Hidden Markov Modeling with HMMTeacher.

Is it possible to learn and create a first Hidden Markov Model (HMM) without programming skills or understanding the algorithms in detail? In this concise tutorial, we present the HMM through the 2 general questions it was initially developed to answer and describe its elements. The HMM elements include variables, hidden and observed parameters, the vector of initial probabilities, and the transition and emission probability matrices. Then, we suggest a set of ordered steps, for modeling the variables and illustrate them with a simple exercise of modeling and predicting transmembrane segments in a protein sequence. Finally, we show how to interpret the results of the algorithms for this particular problem. To guide the process of information input and explicit solution of the basic HMM algorithms that answer the HMM questions posed, we developed an educational webserver called HMMTeacher. Additional solved HMM modeling exercises can be found in the user's manual and answers to frequently asked questions. HMMTeacher is available at https://hmmteacher.mobilomics.org, mirrored at https://hmmteacher1.mobilomics.org. A repository with the code of the tool and the webpage is available at https://gitlab.com/kmilo.f/hmmteacher.

Extracellular Vesicles Secreted by Pre-Hatching Bovine Embryos Produced In Vitro and In Vivo Alter the Expression of IFNtau-Stimulated Genes in Bovine Endometrial Cells.

The embryo-maternal interaction occurs during the early stages of embryo development and is essential for the implantation and full-term development of the embryo. In bovines, the secretion of interferon Tau (IFNT) during elongation is the main signal for pregnancy recognition, but its expression starts around the blastocyst stage. Embryos release extracellular vesicles (EVs) as an alternative mechanism of embryo-maternal communication. The aim of the study was to determine whether EVs secreted by bovine embryos during blastulation (D5-D7) could induce transcriptomic modifications, activating IFNT signaling in endometrial cells. Additionally, it aims to assess whether the EVs secreted by embryos produced in vivo (EVs-IVV) or in vitro (EVs-IVP) have different effects on the transcriptomic profiles of the endometrial cells. In vitro- and in vivo-produced bovine morulae were selected and individually cultured for 48 h to collect embryonic EVs (E-EVs) secreted during blastulation. E-EVs stained with PKH67 were added to in vitro-cultured bovine endometrial cells to assess EV internalization. The effect of EVs on the transcriptomic profile of endometrial cells was determined by RNA sequencing. EVs from both types of embryos induced several classical and non-classical IFNT-stimulated genes (ISGs) and other pathways related to endometrial function in epithelial endometrial cells. Higher numbers of differentially expressed genes (3552) were induced by EVs released by IVP embryos compared to EVs from IVV (1838). Gene ontology analysis showed that EVs-IVP/IVV induced the upregulation of the extracellular exosome pathway, the cellular response to stimulus, and the protein modification processes. This work provides evidence regarding the effect of embryo origin (in vivo or in vitro) on the early embryo-maternal interaction mediated by extracellular vesicles.

MicroRNAs from Extracellular Vesicles Secreted by Bovine Embryos as Early Biomarkers of Developmental Competence.

During early development, embryos secrete extracellular vesicles (EVs) that participate in embryo-maternal communication. Among other molecules, EVs carry microRNAs (miRNAs) that interfere with gene expression in target cells; miRNAs participate in embryo-maternal communication. Embryo selection based on secreted miRNAs may have an impact on bovine breeding programs. This research aimed to evaluate the size, concentration, and miRNA content of EVs secreted by bovine embryos with different developmental potential, during the compaction period (days 3.5-5). Individual culture media from in vitro-produced embryos were collected at day 5, while embryos were further cultured and classified at day 7, as G1 (conditioned-culture media by embryos arrested in the 8-16-cells stage) and G2 (conditioned-culture media by embryos that reached blastocyst stages at day 7). Collected nanoparticles from embryo conditioned culture media were cataloged as EVs by their morphology and the presence of classical molecular markers. Size and concentration of EVs from G1 were higher than EVs secreted by G2. We identified 95 miRNAs; bta-miR-103, bta-miR-502a, bta-miR-100, and bta-miR-1 were upregulated in G1, whereas bta-miR-92a, bta-miR-140, bta-miR-2285a, and bta-miR-222 were downregulated. The most significant upregulated pathways were fatty acid biosynthesis and metabolism, lysine degradation, gap junction, and signaling pathways regulating pluripotency of stem cells. The characteristics of EVs secreted by bovine embryos during the compaction period vary according to embryo competence. Embryos that reach the blastocyst stage secrete fewer and smaller vesicles. Furthermore, the loading of specific miRNAs into the EVs depends on embryo developmental competence.

TEcandidates: prediction of genomic origin of expressed transposable elements using RNA-seq data.

Motivation: In recent years, Transposable Elements (TEs) have been related to gene regulation. However, estimating the origin of expression of TEs through RNA-seq is complicated by multi-mapping reads coming from their repetitive sequences. Current approaches that address multi-mapping reads are focused in expression quantification and not in finding the origin of expression. Addressing the genomic origin of expressed TEs could further aid in understanding the role that TEs might have in the cell. Results: We have developed a new pipeline called TEcandidates, based on de novo transcriptome assembly to assess the instances of TEs being expressed, along with their location, to include in downstream DE analysis. TEcandidates takes as input the RNA-seq data, the genome sequence and the TE annotation file and returns a list of coordinates of candidate TEs being expressed, the TEs that have been removed and the genome sequence with removed TEs as masked. This masked genome is suited to include TEs in downstream expression analysis, as the ambiguity of reads coming from TEs is significantly reduced in the mapping step of the analysis. Availability and implementation: The script which runs the pipeline can be downloaded at http://www.mobilomics.org/tecandidates/downloads or http://github.com/TEcandidates/TEcandidates. Supplementary information: Supplementary data are available at Bioinformatics online.

Towards the bridging of molecular genetics data across Xenopus species.

BACKGROUND: The clawed African frog Xenopus laevis has been one of the main vertebrate models for studies in developmental biology. However, for genetic studies, Xenopus tropicalis has been the experimental model of choice because it shorter life cycle and due to a more tractable genome that does not result from genome duplication as in the case of X. laevis. Today, although still organized in a large number of scaffolds, nearly 85% of X. tropicalis and 89% of X. laevis genomes have been sequenced. There is expectation for a comparative physical map that can be used as a Rosetta Stone between X. laevis genetic studies and X. tropicalis genomic research. RESULTS: In this work, we have mapped using coarse-grained alignment the 18 chromosomes of X. laevis, release 9.1, on the 10 reference scaffolds representing the haploid genome of X. tropicalis, release 9.0. After validating the mapping with theoretical data, and estimating reference averages of genome sequence identity, 37 to 44% between the two species, we have carried out a synteny analysis for 2,112 orthologous genes. We found that 99.6% of genes are in the same organization. CONCLUSIONS: Taken together, our results make possible to establish the correspondence between 62 and 65.5% of both genomes, percentage of identity, synteny and automatic annotation of transcripts of both species, providing a new and more comprehensive tool for comparative analysis of these two species, by allowing to bridge molecular genetics data among them.

K2p channels in plants and animals.

Two-pore domain potassium (K2P) channels are membrane proteins widely identified in mammals, plants, and other organisms. A functional channel is a dimer with each subunit comprising two pore-forming loops and four transmembrane domains. The genome of the model plant Arabidopsis thaliana harbors five genes coding for K2P channels. Homologs of Arabidopsis K2P channels have been found in all higher plants sequenced so far. As with the K2P channels in mammals, plant K2P channels are targets of external and internal stimuli, which fine-tune the electrical properties of the membrane for specialized transport and/or signaling tasks. Plant K2P channels are modulated by signaling molecules such as intracellular H(+) and calcium and physical factors like temperature and pressure. In this review, we ask the following: What are the similarities and differences between K2P channels in plants and animals in terms of their physiology? What is the nature of the last common ancestor (LCA) of these two groups of proteins? To answer these questions, we present physiological, structural, and phylogenetic evidence that discards the hypothesis proposing that the duplication and fusion that gave rise to the K2P channels occurred in a prokaryote LCA. Conversely, we argue that the K2P LCA was most likely a eukaryote organism. Consideration of plant and animal K2P channels in the same study is novel and likely to stimulate further exchange of ideas between students of these fields.

Evolution of ion channels in cetaceans: a natural experiment in the tree of life.

Cetaceans represent a natural experiment within the tree of life in which a lineage changed from terrestrial to aquatic habitats. This shift involved phenotypic modifications, representing an opportunity to explore the genetic bases of phenotypic diversity. Among the different molecular systems that maintain cellular homeostasis, ion channels are crucial for the proper physiological functioning of all living species. This study aims to explore the evolution of ion channels during the evolutionary history of cetaceans. To do so, we created a bioinformatic pipeline to annotate the repertoire of ion channels in the genome of the species included in our sampling. Our main results show that cetaceans have, on average, fewer protein-coding genes and a higher percentage of annotated ion channels than non-cetacean mammals. Signals of positive selection were detected in ion channels related to the heart, locomotion, visual and neurological phenotypes. Interestingly, we predict that the NaV1.5 ion channel of most toothed whales (odontocetes) is sensitive to tetrodotoxin, similar to NaV1.7, given the presence of tyrosine instead of cysteine, in a specific position of the ion channel. Finally, the gene turnover rate of the cetacean crown group is more than three times faster than that of non-cetacean mammals.

Extracellular vesicles secreted by equine adipose mesenchymal stem cells preconditioned with transforming growth factor ß-1 are enriched in anti-fibrotic miRNAs and inhibit the expression of fibrotic genes in an in vitro system of endometrial stromal cells fibrosis.

Mare endometrosis is a major reproductive problem associated with low fertility and is characterized by persistent inflammation, TGFß-1 signaling, and consequently, extracellular matrix deposition, which compromises endometrial glands. Mesenchymal stem cell-based products (MSCs), such as extracellular vesicles (EVs), have gained attention due to the regulatory effects exerted by their miRNA cargo. Here, we evaluated the impact of preconditioning equine adipose mesenchymal stem cells with TGFß-1 for short or long periods on the anti-fibrotic properties of secreted extracellular vesicles. MSCs were isolated from six healthy horses and exposed to TGFß-1 for 4, 24, and 0 h. The expression of anti-fibrotic and pro-fibrotic miRNAs and mRNAs in treated cells and miRNAs in the cargo of secreted extracellular vesicles was measured. The resulting EVs were added for 48 h to endometrial stromal cells previously induced to a fibrotic status. The expression of anti-fibrotic and pro-fibrotic genes and miRNAs was evaluated in said cells using qPCR and next-generation sequencing. Preconditioning MSCs with TGFß-1 for 4 h enriched the anti-fibrotic miRNAs (mir29c, mir145, and mir200) in cells and EVs. Conversely, preconditioning the cells for 24 h leads to a pro-fibrotic phenotype overexpressing mir192 and mir433. This finding might have implications for developing an EV-based protocol to treat endometrial fibrosis in mares.

Bovine embryos release extracellular vesicles with differential miRNA signature during the compaction and blastulation stages.

Pre-implantation embryos release extracellular vesicles (EVs) to extracellular environment. In this work it is hypothesized that the EVs miRNA cargo will vary during pre-implantation development due to the constant changes in gene expression that take place through this period. The concentration, size and miRNA cargo of EVs secreted by competent bovine embryos during the period from compaction to blastulation (Day 3-7) were analyzed. For this analysis tow developmental windows were defined: W2 from 8-cells (D3) to morula (D5) and W3 from morula (D5) to blastocyst (D7). For W2, in vitro produced embryos were individually cultured in EVs-depleted medium from D3 to D5; culture media were collected and assigned to Group W2. Morulae were kept in culture up to blastocyst stage to determine the developmental competence. For W3, D5 morulae were collected and cultured individually in EVs-depleted medium up to blastocyst stage; culture media were assigned to Group W3, and blastocysts were kept in culture up to day 11 to define their competence. The mean size of EVs was similar between groups, however, EVs concentration was lower in W2. A total of 140 miRNAs were identified. From them, 79 were differentially expressed between the groups, 28 upregulated and 51 downregulated. miRNAs differentially detected between both developmental windows participate in the regulation of signaling pathways which crucial for embryonic development. It was concluded that the secretion of EVs is regulated by the developmental progress of the embryo during the pre-implantation period.

Bioinformatic analysis predicts that ethanol exposure during early development causes alternative splicing alterations of genes involved in RNA post-transcriptional regulation.

Prenatal ethanol exposure is associated with neurodevelopmental defects and long-lasting cognitive deficits, which are grouped as fetal alcohol spectrum disorders (FASD). The molecular mechanisms underlying FASD are incompletely characterized. Alternative splicing, including the insertion of microexons (exons of less than 30 nucleotides in length), is highly prevalent in the nervous system. However, whether ethanol exposure can have acute or chronic deleterious effects in this process is poorly understood. In this work, we used the bioinformatic tools VAST-TOOLS, rMATS, MAJIQ, and MicroExonator to predict alternative splicing events affected by ethanol from available RNA sequencing data. Experimental protocols of ethanol exposure included human cortical tissue development, human embryoid body differentiation, and mouse development. We found common genes with predicted differential alternative splicing using distinct bioinformatic tools in different experimental designs. Notably, Gene Ontology and KEGG analysis revealed that the alternative splicing of genes related to RNA processing and protein synthesis was commonly affected in the different ethanol exposure schemes. In addition, the inclusion of microexons was also affected by ethanol. This bioinformatic analysis provides a reliable list of candidate genes whose splicing is affected by ethanol during nervous system development. Furthermore, our results suggest that ethanol particularly modifies the alternative splicing of genes related to post-transcriptional regulation, which probably affects neuronal proteome complexity and brain function.

Mapping the landscape of host-pathogen coevolution: HLA class I binding and its relationship with evolutionary conservation in human and viral proteins.

The high diversity of HLA binding preferences has been driven by the sequence diversity of short segments of relevant pathogenic proteins presented by HLA molecules to the immune system. To identify possible commonalities in HLA binding preferences, we quantify these using a novel measure termed "targeting efficiency," which captures the correlation between HLA-peptide binding affinities and the conservation of the targeted proteomic regions. Analysis of targeting efficiencies for 95 HLA class I alleles over thousands of human proteins and 52 human viruses indicates that HLA molecules preferentially target conserved regions in these proteomes, although the arboviral Flaviviridae are a notable exception where nonconserved regions are preferentially targeted by most alleles. HLA-A alleles and several HLA-B alleles that have maintained close sequence identity with chimpanzee homologues target conserved human proteins and DNA viruses such as Herpesviridae and Adenoviridae most efficiently, while all HLA-B alleles studied efficiently target RNA viruses. These patterns of host and pathogen specialization are both consistent with coevolutionary selection and functionally relevant in specific cases; for example, preferential HLA targeting of conserved proteomic regions is associated with improved outcomes in HIV infection and with protection against dengue hemorrhagic fever. Efficiency analysis provides a novel perspective on the coevolutionary relationship between HLA class I molecular diversity, self-derived peptides that shape T-cell immunity through ontogeny, and the broad range of viruses that subsequently engage with the adaptive immune response.

TnpPred: A Web Service for the Robust Prediction of Prokaryotic Transposases.

Transposases (Tnps) are enzymes that participate in the movement of insertion sequences (ISs) within and between genomes. Genes that encode Tnps are amongst the most abundant and widely distributed genes in nature. However, they are difficult to predict bioinformatically and given the increasing availability of prokaryotic genomes and metagenomes, it is incumbent to develop rapid, high quality automatic annotation of ISs. This need prompted us to develop a web service, termed TnpPred for Tnp discovery. It provides better sensitivity and specificity for Tnp predictions than given by currently available programs as determined by ROC analysis. TnpPred should be useful for improving genome annotation. The TnpPred web service is freely available for noncommercial use.

Lymph Leakage Promotes Immunosuppression by Enhancing Anti-Inflammatory Macrophage Polarization.

Lymphatic vasculature is a network of capillaries and vessels capable of draining extracellular fluid back to blood circulation and to facilitate immune cell migration. Although the role of the lymphatic vasculature as coordinator of fluid homeostasis has been extensively studied, the consequences of abnormal lymphatic vasculature function and impaired lymph drainage have been mostly unexplored. Here, by using the Prox1+/- mice with defective lymphatic vasculature and lymphatic leakage, we provide evidence showing that lymph leakage induces an immunosuppressive environment by promoting anti-inflammatory M2 macrophage polarization in different inflammatory conditions. In fact, by using a mouse model of tail lymphedema where lymphatic vessels are thermal ablated leading to lymph accumulation, an increasing number of anti-inflammatory M2 macrophages are found in the lymphedematous tissue. Moreover, RNA-seq analysis from different human tumors shows that reduced lymphatic signature, a hallmark of lymphatic dysfunction, is associated with increased M2 and reduced M1 macrophage signatures, impacting the survival of the patients. In summary, we show that lymphatic vascular leakage promotes an immunosuppressive environment by enhancing anti-inflammatory macrophage differentiation, with relevance in clinical conditions such as inflammatory bowel diseases or cancer.

GSER (a Genome Size Estimator using R): a pipeline for quality assessment of sequenced genome libraries through genome size estimation.

The first step in any genome research after obtaining the read data is to perform a due quality control of the sequenced reads. In a de novo genome assembly project, the second step is to estimate two important features, the genome size and 'best k-mer', to start the assembly tests with different de novo assembly software and its parameters. However, the quality control of the sequenced genome libraries as a whole, instead of focusing on the reads only, is frequently overlooked and realized to be important only when the assembly tests did not render the expected results. We have developed GSER, a Genome Size Estimator using R, a pipeline to evaluate the relationship between k-mers and genome size, as a means for quality assessment of the sequenced genome libraries. GSER generates a set of charts that allow the analyst to evaluate the library datasets before starting the assembly. The script which runs the pipeline can be downloaded from http://www.mobilomics.org/GSER/downloads or http://github.com/mobilomics/GSER.

How to Grow a Tree: Plant Voltage-Dependent Cation Channels in the Spotlight of Evolution.

Phylogenetic analysis can be a powerful tool for generating hypotheses regarding the evolution of physiological processes. Here, we provide an updated view of the evolution of the main cation channels in plant electrical signalling: the Shaker family of voltage-gated potassium channels and the two-pore cation (K+) channel (TPC1) family. Strikingly, the TPC1 family followed the same conservative evolutionary path as one particular subfamily of Shaker channels (Kout) and remained highly invariant after terrestrialisation, suggesting that electrical signalling was, and remains, key to survival on land. We note that phylogenetic analyses can have pitfalls, which may lead to erroneous conclusions. To avoid these in the future, we suggest guidelines for analyses of ion channel evolution in plants.

Locus-specific analysis of Transposable Elements during the progression of ALS in the SOD1G93A mouse model.

Transposable Elements (TEs) are ubiquitous genetic elements with the ability to move within a genome. TEs contribute to a large fraction of the repetitive elements of a genome, and because of their nature, they are not routinely analyzed in RNA-Seq gene expression studies. Amyotrophic Lateral Sclerosis (ALS) is a lethal neurodegenerative disease, and a well-accepted model for its study is the mouse harboring the human SOD1G93A mutant. In this model, landmark stages of the disease can be recapitulated at specific time points, making possible to understand changes in gene expression across time. While there are several works reporting TE activity in ALS models, they have not explored their activity through the disease progression. Moreover, they have done it at the expense of losing their locus of expression. Depending on their genomic location, TEs can regulate genes in cis and in trans, making locus-specific analysis of TEs of importance in order to understand their role in modulating gene expression. Particularly, the locus-specific role of TEs in ALS has not been fully elucidated. In this work, we analyzed publicly available RNA-Seq datasets of the SOD1G93A mouse model, to understand the locus-specific role of TEs. We show that TEs become up-regulated at the early stages of the disease, and via statistical associations, we speculate that they can regulate several genes, which in turn might be contributing to the genetic dysfunction observed in ALS.

A brain-specific pgc1α fusion transcript affects gene expression and behavioural outcomes in mice.

PGC1α is a transcriptional coactivator in peripheral tissues, but its function in the brain remains poorly understood. Various brain-specific Pgc1α isoforms have been reported in mice and humans, including two fusion transcripts (FTs) with non-coding repetitive sequences, but their function is unknown. The FTs initiate at a simple sequence repeat locus ∼570 Kb upstream from the reference promoter; one also includes a portion of a short interspersed nuclear element (SINE). Using publicly available genomics data, here we show that the SINE FT is the predominant form of Pgc1α in neurons. Furthermore, mutation of the SINE in mice leads to altered behavioural phenotypes and significant up-regulation of genes in the female, but not male, cerebellum. Surprisingly, these genes are largely involved in neurotransmission, having poor association with the classical mitochondrial or antioxidant programs. These data expand our knowledge on the role of Pgc1α in neuronal physiology and suggest that different isoforms may have distinct functions. They also highlight the need for further studies before modulating levels of Pgc1α in the brain for therapeutic purposes.

Distinctive Cellular Transcriptomic Signature and MicroRNA Cargo of Extracellular Vesicles of Horse Adipose and Endometrial Mesenchymal Stem Cells from the Same Donors.

Equine endometrial and adipose mesenchymal stem cells (eMSCs and aMSCs, respectively) were isolated from the same donors of thoroughbred mares. The cells displayed characteristic features of MSCs, including trilineage mesodermal and also neurogenic differentiation. We evaluated the influence of cellular origin on their transcriptome profile. Cellular RNA was isolated and sequenced and extracellular vesicles (EVs) were obtained from conditioned medium of cells cultured in medium depleted of EVs, and their microRNA (miRNA) cargo analyzed by sequencing. Differential expression of mRNAs and EV-miRNA was analyzed, as well as pathways and processes most represented in each cell origin. mRNA reads from all expressed genes clustered according to the cellular origin. A total of 125 up- and 51 downregulated genes were identified and 31 differentially expressed miRNAs. Based on mRNA sequencing, endometrial MSCs strongly upregulated genes involved in the Hippo, transforming growth factor beta, and pluripotency signaling pathways. Alongside with this, pathways involved in extracellular matrix reorganization were the most represented in the miRNA cargo of EVs secreted by eMSCs. The niche from which MSCs originated defined the transcriptomic signature of the cells, including the secretion of lineage-specific loaded EV to ensure proper communication and homeostasis. Identification and testing their biological functions can provide new tools for the therapeutic use of horse MSC.

Mitochondrial acetyl-CoA reversibly regulates locus-specific histone acetylation and gene expression.

The impact of mitochondrial dysfunction in epigenetics is emerging, but our understanding of this relationship and its effect on gene expression remains incomplete. We previously showed that acute mitochondrial DNA (mtDNA) loss leads to histone hypoacetylation. It remains to be defined if these changes are maintained when mitochondrial dysfunction is chronic and if they alter gene expression. To fill these gaps of knowledge, we here studied a progressive and a chronic model of mtDNA depletion using biochemical, pharmacological, genomics, and genetic assays. We show that histones are primarily hypoacetylated in both models. We link these effects to decreased histone acetyltransferase activity unrelated to changes in ATP citrate lyase, acetyl coenzyme A synthetase 2, or pyruvate dehydrogenase activities, which can be reversibly modulated by altering the mitochondrial pool of acetyl-coenzyme A. Also, we determined that the accompanying changes in histone acetylation regulate locus-specific gene expression and physiological outcomes, including the production of prostaglandins. These results may be relevant to the pathophysiology of mtDNA depletion syndromes and to understanding the effects of environmental agents that lead to physical or functional mtDNA loss.

Histidine triad-like motif of the rotavirus NSP2 octamer mediates both RTPase and NTPase activities.

Rotavirus NSP2 is an abundant non-structural RNA-binding protein essential for forming the viral factories that support replication of the double-stranded RNA genome. NSP2 exists as stable doughnut-shaped octamers within the infected cell, representing the tail-to-tail interaction of two tetramers. Extending diagonally across the surface of each octamer are four highly basic grooves that function as binding sites for single-stranded RNA. Between the N and C-terminal domains of each monomer is a deep electropositive cleft containing a catalytic site that hydrolyzes the gamma-beta phosphoanhydride bond of any NTP. The catalytic site has similarity to those of the histidine triad (HIT) family of nucleotide-binding proteins. Due to the close proximity of the grooves and clefts, we investigated the possibility that the RNA-binding activity of the groove promoted the insertion of the 5'-triphosphate moiety of the RNA into the cleft, and the subsequent hydrolysis of its gamma-beta phosphoanhydride bond. Our results show that NSP2 hydrolyzes the gammaP from RNAs and NTPs through Mg(2+)-dependent activities that proceed with similar reaction velocities, that require the catalytic His225 residue, and that produce a phosphorylated intermediate. Competition assays indicate that although both substrates enter the active site, RNA is the preferred substrate due to its higher affinity for the octamer. The RNA triphosphatase (RTPase) activity of NSP2 may account for the absence of the 5'-terminal gammaP on the (-) strands of the double-stranded RNA genome segments. This is the first report of a HIT-like protein with a multifunctional catalytic site, capable of accommodating both NTPs and RNAs during gammaP hydrolysis.