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Abstract Polyribonucleotide phosphorylase (PNPase) is a phosphorolytic RNA exonuclease highly conserved throughout evolution. In Escherichia coli, PNPase controls complex phenotypic traits like biofilm formation and growth at low temperature. In human cells, PNPase is located in mitochondria, where it is implicated in the RNA import from the cytoplasm, the mitochondrial RNA degradation and the processing of R-loops, namely stable RNA–DNA hybrids displacing a DNA strand. In this work, we show that the human PNPase (hPNPase) expressed in E. coli causes oxidative stress, SOS response activation and R-loops accumulation. Hundreds of E. coli RNAs are stabilized in presence of hPNPase, whereas only few transcripts are destabilized. Moreover, phenotypic traits typical of E. coli strains lacking PNPase are strengthened in presence of the human enzyme. We discuss the hypothesis that hPNPase expressed in E. coli may bind, but not degrade, the RNA, in agreement with previous in vitro data showing that phosphate concentrations in the range of those found in the bacterial cytoplasm and, more relevant, in the mitochondria, inhibit its activity.

NF-Y is a trimeric Transcription Factor -TF- which binds with high selectivity to the conserved CCAAT element. Individual ChIP-seq analysis as well as ENCODE have progressively identified locations shared by other TFs. Here, we have analyzed data introduced by ENCODE over the last five years in K562, HeLa-S3 and GM12878, including several chromatin features, as well RNA-seq profiling of HeLa cells after NF-Y inactivation. We double the number of sequence-specific TFs and co-factors reported. We catalogue them in 4 classes based on co-association criteria, infer target genes categorizations, identify positional bias of binding sites and gene expression changes. Larger and novel co-associations emerge, specifically concerning subunits of repressive complexes as well as RNA-binding proteins. On the one hand, these data better define NF-Y association with single members of major classes of TFs, on the other, they suggest that it might have a wider role in the control of mRNA production.

Chromatin immunoprecipitation followed by next-generation sequencing (ChIP-Seq) has opened new avenues of research in the genome-wide characterization of regulatory DNA-protein interactions at the genetic and epigenetic level. As a consequence, it has become the de facto standard for studies on the regulation of transcription, and literally thousands of data sets for transcription factors and cofactors in different conditions and species are now available to the scientific community. However, while pipelines and best practices have been established for the analysis of a single experiment, there is still no consensus on the best way to perform an integrated analysis of multiple datasets in the same condition, in order to identify the most relevant and widespread regulatory modules composed by different transcription factors and cofactors. We present here a computational pipeline for this task, that integrates peak summit colocalization, a novel statistical framework for the evaluation of its significance, and motif enrichment analysis. We show examples of its application to ENCODE data, that led to the identification of relevant regulatory modules composed of different factors, as well as the organization on DNA of the binding motifs responsible for their recruitment.

The transcription factor SOX2 is important for brain development and for neural stem cells (NSC) maintenance. Sox2-deleted (Sox2-del) NSC from neonatal mouse brain are lost after few passages in culture. Two highly expressed genes, Fos and Socs3, are strongly downregulated in Sox2-del NSC; we previously showed that Fos or Socs3 overexpression by lentiviral transduction fully rescues NSC’s long-term maintenance in culture. Sox2-del NSC are severely defective in neuronal production when induced to differentiate. NSC rescued by Sox2 reintroduction correctly differentiate into neurons. Similarly, Fos transduction rescues normal or even increased numbers of immature neurons expressing beta-tubulinIII, but not more differentiated markers (MAP2). Additionally, many cells with both beta-tubulinIII and GFAP expression appear, indicating that FOS stimulates the initial differentiation of a “mixed” neuronal/glial progenitor. The unexpected rescue by FOS suggested that FOS, a SOX2 transcriptional target, might act on neuronal genes, together with SOX2. CUT&RUN analysis to detect genome-wide binding of SOX2, FOS, and JUN (the AP1 complex) revealed that a high proportion of genes expressed in NSC are bound by both SOX2 and AP1. Downregulated genes in Sox2-del NSC are highly enriched in genes that are also expressed in neurons, and a high proportion of the “neuronal” genes are bound by both SOX2 and AP1.

Transcriptomics studies have been facilitated by the development of microarray and RNA-Seq technologies, with thousands of expression datasets available for many species. However, the quality of data can be highly variable, making the combined analysis of different datasets difficult and unreliable. Most of the microarray data for Medicago truncatula, the barrel medic, have been stored and made publicly accessible on the web database Medicago truncatula Gene Expression atlas (MtGEA). The aim of this work is to ameliorate the quality of the MtGEA database through a general method based on logical and statistical relationships among parameters and conditions. The initial 716 columns available in the dataset were reduced to 607 by evaluating the quality of data through the sum of the expression levels over the entire transcriptome probes and Pearson correlation among hybridizations. The reduced dataset shows great improvements in the consistency of the data, with a reduction in both false positives and false negatives resulting from Pearson correlation and GO enrichment analysis among genes. The approach we used is of general validity and our intent is to extend the analysis to other plant microarray databases.

Fine regulation of exocytosis and endocytosis plays a basic role in pollen tube growth. Excess plasma membrane secreted during pollen tube elongation is known to be retrieved by endocytosis and partially reused in secretory pathways through the Golgi apparatus. Dissection of endocytosis has enabled distinct degradation pathways to be identified in tobacco pollen tubes and has shown that microtubules influence the transport of plasma membrane internalized in the tip region to vacuoles. Here, we used different drugs affecting the polymerization state of microtubules together with SYP21, a marker of prevacuolar compartments, to characterize trafficking of prevacuolar compartments in Nicotiana tabacum pollen tubes. Ultrastructural and biochemical analysis showed that microtubules bind SYP21-positive microsomes. Transient transformation of pollen tubes with LAT52-YFP-SYP21 revealed that microtubules play a key role in the delivery of prevacuolar compartments to tubular vacuoles.

Large-scale initiatives aiming to recover the complete sequence of thousands of human genomes are currently being undertaken worldwide, concurring to the generation of a comprehensive catalog of human genetic variation. The ultimate and most ambitious goal of human population scale genomics is the characterization of the so-called human “variome,” through the identification of causal mutations or haplotypes. Several research institutions worldwide currently use genotyping assays based on Next-Generation Sequencing (NGS) for diagnostics and clinical screenings, and the widespread application of such technologies promises major revolutions in medical science. Bioinformatic analysis of human resequencing data is one of the main factors limiting the effectiveness and general applicability of NGS for clinical studies. The requirement for multiple tools, to be combined in dedicated protocols in order to accommodate different types of data (gene panels, exomes, or whole genomes) and the high variability of the data makes difficult the establishment of a ultimate strategy of general use. While there already exist several studies comparing sensitivity and accuracy of bioinformatic pipelines for the identification of single nucleotide variants from resequencing data, little is known about the impact of quality assessment and reads pre-processing strategies. In this work we discuss major strengths and limitations of the various genome resequencing protocols are currently used in molecular diagnostics and for the discovery of novel disease-causing mutations. By taking advantage of publicly available data we devise and suggest a series of best practices for the pre-processing of the data that consistently improve the outcome of genotyping with minimal impacts on computational costs.

In order to study the role played by cellular RNA pools produced by homologous genomic loci in defining the transcriptional state of a silenced gene, we tested the effect of non-functional alleles of the white gene in the presence of a functional copy of white, silenced by heterochromatin. We found that non-functional alleles of white, unable to produce a coding transcript, could reactivate in trans the expression of a wild type copy of the same gene silenced by heterochromatin. This new epigenetic phenomenon of transcriptional trans-reactivation is heritable, relies on the presence of homologous RNA's and is affected by mutations in genes involved in post-transcriptional gene silencing. Our data suggest a general new unexpected level of gene expression control mediated by homologous RNA molecules in the context of heterochromatic genes.

p63 is a transcription factor required for the development and maintenance of ectodermal tissues in general, and skin keratinocytes in particular. The identification of its target genes is fundamental for understanding the complex network of gene regulation governing the development of epithelia. We report a list of almost 1000 targets derived from ChIP on chip analysis on two platforms; all genes analyzed changed in expression during differentiation of human keratinocytes. Functional annotation highlighted unexpected GO terms enrichments and confirmed that genes involved in transcriptional regulation are the most significant. A detailed analysis of these transcriptional regulators in condition of perturbed p63 levels confirmed the role of p63 in the regulatory network. Rather than a rigid master-slave hierarchical model, our data indicate that p63 connects different hubs involved in the multiple specific functions of the skin.