RAP-mediated control of transcription elongation is a phenomenon that is manifested by the cross-talk of specific sequences within the nascent RNA interacting with the elongating RNA polymerase. This phenomenon is not restricted to yeast RNA Pol II, as we also observed similar activities with lymerase . RAPs idnscripts . TherefoS. cerevisiae strains were grown in YPD (2% Glucose) to early exponential growth phase (OD600 0.8–1) and harvested by centrifugation followed by freezing in liquid nitrogen. Strains with RAP-containing transcripts under the control of the GAL-10 promoter were grown in 2% raffinose to early exponential phase followed by addition of 2% galactose and harvest of cells after 10, 30 and 60 minutes. In case of the essential CBK1 locus cells were grown in the presence of 2% galactose followed by addition of 2% glucose. Cells containing the endogenously expressed reporter construct were also grown in medium with raffinose followed by addition of galactose. Alternatively, reporter cells were exclusively grown in medium with 2% galactose. Anchor Away strains Nrd1-Frb-KanMX6 and Rat1-Frb-KanMX6 were grown to early exponential phase and treated with 1μg/ml rapamycin (LC-Laboratories) for 60 minutes or left untreated.Strains used in this study are listed in S. cerevisiae in an in vitro binding reaction as described in [4SO4, 10 μM ZnCl2, 1 mM KCl, 10 mM DTT, 5% glycerol and 10 mM MgCl2.The genomic library was created as described in  with genribed in . For theGGAATTCGGAGCGGGACTGG, fixRV_multi: CGGGATCCTCGGGGCTGACTTG (Sigma)). Library preparation (adapter ligation step) and deep sequencing were performed at the VBCF NGS Unit (www.vbcf.ac.at). The SELEX library was sequenced using a GAIIx with paired-end 76 base-pair reads. 3’ adaptors were removed using cutadapt (doi: 10.14806/ej.17.1.200) and 5’ sequencing primers and barcodes were removed with a custom script. Read pairs of which either read was less than 18nt long were discarded. After primer removal and demultiplexing, paired-end reads were aligned to the sacCer2 genome as downloaded from the UCSC genome browser (doi:10.1101/gr.229102) using bowtie2 (doi:10.1038/nmeth.1923) using the settings–score-min G,14,8 -N 1–reorder -p 6–dovetail–local. Mapped mate pairs were filtered such that the distance between 5’ ends were no less than 20 and no more than 500 bases apart on the same chromosome.The DNA library from SELEX cycle 6 amplified by 16 cycles of PCR with Pfu polymerase (New England Biolabs) using primers with fixed part and added barcode for multiplexing according to  . First, natMX::GAL-10prGFP was amplified by PCR using primers with homologous overhangs for genomic integration  overlapping to GFP (5’ end) and HIS3 (3’ end) and submitted for synthesis to Genewiz®. All strains were controlled for integration by PCR. Control primers are indicated in The RAP-reporter strain was created by sequential homologous recombination into the region between the egration . PlasmidIPP1 transcript [Cells were grown to early exponential phase and harvested by centrifugation. RNA was isolated using Phenol-Chloroform extraction essentially as described before ,57. ConcNorthern Blotting was performed as described before . PurifieChIP was performed as described . Cells wAAUGGACUCGUAUCACACCCGACAA, Thermo Scientific®) were phosphorylated using T4PNK (NEB®). After extraction by Phenol-Chloroform RNA ligation was performed at 17°C over night using T4 RNA ligase (NEB®). The ligation reaction was purified by PCl-extraction and cDNA was synthesized using random nonamers and Protoscript RT II (NEB®). For amplification target-specific forward primers and an adapter-specific reverse primer were used. PCR-products were cloned into pGEM-T Easy vector (Promega®) and sequenced.6μg of DNA-free total RNA were used for dephosphorylation by T4PNK (NEB®) in Buffer: 400mM Tris-HCl pH6.5, 400mM MgAc, 20mM Mercaptoethanole. Dephosphorylated, purified RNA was phosphorylated by Calf intestinal phosphatase (NEB®). 5pmol of 3’ adaptor (sourceforge.net/projects/bbmap/). Reads were mapped using segemehl version 2.0 [https://www.r-project.org/).For the correlation of transcript abundance of RAP hosting genes the mRNA-seq data set from Churchman et al. was used  . Read qusion 2.0 . Multimasion 2.0 . TPM-valsion 2.0 . Genes whttp://www.yeastgenome.org) and SUTs& CUTs [GO term enrichment analysis for all protein coding genes containing at least one RAP in contrast to the remaining yeast genes was performed using GOrilla . EnrichmTs& CUTs . OverlapTo uncover putative enriched sequence motifs associated with RAPs the Homer software suite was applied . An impoFor the analysis of RNA Pol II occupancy NET-seq data from Churchman et al.  and CRAC-seq data from Milligan et al. (SRA accession numbers SRR2056997 and SRR2056998) were used ,47. ReadTo analyze the effect of RAPs onto RNA Pol II occupancy from a RAP perspective, the ratios of mean RNA Pol II occupancy upstream and downstream of each RAP from within annotated genes were evaluated based on a Poisson test if significantly different from 1 . If not, the ratio was set to 0. To obtain a background distribution each RAP was randomly moved to 100 genes preserving its position relative to the gene boundaries. The analysis was repeated with this randomized background data set. The distributions of log2 ratios between the upstream and downstream gene body were tested for equality with a nonparametric Kolmogorov-Smirnov test.S1 Fig(A) Class distribution of RAPs. Analysis of the occurrence of RAPs in different genomic contexts. For each category the number of RAPs overlapping at least one base with any annotated feature was counted. Since the feature and the RAPs differ in length the expected count was calculated by randomizing the RAP location preserving its length, and the analysis was re-done 100,000 times. The lower panels show the log2-transformed enrichment of observed counts/expected counts. Categories are marked according to the empirically determined p-value, <*> and <**> represent significance levels p-value <0.01 and <0.001, respectively. (B) and (C) Gene Ontology enrichment analysis for all protein coding genes harboring a RAP. All terms and their relation from the domain of biological process with a p-value below 10^(-6) are depicted. The RAP containing gene set is highly enriched in genes associated with regulatory function, especially transcription regulation via RNA polymerase II.(PDF)Click here for additional data file.S2 FigMAL11 and MAL13 on chromosome VII (Chr VII:1072353–1072472). After integration of natMX::GAL1prGFP (amplified from plasmid pYMN-25), a HIS3 cassette was integrated downstream of the GFP followed by recombination of a URA3STOP cassette enabling selection for growth on medium lacking uracil. The URA3STOP cassette could be exchanged for any RAP or control sequence. Resulting transformants can be selected for growth on medium lacking histidine or containing 5-FOA. (B) Location of primer pairs for qRT-PCR. (C) Expression profile of the GFPHIS3 reporter during growth in medium with 2% galactose as a sole carbon source. Expression levels are equally distributed over the fusion transcript. (D) The GFPHIS3 fusion transcript can be induced via the GAL1-10 promoter. Cells were grown in 2% raffinose to early exponential phase and galactose was added to a final concentration of 2%. Aliquots were taken at indicated time points. (E) Effect of CYC8-RAP in the reporter construct. Cells containing the reporter construct with (GFPRAPHIS3) or without (GFPHIS3) the full-length RAP of the CYC8 locus  The reporter system for detection RAP effects was created by sequential homologous recombination at a non-transcribed region between ocus see  Tables wmers see . Values (PDF)Click here for additional data file.S3 Figrrp6Δ strains are shown in parallel. No antisense transcripts can be observed at the sites of the analyzed RAP-containing genes.Browser pictures resulted from high resolution tiling microarrays (8bp) taken from the data set by Xu et al. (36). cDNA synthesis was performed using a combination of oligodT and random primers in this setup. Signal intensities for both DNA strands (W and C) for the different profiled samples are shown on the y-axis. Three independent results from BY wild type and (PDF)Click here for additional data file.S4 FigACT1, PDA1, RPN2, PGK1, STE5 and CET1) not containing RAPs. Reverse transcription was performed with random nonamers and expression levels were quantified by qRT-PCR using amplicons as indicated in the respective figures. Values were normalized to the first 5’ amplicon. (G) to (I) RAP-containing genes PRY3, SRV2 and TUB4 do not show any drop of transcript levels downstream of the RAPs.(A) to (F) Transcript levels of 6 selected target genes ((PDF)Click here for additional data file.S5 FigGAL1 locus under the control of its endogenous GAL1-10 promoter. (B) Northern Blot using the probes indicated in A).(A) Schematic of position of Northern Blot probes at the (PDF)Click here for additional data file.S6 FigMOT3, CBK1 and PUF3 under the control of the inducible GAL1-10 promoter. Positions of the RAPs and the used gene specific forward primers are indicated. PCR products resulting from amplification with the gene specific primer and the adapter specific primers are shown below the sequences. For MOT3 the transcripts lengths range from ~200-1100bp with a forward primer starting at 186bp, the majority having a size of 500bp. For PUF3 the lengths range from ~100-1000bp when forward primer 127fw is used and from 100–750 when primer 565fw is used. CBK1 shows a size distribution of 100-500bp with primer 81fw. The PCR products were cloned into the p-GEM vector (Promega) and 7–11 clones were sent for sequencing to confirm specificity. The frequency of the identified 3’ends of these clones are indicated.(A) to (C) 3’RACE results for (PDF)Click here for additional data file.S7 Figrrp6Δ was analyzed. The short unstable transcripts detected with the upstream probe1 are similarly expressed in wild type and mutant strain. (B) qRT-PCR analyzing the same samples used for Northern Blotting show similar expression patterns in wild type and rrp6Δ mutant. (C) to F) qRT-PCRs of MOT3, CYC8, CBK1 and SSL2 wild type and mutant cells. Reverse transcription of DNA-free RNA was performed using random nonamers and quantification was done using primers upstream and downstream  of the RAP similar as in (A) Northern Blot as presented in (PDF)Click here for additional data file.S8 FigBUD8 and TUP1). The MOT3 and SSM4 loci seem to have a pause site immediately upstream of the RAP.(A) to (J) Chromatin Immunoprecipitation of RNA Pol II (Rpb1) at RAP-containing target genes. Similar amplicons as for analysis of expression profiles were used see  for qRT-(PDF)Click here for additional data file.S9 Figst decile. The read signal in each decile is further normalized with the total read signal for each gene. Both data sets show that the average RNA Pol II occupancy is highest at the 5’ end of ORFs.(A) Relative RNA Pol II occupancy across the gene body as seen in the CRAC-seq and NET-seq data . All the positions in all the annotated genes are bin into ten deciles, so that the first 10% of the gene bases are grouped in the 1(PDF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.


The affinity of Gag  for cellular RNAs at physiological ionic strength is not much higher than that for the gRNA. However, binding to the gRNA is more salt-resistant, implying that it has a higher non-electrostatic component. We have previously studied the spacer 1 (SP1) region of Gag and showed that it can undergo a concentration-dependent conformational transition. We proposed that this transition represents the first step in assembly, i.e., the conversion of Gag to an assembly-ready state. To explain selective packaging of gRNA, we suggest here that binding of Gag to gRNA, with its high non-electrostatic component, triggers this conversion more readily than binding to other RNAs; thus we predict that a Gag–gRNA complex will nucleate particle assembly more efficiently than other Gag–RNA complexes. New data shows that among cellular mRNAs, those with long 3′-untranslated regions (UTR) are selectively packaged. It seems plausible that the 3′-UTR, a stretch of RNA not occupied by ribosomes, offers a favorable binding site for Gag.Like other retroviruses, human immunodeficiency virus type 1 (HIV-1) selectively packages genomic RNA (gRNA) during virus assembly. However, in the absence of the gRNA, cellular messenger RNAs (mRNAs) are packaged. While the gRNA is selected because of its  Human immunodeficiency virus type 1 (HIV-1), the causative agent of acquired immunodeficiency syndrome (AIDS), is a retrovirus. Retroviruses are assembled from a few thousand copies of a single structural polyprotein, termed “Gag”. The particles contain much smaller numbers of other virus-coded polyproteins and RNA, and are surrounded by a lipid bilayer derived from the plasma membrane of the virus-producing cell. After the viral particle is released from the cell, a series of proteolytic cleavages transform it from the non-infectious “immature” form to the infectious “mature” form; however, this review will only deal with immature particle assembly.The major domains of the Gag protein are matrix (MA), capsid (CA), nucleocapsid (NC), and p6. There are also two short “spacer” regions, SP1 and SP2, flanking the NC domain. Briefly, the MA domain is involved in the interaction of Gag with membranes, but can also bind to RNA; the CA domain mediates most of the protein-protein interactions between Gag molecules; NC, which is positively charged and contains two small zinc fingers, plays a major role in interactions of Gag with nucleic acids; and p6 contributes to the release of the assembled particle from the host cell by interacting with the cellular endosomal sorting complexes required for transport (ESCRT) machinery.cis-acting RNA element  that begins in the 5′-untranslated region (UTR) and extends into the Gag open reading frame (ORF) [The genomic RNA (gRNA) of HIV-1 is ~9.7 kb long. All retrovirus particles contain a dimer of gRNA, in which two molecules of the same polarity are joined together by a limited number of base-pairs . Packagime (ORF) ,4. Unforme (ORF) ,5, it isme (ORF) . By analAs in most, if not all viruses that package single-stranded RNA, the assembly of HIV-1 virions requires the presence of RNA. The RNA is incorporated during, not after, the process of capsid formation. Thus, the gRNA not only carries genetic information, but also contributes to the assembly of the structural proteins into a virion. However, it is important to realize that this is not a unique property of the gRNA. In the absence of the gRNA, the HIV-1 Gag, as well as the Gag from other retroviruses, assembles in cultured cells into virus-like particles (VLPs) . The amoA very significant clue to this riddle was uncovered by Zhang et al., who reported that if the NC domain of Gag is replaced by a protein that can form dimers (leucine zippers) without binding to nucleic acids, then the resulting chimeric protein assembles well in mammalian cells . These pAlthough NC may play a predominant role in the interactions of Gag with RNAs, there are many kinds of evidence showing that the MA domain also binds RNAs. Thus, the binding of the MA domain to RNA in reticulocyte lysates apparently prevents the association of Gag with (and assembly at) cytoplasmic membranes . In vitrAlthough binding of Gag to RNA is mostly driven by the interactions between the NC domain and the nucleic acid, there is some evidence that Gag–Gag interactions play an important role in stabilizing Gag–RNA complexes. For example, it has been shown that deletion of the C-terminal domain (CTD) of CA (responsible for Gag dimerization in solution) decreases the stability of Gag–RNA complexes, both in the cytoplasm and at the plasma membrane . This deAs noted above, RNA is unnecessary for particle assembly if the Gag protein contains a dimerizing zipper domain ,11. ThisIn general, the packaging signals of retroviruses are located in and near the 5′-UTR of the gRNA and are several hundred bases long. Surprisingly, the Ψ of HIV-1 is still not very well defined; as far as is known, there is no single, contiguous stretch of the gRNA which confers selective packaging upon a heterologous RNA .  that are small compared to the length of the HIV-1 gRNA ,33,34. IAnother inherent complexity in these measurements arises from the fact that Gag contains positively charged stretches: both the MA and NC domains are polycationic and will therefore have significant affinity for any nucleic acid. In fact, this non-specific RNA binding is not merely a complication for the investigator, but is part of the reason why selective packaging defies a facile explanation: the gRNA in the cell is in the presence of a large excess of other RNAs. In fact, cellular messenger RNAs (mRNAs) are packaged in cells containing Gag but lacking Ψ-containing RNA ,35; thusS-transferase-tagged Gag by electrophoretic mobility shift assay in a >1000-fold excess (by mass) of tRNAs [One experimental strategy that has been employed to “block out” the non-specific component of Gag–RNA binding in vitro is to measure binding to the RNA of interest in the presence of an excess of irrelevant competitor RNA, such as tRNA ,36. ThisAnother strategy was used by Webb et al. to eliminate non-specific binding . They mehttps://www.R-project.org) and Bioconductor [2 (fold change) in each bin and the log10 (median 3′-UTR length) for the mRNAs in the bin. As can be seen, the mRNAs that are encapsidated with higher-than-average efficiency possess, on average, a significantly longer 3′-UTR than other mRNAs.As mentioned before, in the absence of the gRNA, HIV-1 Gag, as well as murine leukemia virus (MLV) Gag, packages cellular mRNAs without any obvious specificity . Howeveronductor . The rawonductor . As we dt-test between the UTR lengths in the “average fold-change” group and the “high fold-change” group was highly significant, with a p-value < 10−15. This relationship was observed for both HIV-1 and MLV Gag, suggesting that this apparent selectivity for long 3′-UTR RNAs is a general property of the retroviral assembly pathway, not unique to HIV-1 (a member of the lentivirus genus) or MLV (a gammaretrovirus). One possible explanation is that the 3′-UTR is a stretch of mRNA that is unoccupied by ribosomes; perhaps this provides sites to which Gag can bind in the absence of gRNA. Thus, RNAs with longer 3′-UTRs have a larger number of available Gag binding sites than RNAs with shorter 3′-UTRS. It is interesting to notice that the gRNA of retroviruses has an extremely long 3′-UTR.The data was also analyzed by selecting the 1000 mRNAs with the highest fold change, the 1000 species with the average fold-change, and the 1000 species with the lowest fold-change, i.e., those with the most preferential, average, and most excluded encapsidation efficiencies. 3 is the primer for DNA synthesis during HIV-1 infection, and so this specific tRNA must be packaged in the particle. Moreover, it is a relatively low-abundance tRNA in the cell, and must therefore be packaged selectively. In turn, the synthetase is apparently packaged because it interacts with sequences in the CA domain of Gag [In addition to the gRNA, wild-type viral particles contain host cell RNAs which can make up to one half of the total virion RNA mass. Most of these RNAs are tRNAs and non-coding RNAs and they are short (≤300 nts) compared to the length of the gRNA. Although it has been shown that, in the cytoplasm, the MA domain of Gag preferentially binds to tRNAs , there in of Gag . A numben of Gag ; there an of Gag ). Interen of Gag .It is a well-known fact that in retroviruses the gRNA forms a stable dimer inside the mature virion ,2. From Transcripts representing HIV-1 gRNA can be dimerized in vitro in the absence of Gag and under very specific temperature and solution conditions. However, the rate of in vitro RNA dimerization can be increased by the presence of NC. A recent study in cell culture showed that it is very likely that RNA dimerization occurs close to the plasma membrane and that it requires the presence of Gag . Gag, liNikolaitchik and co-workers showed that RNA dimerization is crucial for efficient RNA packaging and that HIV-1 regulates genome packaging by RNA copy number and not by RNA mass . AnotherAlthough, for a long time, it was believed that the most important element within Ψ was stem-loop 3 (SL3), a recent study from Abd El-Wahab et al. showed that, in the presence of Gag, a bulge in SL1 (the stem-loop required for HIV-1 gRNA dimerization) is more protected from chemical and biochemical modifications than other regions of Ψ, including SL3 . Abd El-Earlier in this review, we described our findings on the SP1 domain of Gag, which led us to suggest that a conformational change in SP1 switches Gag into an assembly-ready state . In some+ and Ψ− RNAs, both in vitro and in cell culture. Testing this hypothesis should enable us to understand how the strength of Gag–Gag interactions controls assembly, both in vitro and in cell culture, and how the modulation of these interactions leads to selective packaging of the gRNA. Furthermore, the existence of a Ψ-dependent nucleation complex might suggest new therapeutic strategies that would inhibit packaging of the gRNA.In order to determine the validity of this hypothesis one would need to be able to measure Gag–Gag interactions in the presence and absence of RNA . More importantly, one would have to determine if the strength of Gag–Gag interactions or the conformation of Gag in Gag–RNA complexes depends on the sequence of the bound RNA . This hypothesis could be tested by measuring the rate of VLP assembly, or virus budding, in the presence of Ψ


Latin America and the Caribbean (LAC) has experienced an unprecedented mining boom since the mid-2000s with unknown effects on sexual and reproductive health (SRH). This study takes the essential first steps of summarizing the available literature regarding SRH in mining contexts in LAC, identifying critical gaps in knowledge, and discussing main implications for future research.We conducted a scoping review with a systematic search of health literature in four databases, reference lists of selected papers, and citations in Google Scholar.The systematic search yielded 592 primary references and 16 articles from LAC. The 11 papers finally selected were conducted in gold-mining contexts in Brazil, Venezuela, Guyana, Peru, and Colombia, between 1995 and 2016. Ten studies centered on measuring HIV/STD prevalence among mineworkers and other populations; few examined associated risk factors. Eight studies reported high HIV/STD prevalence in the study population. None of the studies explored broader SRH issues.Available research is scarce and provides limited evidence on SRH in LAC mining contexts. Critical gaps include little knowledge on (1) broader SRH impacts besides HIV/STDs, (2) SRH in settings different from gold-mining contexts in Amazon countries, (3) mechanisms shaping SRH in LAC mining contexts, and (4) effective interventions in these scenarios. Future research must consider the distinctive demographic, environmental, socioeconomic, and gender dynamics triggered by the mining economy in the analysis of the relationship between mining and SRH, particularly in a period of extractive boom. Consistent evidence characterizes mining as one of the world’s most hazardous activities because of the high risk of occupational accidents and toxicological exposure among mineworkers and communities . ConversThe study of the relationship between mining and SRH in Latin America and the Caribbean (LAC) is pertinent, as the region has experienced an unprecedented mining boom since the mid-2000s. The increased global demand for and rising prices of minerals , along wDespite the high dividends from the mining sector commonly reported in the region  , 21, 22,The mining boom in LAC takes place in a scenario of pending challenges in SRH. Estimations of either intimate partner violence or non-partner sexual violence against women vary between 24% in the Southern LAC and 41% in the Andean LAC—higher than the global estimation of 35% . The regThe recent mining boom in the region, in a scenario of profound SRH inequities, provides a strong rationale for examining to what extent the mining dynamics have impacted SRH at the local level. To this end, it is necessary to establish first what is currently known about the relationship between mining and SRH in the region. This scoping literature review takes the essential first steps of examining and summarizing the available literature regarding SRH in LAC mining contexts, identifying critical gaps in knowledge, and discussing primary implications for future research.A scoping review is a systematic process intended to explore the extent and nature of research on a particular topic, identify gaps in knowledge, and summarize and disseminate findings . One of -Virtual Health Library), and two LAC databases, LILACS  and SciELO (Scientific Electronic Library Online). We scanned reference lists and citations of the selected papers in Google Scholar to identify additional studies.We conducted a scoping review with a systematic search of health literature during May 2017, in PubMed and PaHO-VHL . First, “mining” and “mining industry.” Second, “sexual health,” “reproductive health,” “maternal health,” “HIV,” “sexually transmitted diseases,” “sexually transmitted infections,” “fertility,” and “sexual behavior.” To narrow the search in PubMed and PaHO-VHL, we used the term of exclusion “data mining.”We included only references placed in LAC countries, with no limitation by the date of publication. We excluded articles addressing exclusively environmental and occupational health , health issues different from SRH , and non-health issues. We excluded non-primary research papers . The selection process included the elimination of duplicates, selection of references based on titles and abstracts, identification of full-text papers, selection of additional studies from reference lists and citations in Google Scholar, and final selection of full-text primary research articles Fig. .Fig. 1ScFinally, we summarized and discussed the selected articles based on the country and sub-region of the study, key features of the mining operations , the purpose of the study, population and sample size, and main findings on SRH.The scoping review yielded 592 primary references and retrieved 16 articles placed in LAC countries that focused on SRH  or an individual sociodemographic variable (the mineworker occupation). Astete et al. [The study of the relationship between mining and SRH in LAC has overlooked the context where mining takes place. For most of the studies reviewed, the construct e et al.  and Mirae et al.  also cone et al.  exploredThe mining context in a period of extractive boom has been commonly characterized by disruptive demographic, environmental, socioeconomic, and gender dynamics triggered by the mining economy. Significant workforce migration to mining places  and high mining wages in mining communities relate to increases in the local demand and prices of land, housing, basic goods, and services, as described in countries like Peru . The incThese dynamics determine a context of unbalanced gendered power relations, where women are disproportionally vulnerable to the environmental, socioeconomic, and health impacts of mining . As revihigh-risk environment approach which enables structural-level analyses and interventions. The study by Orellana et al. [ecosocial levels of influence and discussed the risk environment as an appropriate category of analysis about the spread of HIV/STDs among indigenous communities. Even though the authors did not explore broad aspects of the SRH—likely occurring in these same risk environments, this approach serves as a point of reference for further exploration about the way SRH is shaped in LAC mining contexts.The simultaneous dynamics often described in mining contexts challenge the way we address the links between mining and SRH in research and intervention. Some studies in South African mining settings showed that prevention interventions typically based on biomedical and behavioral approaches had little impact in reducing or even controlling the spread of HIV , 8, 55. a et al.  coinciderisk environment instead of high-risk population, as proposed by Desmond et al. [To address the gaps in knowledge identified, new research efforts must consider key conceptual and methodological challenges. First, they require comprehensive approaches for SRH that include, in addition to the study of HIV and STDs, broader SRH aspects related to sexual violence against women, adolescents and children, intimate partner violence, reproductive decision-making, use of contraception, early marriage, adolescent pregnancy, abortion, maternal health, and access and use of SRH services. Second, future studies must consider the mining context as a critical component of analysis, by examining the demographic, environmental, socioeconomic, and gender dynamics triggered by the mining economy, involved in the production of SRH risk. This also requires taking into account the particularities of the mining operations, i.e., minerals of extraction, stages of mining, scales, and status of formality/legality. Empirical research should consider the use of categories like d et al.  and Oreld et al. ; the usen = 300), in the copper region of Tarapacá, Chile. Mineworkers showed high levels of knowledge in HIV issues, high demand for commercial sex , and little access to condoms (81.4% referred non-availability of condoms in the mining place). These findings, similar to those from Seguy et al. [We conducted a scoping literature review looking for peer-reviewed publications. We did not examine gray literature, e.g., dissertations, technical reports, or conference papers. Despite potential drawbacks on availability and quality, gray literature might provide insights on how other researchers and stakeholders  have approached the relationship between mining and SRH in LAC. For instance, Organización Internacional del Trabajo, Gobierno de Chile, and SEREMI Tarapacá  presentey et al. , also taThis scoping review clarifies the current knowledge about SRH in LAC mining contexts and joins the recent reviews by Drewry, Shandro, and Winkler  and Pere


Mutations in genes that encode RNA-binding proteins (RBPs) have emerged as critical determinants of neurological diseases, especially motor neuron disorders such as amyotrophic lateral sclerosis (ALS). RBPs are involved in all aspects of RNA processing, controlling the life cycle of RNAs from synthesis to degradation. Hallmark features of RBPs in neuron dysfunction include misregulation of RNA processing, mislocalization of RBPs to the cytoplasm, and abnormal aggregation of RBPs. Much progress has been made in understanding how ALS-associated mutations in RBPs drive pathogenesis. Here, we focus on several key RBPs involved in ALS—TDP-43, HNRNP A2/B1, HNRNP A1, FUS, EWSR1, and TAF15—and review our current understanding of how mutations in these proteins cause disease. RBPs play a central role in mediating post-transcriptional gene expression. They directly bind to RNA and control all aspects of RNA processing, including RNA synthesis, maturation, localization, translation, and decay. A genome-wide survey of RBPs in the developing mouse nervous system revealed that two-thirds of RBPs are expressed in a cell-type specific manner , spinal muscular atrophy (SMA), multisystem proteinopathy (MSP) and frontotemporal lobar degeneration (FTLD). In adults, ALS is the most common motor neuron disorder and is characterized by progressive loss of upper and lower motor neurons, which leads to fatal paralysis  in 2006. It was identified as a major component of ubiquitinated cytoplasmic inclusions in spinal motor neurons from ALS patients  A1 is a member of the hnRNP family, a group of related proteins known to associate with nascent mRNA , a group of pleiotropic neurodegenerative disorders that includes ALS. Kim et al.  mutation was identified . However, in the case of Alzheimer’s disease, a simple link between hnRNP protein levels and disease has been established. Along with hnRNP A2/B1, hnRNP A1 was shown to be depleted in Alzheimer’s patient brains  in target transcripts  and B1 (353 amino acids), both transcribed from the HNRNPA2B1 (p. D290V) has been reported in a family with MSP, but not specifically ALS    in 735 individuals diagnosed with ALS and discovered several mutations in patients with familial ALS but not in healthy individuals: M368T, G391E, R408C, and G473E  increased the number of TAF15-positive cytoplasmic foci in the dendrites and axons of embryonic rat cultured neurons above wild-type levels  can offer insight into common RNA targets. PAR-CLIP studies performed for FET proteins in human cell lines showed that the family members bind many of the same mRNAs: the RNA targets of FUS, EWSR1, and TAF15 overlapped with ~32, 69, and 48%, respectively, of RNA targets of the other two members , there are clear overlaps in physiological and pathological functions. Considering how the functions of these proteins relate to the more global properties of the nerves highlights one of the more perplexing conundrums in the field of ALS: why the neuron? Indeed, all of the proteins discussed here are ubiquitously expressed, and yet patients typically exhibit symptoms in only a few specific tissues, usually the central nervous system. By considering the properties of ALS-linked RBPs, we can speculate on why this may be the case.We propose two non-exclusive hypotheses. First, nearly every protein discussed above is involved in RNA transport. The upper and lower motor neurons are the longest cells in the body and proper function requires the transport of RNP granules over nearly a meter of distance. Perhaps, due to their unique physiology, motor neurons are most susceptible to subtle disturbances in the RNA transport machinery. Second, most of the proteins discussed in this review are directly or indirectly involved in RNA splicing. RNA splicing is most prevalent in cells of the nervous system than in any other cell types (Yeo et al.


Viruses have been shown to be equipped with regulatory RNAs to evade host defense system. It has long been known that rabies virus (RABV) transcribes a small regulatory RNA, leader RNA (leRNA), which mediates the transition from viral RNA transcription to replication. However, the detailed molecular mechanism remains enigmatic. In the present study, we determined the genetic architecture of RABV leRNA and demonstrated its inhibitory effect on replication of wild-type rabies, DRV-AH08. The RNA immunoprecipitation results suggest that leRNA inhibits RABV replication via interfering the binding of RABV nucleoprotein with genomic RNA. Furthermore, we identified heat shock cognate 70 kDa protein (Hsc70) as a leRNA host cellular interacting protein, of which the expression level was dynamically regulated by RABV infection. Notably, our data suggest that Hsc70 was involved in suppressing RABV replication by leader RNA. Finally, our experiments imply that leRNA might be potentially useful as a novel drug in rabies post-exposure prophylaxis. Together, this study suggested leRNA in concert with its host interacting protein Hsc70, dynamically down-regulate RABV replication. During the last two decades, tremendous evidence has unveiled regulatory non-coding RNA (ncRNA) as a leading actor in the RNA world throughout nearly all organisms ranging from viruses to humans . In eukaRhabdoviridae family including VSV [LeRNA has been discovered in RABV for more than 30 years, but its function and the underlying mechanism are still poorly understood –16. RABVding VSV . Variousding VSV . LeRNAs ding VSV . Previouding VSV . Similarding VSV . When smding VSV . A membeding VSV , 25. It ding VSV . La protding VSV . It is ading VSV , 28. Mording VSV . Thus, tding VSV . Althougin vitro and in vivo function of leRNA on RABV replication of DRV-AH08, a wild type RABV strain, and the detailed molecular mechanisms. To this end, we applied aptamer mediated RNA-protein precipitation assay coupled with mass spectrometric analysis and identified a host cell interacting protein of leRNA, Hsc70. This protein has been shown to bind to AU-rich elements (AREs) in the 3′-untranslated region of specific mRNAs to enhance their stability [in vivo experiments, we showed that leRNA regulates RABV replication via interacting with Hsc70 and knockdown of the Hsc70 resulted in the suppression of RABV replication. In addition, our in vitro and in vivo experiments implied that leRNA might be potentially useful as a novel drug in rabies post-exposure prophylaxis.The aim of this study is to investigate the tability  and disptability , autophatability , synaptitability , and nottability . ThroughTo determine the precise sequence and length of leRNA, the total RNAs were extracted from mouse brain infected with RABV DRV-AH08 strain, a wild-type RABV. The RNA was subjected to sequencing of the 3′ and 5′ ends of leRNA. Firstly, the total RNAs were added with Poly(A) tail through Poly(A) Polymerase and reversely transcribed by RT primers containing adapter 1 before oligo d(T) Figure . Then thTo further validate the length of leRNA, total RNA was extracted from DRV-AH08-infected SK-N-SH cells at 12 h post-infection, and ligated an adaptor named 3′ SR to the 3′ end of the total RNA. Specific reverse transcript primer complementary with 3′ SR was used for reverse transcription. After PCR amplification by Illumina sequence primer, the DNA fragments were subjected to high throughput sequencing Figure . AnalysiSimilarly, for the 5′ end library, cDNA of leRNA was ligated with adapter 2 containing NNNN overhang to match the 3′ terminal of the cDNA Figure , then amhttp://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi) show a typical hairpin structure for the 56 nt, 58 nt and 64 nt leRNA from DRV-AH08  and scrambled RNA (64 nt) were cloned into pAAV-U6 between r Figure . As showA Figure  and 2E. A Figure  and 2G, A Figure  and 2I tin vivo, more than six mice were simultaneously injected with rAAV-U6-leRNA AAV virus and rAAV-U6-control RNA AAV virus  in the left and right hippocampus, respectively, followed by challenge with DRV-AH08. The injection of AAV viruses expressing leRNA or control RNA were indicated by the mCherry fluorescence in the hippocampus  was measured in SK-N-SH cells infected with DRV-AH08 12 h and 30 h post-infection in the presence of rAAV-U6-leRNA AAV virus or rAAV-U6-control RNA AAV virus. The level of vRNA was significantly decreased by leRNA overexpression as compared with that by control RNA (40%) at 12 h Figure  and 30 hAs it has been shown that RABV N protein binds preferentially to the leRNA than the genomic RNA , we invein vitro transcribed tRSA-leRNA was mixed with SA beads (Streptavidin beads), followed by incubation with mouse brain lysates   mediateds Figure . After Ss Figure . By thiss Figure  , followed by rAAV-U6-leRNA AAV virus or rAAV-U6-control RNA AAV virus injection in the same site 1 h post-exposure respectively. The clinical signs and death were monitored for 25 days. As shown in Figure in vivo. Taken together, leRNA could inhibit DRV-AH08 replication post-infection both in vitro and in vivo, implying a potential application of leRNA in rabies post-exposure prophylaxis.Considering the inhibitory effect of leRNA on RABV replication, it is of great necessity to explore the potential application of leRNA in rabies post-exposure prophylaxis. To this end, SK-N-SH cells were firstly infected by DRV-AH08, and then transfected with s Figure . The meas Figure . Importas Figure  and 7D. During the endless arms’ race between the host and the virus, both belligerent parties have equipped ncRNA to fight with each other. While the role of host ncRNA during antiviral defense has been well documented, little is known about the molecular function of viral ncRNA during infection. It has long been known that negative-strand RNA viruses, such as VSV and RABV, transcribe a small RNA from the 3′ end of their genome , 40. PreIt has been reported that leRNA plays an important role in regulating RABV replication and transcription . LeRNA iIn the present study, a host protein, Hsc70, has been identified to interact with RABV leRNA. Using tRSA aptamer-mediated RNA-binding protein precipitation experiment and EMSA assay, it is demonstrated that leRNA directly binds to Hsc70. The RNA-Protein complex modelling suggests that the 51–59 nt of leRNAs are likely packaged by a positively charged amino acids rich N-terminal nucleotide-binding pocket (NBD) in Hsc70. Interestingly, the sequence from 51- 59 nt of leRNA was an AU rich domain, consistent with previous data showing that Hsc70 binds to AU-rich elements (AREs) . As Hsc7in vitro and in vivo, the potential to use leRNA as a novel drug in rabies post-exposure prophylaxis was determined. Currently a combination of rabies vaccination and rabies immunoglobulin was given to people severely exposed to rabies according to WHO recommendations [in vivo by AAV vector, it could be further optimized by increasing the leRNA at the site of infection such as direct electroplating leRNA.Since over-expression of leRNA specifically inhibits RABV replication ndations . Howeverndations . RNA-basndations –53. Compin vitro transcribed to obtain bait RNA which is used in RNA binding protein pull-down assay. The DRV-AH08 N protein with Fc tag and P protein were cloned into pcDNA-3.1, respectively. The genome fragment (1–1514 bp) was also cloned into pcDNA-3.1 flanked by hammerhead ribozyme (HamRz) and hepatitis delta virus ribozyme (HdvRz) sequence cDNA.The sequence of Hsc70 shRNA (GGTGTGCTT ATTCAGGTTTATGAAGCTTGATAAACCTGAATAAG CACACC), shGFP (TTGATGCCGTTCTTCTGCTTGT CGAAGCTTGGACAAGCAGAAGAACGGCATCAA), scrambled leader RNA (AGGACTCTATGTATTAGCA CAAGAACAACTAACTATCAAACGTAAAGACAAG AGGATCAAACAC) and leRNA  were constructed into pAAV-U6 vector containing U6 promoter and mCherry reporter to obtain pAAV-U6-shHsc70, pAAV-U6-shGFP, pAAV-U6-scrambled RNA and pAAV-U6-leRNA plasmids. The RNA bait was cloned into pcDNA3-tRSA  and then 2. Human SK-N-SH cell line was grown in Minimum Essential Medium  supplemented with 15% FBS.Mouse neuroblastoma (NA) cells and HEK-293T cells were cultured in RPMI 1640 medium (Gibco) containing 10% fetal bovine serum  at 37°C with 5% CODRV-AH08 is a wild type rabies virus (RABV) isolated from a pathogenetic dog in Anhui province, China . Virus s® Select Master Mix kit (Life technologies) were used. For the quantification of leRNA, cDNAs were synthesized with tagged primer (CCAGATGCTTGGCGTCCTGCTTTA CAAATGACGCTGTC) with an 18-nucleotide (nt) tag that was unrelated to RABV. qRT-PCR was performed using primers leRNA-F (CCAGATGCTTGGCGTCCT) and leRNA-R (ACGCTTAACAACAAAATC). However, these primers could also amplify the antigenomic RNA, of which the level is negligible compared to that of genomic RNA [Total RNA were extracted from cells using TROZIL (Invitrogen). The cDNA of leRNA and viral genomic RNA (vRNA) were reversely transcribed by AMV reverse transcriptase (Takara) and specific primers. For quantification, 1 μl of cDNA, 5 pM of primers and SYBRomic RNA . For thein vitro transcription using AmpliScribe™ T7-Flash™ Transcription Kit (Epicentre Biotechnologies). The in vitro transcribed RNAs were checked by electrophoresis and spectrometry, and then stored at –80°C.The sense and anti-sense scrambled RNA and leRNA were synthesized from Genescript (China) and cloned into pcDNA-3-T7 vector after annealing. The plasmids were linearized by enzyme digestion, and then subjected to in vitro synthetic RNAs were denatured at 65°C for 5min, and then applied to 50 μl streptavidin beads (Thermo) in 800 μllysis buffer  with protease inhibitor (Roche) and RNasin  after cooling to room temperature. The mixer were incubated on a rotation shaker at 4°C for 2–2.5 h. Total mouse brain lysate was collected by centrifuging at 4°C for 10 min at 16,000 g after grinded in lysis buffer  with protease inhibitor (Roche) and RNasin . The supernatants were incubated with Yeast RNA (Sigma) and Egg white avidin  to block endogenous biotinylated proteins and non-specific RNPs, and were cleared by centrifuge. Next, the cleared lysates were incubated with RNA-beads in a new tube on a rotation shaker at 4°C for 2.5–3 h. The supernatant was discarded after centrifugation for 2 min at 4°C. After washing with fresh lysis buffer for 5 times, the captured proteins were eluted and analyzed by SDS–PAGE with silver staining. The specific RNA binding protein bands were cut from the gel and subjected tocommercial mass spectrometry analysis .The RNA binding protein pull-down assay was performed as described . BrieflyN protein with Fc tag, P protein and DRV-AH08 genomic fragment (1–1514 bp) (Described above) were co-expressed with the leRNA or control RNA in HEK-293T cells. The cells were harvested by trypsinization and resuspended in RIPA buffer (with 200 U/ml RNase inhibitor and protease inhibitor), kept on ice for 20 min (with frequent mixing). Centrifuged at 4°C, 10000 g for 10 min. Take 100 μlof the supernatant for the detection of input protein and input RNA. The proteins in supernatant were treated with protein A/G (a recombinant fusion protein that combines IgG binding domains of both Protein A and Protein G) beads (40 μl) (Santa cruz) and incubated for 2 h at 4°C with gentle rotation. The unbound material (proteins) was washed- off for 5 times by RIPA buffer. Isolate N protein coprecipitated RNAs by resuspending beads in TRIZOL RNA extraction reagent (1 ml). Reverse transcription of DNase treated RNA to cDNA and analysis by qRT-PCR.in vitro and in vivo. Three kinds of control RNAs for leRNA with hairpin structure but no homologous with the genome of human, mouse and rabies virus, had been used. One of the control RNAs (CACATGAAGCAGCACGACTTC TTGAAGCTTGAAGAAGTCGTGCTGCTTCATGTG) has been cloned into pAAV-U6 to get pAAV-U6-cotrol RNA plasmid and rAAV-U6-cotrol RNA AAV virus, one of the control RNAs (UUCUCCGAACGUGUCACGUAC GUGACACGUUCGGAGAA) has been synthesized and used in transfection, and another control RNA was scrambled leader RNA (AGGACTCTATGTATTAG CACAAGAACAACTAACTATCAAACGTAAAGACAA GAGGATCAAACAC) has been cloned into pAAV-U6 to get pAAV-U6-scrambled RNA plasmid and used in transfection.For shRNA, scrambled leader RNA and leRNA, the sense and anti-sense ssDNA oligonucleotides were synthesized and inserted into pAAV-U6 after annealing. The AAV virus rAAV-U6-shRNA and rAAV-U6-leRNA were produced in HEK-293T cells as previously described  to expre2, 20 mM DTT, 50% v/v glycerol, 0.5 mg/mL BSA). Electrophoresis was carried out on 8% w/v polyacrylamide non-denaturing gel in 0.5 × TG buffer . Gels were stained using SYBR® Gold Nucleic Acid Gel Stain . Scrambled RNA was also incubated with Hsc70 for 30 min at room temperature in binding buffer too.Hsc70 and the control protein 4BOW with 6x His tag were expressed in prokaryotic expression system and purified. LeRNA was incubated with Hsc70 and 4BOW respectively for 30 min at room temperature in binding buffer  for 4 h at 37°C in PBS. Images were obtained by epifluorescence microscope .Cells infected with DRV-AH08 were fixed with 80% ice-cold acetone for 30 min at room temperature, Then cells were incubated with FITC conjugated anti-rabies monoclonal antibody  for 1 h at 37°C after washing for three times with PBS. Adult mice were anesthetized and perfused transcardially with 4% PFA/PBS. The brains were dissected out and post-fixed in 4% PFA in PBS overnight at 4°C. Brain sections (30μm) were prepared with vibratome, and then incubated with mouse anti-P monoclonal antibody in PBS with 0.2% Triton X-100 and 1% BSA at 4°C overnight. Signals were developed by incubating sections with Alexa FluorThe Control RNA-expressing rAAV and leRNA-expressing rAAV were stereotactically injected to the mouse hippocampus or intramuscularly injected to the mouse hind limb. Rabies virus (DRV-AH08) was injected intramuscularly. Mice were anaesthetized and perfused with PBS, and brains were fixed with 4% paraformaldehyde (PFA). All of the animal experiments were approved by the Research Ethics Committee, Huazhong Agricultural University, Hubei, China (HZAUMO2015-0016) and in accordance with the Guide for the Care and Use of Laboratory Animals from Research Ethics Committee, Huazhong Agricultural University.http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi).RNA fold webserve was used to build the leRNA secondary structure. After the sequence of leRNA was imported to the Webserver, the suggested minimum free energy RNA secondary structure was returned . The statistical significance of survival rates was determined by the log-rank test and Kaplan-Meier survival analysis. Differences were considered statistically significant when P < 0.05.The significant differences of virus titer, protein expression, RNA levels, and mean fluorescence intensity were analyzed using Student


The recovery of ancient RNA from archeological material could enable the direct study of microevolutionary processes. Small RNAs are a rich source of information because their small size is compatible with biomolecular preservation, and their roles in gene regulation make them likely foci of evolutionary change. We present here the small RNA fraction from a sample of archeological barley generated using high-throughput sequencing that has previously been associated with localized adaptation to drought. Its microRNA profile is broadly similar to 19 globally distributed modern barley samples with the exception of three microRNAs , all of which are known to have variable expression under stress conditions. We also found retrotransposon activity to be significantly reduced in the archeological barley compared with the controls, where one would expect the opposite under stress conditions. We suggest that the archeological barley’s conflicting stress signals could be the result of long-term adaptation to its local environment. As domesticated plants were transported from their areas of origin they continued to evolve and adapt under domestication to these new environments. Such adaptations included to changes in seasonality  and to lvrs1.a1 allele inactive, resulting in development of spikelets lateral to the two central spikelets and hence six rows of grain. In all cultural stages, the barley at Qasr Ibrim shows a two-row phenotype combined with the genetic basis of a six-row type. We confirmed the genetic basis of the six-rowed type in the Qasr Ibrim barley by sequencing the vrs1  and found both to be consistent with the respective six-rowed alleles. This suggests a reassertion of two-row over six-row through a novel mechanism that is indicative of a strong selection pressure. The most likely pressure in this case was water stress given the aridity of the site and extremely short growing season determined by basin irrigation , the total RNA content of the archeological barley was determined to be three times that of the DNA content with an average of 1.45 µg per seed DNA content and 4.45 µg per seed RNA content. A total of 59,702,667 reads were generated from the archeological barley using the Illumina HiSeq 2500 platform, and an average of 696,171 reads were generated from each of the control samples using the Illumina MiSeq platform. We opted for a single-sample run using the HiSeq for the archeological sample to preempt low copy numbers of microRNA as a result of diagenesis, and to compensate for possible overrepresentation of other RNA breakdown products. Of the archeological reads generated, 59% fell into the small regulatory RNA size range, whereas the same average figure was 17% for the modern barley. Metagenomic analysis established that 93% of assignable reads were barley in the archeological data set  online, The composition of the barley small RNA was similar between control samples, but the archeological sample was markedly different. The majority of reads from the archeological sample were assigned as ribosomal at 71% compared with a mean of 23% in the control samples. tRNA and mRNA were all likewise highly overrepresented in the archeological sample proportionally to miRNA, compared with all control samples  online. 1938 HiSeq reads from a total of 59,702,667 reads were identified as known miRNAs from the archeological barley, representing 23 families referenced against miRBase . 22053 MP values <0.0001), the profiles of archeological and control samples however differ significantly; linear regression shows a low degree of correlation . This is due to notable differences in four miRNA families: miR159, miR319, and miR5139 both highly abundant in the archeological sample compared with the controls, and miR396, highly abundant in the control samples compared with the archeological sample . The similarity of these profiles suggests that these two data sets can be directly compared meaningfully, and supports the notion that RNA frequencies can be usefully retrieved from the archeological record.While the profiles between control samples show high levels of similarity  genes . GRF conr2 = 0.00059, in the ancient, and 0.00541 in the modern, To determine whether it is more likely that the miRNA expression profiles in the archeological barley represent an immediate stress response to the environment or a more stable adaptive state, we assessed the inferred state of stress by examining evidence of retroelement activity. Increased retroelement activity under a variety of biotic and abiotic stress in plants has been well documented . A totalThese data suggest retroelement activity in the archeological sample is on a comparable order of magnitude to the controls, often lower, and therefore not indicative of an increased stress response relative to the controls. Previous work relating to the same sample  has showvrs1 allele diverging from the expected phenotype at multiple strata observed at Qasr Ibrim, combined with all other similar studies showing an expected match between phenotype and vrs1 allele  was determined by the Nile flood and the ensuing time it took for the soil to dry out again, about 120 days  was supplied by Dr Clapham. 15 accessions of modern barley were supplied by the USDA: PI 642783 (Giza), PI 182613 (Japan), PI 392528 (South Africa), PI 510559 (Peru), PI 524707 (Egypt), PI290743 (Kenya), PI 100121 (India), PI 65216 (China), PI 524708 (Egypt dryland), PI 168904 (Argentina), PI 490393 , PI 642787 (Giza), CIho5059 , and GSHO842 (Japan). Two accessions were supplied by the John Innes Centre: B9604 (Iraq) and B7297 (Iran). Two accessions were supplied by NIAB: Tipple and Line 14 hulled (both U.K.).All ancient nucleic acids were handled in a dedicated ancient DNA laboratory. RNA was isolated using a modified protocol of the Ambion MirVana RNA isolation kit. Six grains from the same archeological context were extracted in bulk as a single sample. Modifications included an extended incubation time in 2% CTAB buffer (5 days at 37°C) followed by a single chloroform extraction and isopropanol precipitation, before resuspending the RNA pellet in the supplied binding buffer and continuing the protocol as per the manufacturer’s instructions. Because the vast majority of archeological RNA fragments already exist within the target size for column based enrichment of small fragments (<300 nt), this step was deemed unnecessary and omitted to avoid further loss of molecules. RNA was extracted from control samples in a separate, nonPCR laboratory using a modified protocol of the Ambion MirVana RNA isolation kit. Crushed seeds were incubated for 1 h at 65°C in 2% CTAB buffer followed by a single chloroform extraction and ispropanol precipitation. Due to the high starch content of modern grains compared with the archeological, RNA pellets were dissolved in SSTE buffer to remove polysaccharides, and RNA reprecipitated with isopropanol. Pellets were resuspended in the supplied binding buffer and the protocol was completed according to manufacturer’s instructions. Small RNA fragments (<300 nt) were enriched using the manufacturer’s column-based method. Since mechanical size enrichment of this type has not been reported to introduce sequence bias, we do not anticipate this modification influenced the results. Further, we do not see any order-of-magnitude differences in reported relative expression from similarly sized transcripts such as transposable elements or ribosomal transcripts, suggesting the differences in protocol introduce minimal, if any, bias into our molecular classification. RNA for all samples were quantified using the Qubit RNA assays.A singleplex library was created for the archeological sample using an Air Small RNA Library Preparation Kit  according to manufacturer’s instructions, and run as a single lane on the Illumina HiSeq platform by BGI . Control sample libraries were made using a NextFlex Small RNA Sequncing Kit  due to multiplexing allowances, again following the manufacturer’s protocol. We used standard indexing primers for multiplexing, and run on the Illumina MiSeq platform using a v2 reagent kit. All library enrichment took place using eight PCR cycles, and all sequencing runs took place with 50 rounds of sequencing.Primary data processing  was carried out using scripts of the author’s design. The majority of pattern matching exercises were performed using bowtie version 0.12. Separate bowtie indexes were created for each molecule type from corresponding fasta files: complete barley genome cultivar Morex, mature plant miRNA from mirbase, complete graminae retroelements from Michigan State University’s Plant Repeat Database, and individual genes of interest  from NCBI. In all cases of bowtie pattern matching, a single mismatch was allowed in the archeological sample but only where a positive-strand C > U or negative strand G > A base modification occurred. This was to compensate for postmortem deamination known to occur in ancient nucleic acids . For theNo comprehensive list of primary miRNA transcripts exists, so a database was created based on homologous premiRNA stem loop sequences. Pri-miRNA sequence data for all available plant species was downloaded from mirbase and subjected to BLAST searching against a database created from the barley cultivar Morex genome (contigs of ∼100–800 nt each). PremiRNA sequences matching to the Morex genome under standalone BLASTn parameters were considered to be homologous if originated in other plant species. Morex contigs to which homologous stem-loop sequences matched were considered to be representative of miRNA primary transcripts. These contigs were extracted and converted to a bowtie index to which the Illumina sequences of archeological and control samples were matched, again allowing a single deamination mismatch in the archeological sample.For metagenomic analysis, sequences were processed using standalone blastn, using the complete nucleotide (nt) database and preset parameters for short sequences (“blastn-short”), which reduces word size values to 7 over the default 11. This was deemed the most appropriate method due to the ultrashort nature of sequences being evaluated.Phylogenetic Intersect Analysis (PIA) was performed on a subset of reads to verify the expected levels of endogenous miRNAs, by proxy of confidence in their taxonomic assignations. Plant small RNA fractions are typically ultrashort and highly conserved, and this poses challenges for discriminant phylogenetic analyses such as PIA. Essentially, many sRNA sequences are so highly conserved, and by virtue of their sizes are often coincidentally present in nonplants, they are unresolvable from almost a basal level on the phylogenetic tree. To account for this, we took 10,000 redundant sequences and ran them through the PIA pipeline as normal. We disregarded those sequences whose second BLAST hit had an identical score to the first, then filtered the remaining reads by disregarding those with an unknowable classification intersect value. We then remapped the remaining reads to the barley genome. Finally, we assessed the number of exogenous reads by isolating those meeting two criteria: not mapping to the genome, and not consistent with a putative barley assignation under PIA . We then applied redundancy frequencies to these data to calculate the overall proportions of endogenous and exogenous reads. We found the endogenous content of the barley to be at least 87.6%, and the accuracy of PIA to be 80.6%, both values being consistent with our previous estimates .VRS1 and INT-C genes  was found in the Qasr Ibrim barley across samples, which is the vrs1 allele associated with the six row phenotype.Barley architecture is controlled by both -C genes . To checentotype  despite GC content for sequences matching conserved miRNAs  online wT1/2) using the equation:To estimate the half-life of RNA at the site, we used a per-seed calculation from modern and archeological barleys since per-mass calculations would be skewed due to desiccation. We observed a mean RNA value of 4.45 μg per seed in the archeological, and 65.2 μg per seed in the modern sample of the closest geographical match (Giza). We then calculated the RNA half-life . Frequencies of conserved miRNA families were normalized between the control samples by dividing the frequency of each miRNA family in each sample by the ratio of total miRNA frequency to total reads in that sample, and then multiplying that value by the mean ratio of total miRNA frequencies to total reads (0.00165). The adjusted values were then used to normalize the miRNA frequencies of the archeological sample, by calculating the mean adjusted total miRNA frequency per control (1145.5) and dividing this by the total miRNA frequency of the archeological sample to give an adjustment ratio of 0.59. Raw frequencies of miRNA families in the archeological sample were multiplied by this ratio to give an adjusted frequency. Comparisons of modern and archeological were simplified for graphical representation by calculating the normalized mean of all control samples for each miRNA family and plotting these values against normalized archeological reads for each miRNA family. Standard deviations between controls were expressed as error bars .r) of normalized miRNA counts for each miRNA, and obtained the average value. The likelihood kernel was then based on a Normal approximation of an extended Poisson process using a variance equal to the product of the mean and the ratio (r), using equation 5.1a.a of Likelihood kernels were generated using a standard Poisson process for figures S4 and S5 where low copy numbers occurred (<50). Large RNA count numbers are known to deviate from pure Poisson processes , but extMolecular Biology and Evolution online.Supplementary data are available at Supplementary DataClick here for additional data file.