Novel insights into human RNA polymerase III transcription: non canonical termination and biogenesis of potential regulatory RNAs

Transcription by human RNA polymerase III has been studied extensively to date, but many processes still need to be fully elucidated to get a more comprehensive picture. Here, we redefined the minimal sequence requirements for human RNA polymerase III termination and we investigated a novel kind of post-transcriptional gene silencing carried out by antisense AluRNAs derived from the 3’-UTR of human mRNAs. Starting from the observation that ̴ ¼ of human tRNA genes are enriched in canonical terminators located at ≥50 bp from the 3’-end of the tRNA coding sequence, we identified several novel non canonical terminators located further upstream at which RNA polymerase III terminates with different efficiencies, generally lower than those observed for termination at the canonical ≥T4 sites. Considering that more than one million of type 2 promoter elements are scattered through the human genome (tDNAs, Alu, MIR, etc), read-through at non-canonical sites could end up in the biogenesis of primary RNAs, with potential regulatory functions as previously observed for RNA polymerase III-derived miRNA, embedded in the 3’trailer of predicted type 2 promoter transcripts. Relaxed RNA polymerase III terminators could thus represent the evolution of a more sophisticated regulatory mechanism for the biogenesis of small RNAs. Additionally, we found that an antisense AluY RNA, transcribed from the 3’UTR of TERF2 mRNA is likely to trigger the silencing of its cognate mRNA by targeting its complementary sequence and speculated that the target specificity resides in its unique 3’trailer.La trascrizione da parte della RNA polimerasi III umana è stata studiata estesamente negli ultimi anni, ma l’impressione è che molti processi debbano ancora essere chiariti. Nel presente lavoro abbiamo ridefinito i requisiti minimi di sequenza per avere terminazione da parte della RNA polimerasi III umana e investigato un nuovo modello di silenziamento genico post-trascrizionale mediato da Alu RNA antisenso trascritti dalla 3’UTR di mRNA umani. Partendo dall’osservazione che circa un quarto dei geni umani codificanti tRNA è arricchito in terminatori canonici posizionati a più di 50 pb dall’ estremità 3’ del tRNA maturo, abbiamo identificato nuovi terminatori non canonici, situati più a monte, a livello dei quali la RNA polimerasi III termina diversamente e con efficienza inferiore rispetto a quanto osservato al terminatore canonico ≥T4. Considerando che il genoma umano contiene più di un milione di promotori di tipo 2 (tDNAs, Alu, MIR, etc), il parziale riconoscimento di terminatori non canonici e la conseguente trascrizione di sequenze a valle potrebbe risultare nella sintesi di RNA, situati nel 3’trailer, con possibili funzioni regolative come già osservato nel caso di miRNA trascritti dalla RNA polimerasi III. L’uso di terminatori più deboli da parte della RNA polimerasi III umana potrebbe quindi rappresentare lo sviluppo di un sofisticato meccanismo regolativo finalizzato alla biogenesi di piccoli RNA. In secondo luogo, abbiamo scoperto che un AluY RNA antisenso, trascritto come parte della 3’UTR di TERF2 mRNA, è in grado di indurre il silenziamento del corrispondente mRNA e ipotizzato che la specificità nei confronti del proprio bersaglio risieda nel 3’trailer dell’Alu RNA

Posterior reversible encephalopathy syndrome: role of transorbital ultrasound

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Persian Walnut (Juglans regia L.) Bud Dormancy Dynamics in Northern Patagonia, Argentina

Temperate deciduous fruit trees survive winter temperatures by entering a dormant phase in their aerial meristematic organs. Release from bud dormancy occurs after chill requirements (CR) have been satisfied, whereas bud burst/flowering follows heat requirement (HR) fulfillment. The physiological basis behind these metrics remains elusive. In this study, we are presenting the first multidisciplinary dormancy progression analysis in northern Patagonia, linking (1) forcing/field phenology, (2) bud anatomical development, and (3) soluble sugar (sucrose, glucose, and fructose) dynamics in Juglans regia L. CR and HR were determined for ‘Chandler’ and ‘Franquette,’ two walnut cultivars with markedly different CR, in artificial chill/forced heat trials (three seasons) and in-field chill/forced heat tests (five seasons) using excised twigs either with or without apical buds (non-decapitated and decapitated). The soluble sugar dynamics of ‘Chandler’ (high-performance liquid chromatography) and the anatomical changes of the buds (light microscopy) of the two cultivars were analyzed during endo-ecodormancy progression in one and two seasons, respectively. The CR defined by artificial chill tests proved to be an overestimation compared to the field determinations. Moreover, HR was the main driver in the phenology dynamics, as expected for a high-chill region. ‘Chandler’ showed an average of 10.3 field chill portions (CP) and 2,163 Growing Degree Hours (GDH°C) less than ‘Franquette’ for dormancy release and bud burst, respectively. These results were consistent with the transition of the shoot apex from the vegetative to the reproductive phase and the soluble sugar profile. The decrease in sucrose between 15 and 30 days after CR fulfillment could be a reliable biological marker for endodormancy release in walnut, while the increase in fructose and glucose is likely an osmolyte and cellulosic carbon source in pre-sprouting. In addition, we discuss the effect of paradormancy thanks to our apical bud experiment (with or without). Our results improve the current understanding of endo-ecodormancy progression in walnut and provide insightful results for walnut production (i.e., cultivation practices such as pruning) as well as for further application in dormancy modeling, to infer the ideotypes that should be bred for future climate conditions.Fil: del Barrio, Ricardo Alfredo. Universidad Nacional de Río Negro. Sede Andina; ArgentinaFil: Orioli, Gustavo Adolfo. Universidad Nacional del Sur. Departamento de Agronomía; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Brendel, Andrea Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto Argentino de Oceanografía. Universidad Nacional del Sur. Instituto Argentino de Oceanografía; Argentina. Universidad Nacional del Sur. Departamento de Agronomía; ArgentinaFil: Lindström, Lilia Ivone. Universidad Nacional del Sur. Departamento de Agronomía; ArgentinaFil: Pellegrini, Cecilia. Universidad Nacional del Sur. Departamento de Agronomía; ArgentinaFil: Campoy, José Antonio. Max Planck Institute Of Biochemistry.; Alemani

Widespread occurrence of non-canonical transcription termination by human RNA polymerase III

Human RNA polymerase (Pol) III-transcribed genes are thought to share a simple termination signal constituted by four or more consecutive thymidine residues in the coding DNA strand, just downstream of the RNA 3′-end sequence. We found that a large set of human tRNA genes (tDNAs) do not display any T≥4 stretch within 50 bp of 3′-flanking region. In vitro analysis of tDNAs with a distanced T≥4 revealed the existence of non-canonical terminators resembling degenerate T≥5 elements, which ensure significant termination but at the same time allow for the production of Pol III read-through pre-tRNAs with unusually long 3′ trailers. A panel of such non-canonical signals was found to direct transcription termination of unusual Pol III-synthesized viral pre-miRNA transcripts in gammaherpesvirus 68-infected cells. Genome-wide location analysis revealed that human Pol III tends to trespass into the 3′-flanking regions of tDNAs, as expected from extensive terminator read-through. The widespread occurrence of partial termination suggests that the Pol III primary transcriptome in mammals is unexpectedly enriched in 3′-trailer sequences with the potential to contribute novel functional ncRNA

Identification and removal of low-complexity sites in allele-specific analysis of ChIP-seq data

Motivation: High-throughput sequencing technologies enable the genome-wide analysis of the impact of genetic variation on molecular phenotypes at unprecedented resolution. However, although powerful, these technologies can also introduce unexpected artifacts. Results: We investigated the impact of library amplification bias on the identification of allele-specific (AS) molecular events from high-throughput sequencing data derived from chromatin immunoprecipitation assays (ChIP-seq). Putative AS DNA binding activity for RNA polymerase II was determined using ChIP-seq data derived from lymphoblastoid cell lines of two parent-daughter trios. We found that, at high-sequencing depth, many significant AS binding sites suffered from an amplification bias, as evidenced by a larger number of clonal reads representing one of the two alleles. To alleviate this bias, we devised an amplification bias detection strategy, which filters out sites with low read complexity and sites featuring a significant excess of clonal reads. This method will be useful for AS analyses involving ChIP-seq and other functional sequencing assays. Availability: The R package absfilter for library clonality simulations and detection of amplification-biased sites is available from http://updepla1srv1.epfl.ch/waszaks/absfilter Contact: [email protected] or [email protected] Supplementary information: Supplementary data are available at Bioinformatics onlin

Ipsilateral motor evoked potentials in a patient with unihemispheric cortical atrophy due to Rasmussen encephalitis

The role of the ipsilaterally descending motor pathways in the recovery mechanisms after unilateral hemispheric damage is still poorly understood. Motor output reorganization was investigated in a 56-year-old male patient with acquired unilateral hemispheric atrophy due to Rasmussen encephalitis. In particular, the ipsilateral corticospinal pathways were explored using focal transcranial magnetic stimulation. In the first dorsal interosseous and wrist extensors muscles, the median amplitudes of the ipsilateral motor evoked potentials induced by transcranial magnetic stimulation in the patient were higher than those of 10 age-matched healthy control subjects. In the biceps brachii muscle, the median amplitudes of the ipsilateral motor evoked potentials were the second largest in the patient compared to the controls. This study demonstrated a reinforcement of ipsilateral motor projections from the unaffected motor cortex to the hemiparetic hand in a subject with acquired unihemispheric cortical damage

Enamel Formation Genes Influence Enamel Microhardness Before and After Cariogenic Challenge

There is evidence for a genetic component in caries susceptibility, and studies in humans have suggested that variation in enamel formation genes may contribute to caries. For the present study, we used DNA samples collected from 1,831 individuals from various population data sets. Single nucleotide polymorphism markers were genotyped in selected genes (ameloblastin, amelogenin, enamelin, tuftelin, and tuftelin interacting protein 11) that influence enamel formation. Allele and genotype frequencies were compared between groups with distinct caries experience. Associations with caries experience can be detected but they are not necessarily replicated in all population groups and the most expressive results was for a marker in AMELX (p = 0.0007). To help interpret these results, we evaluated if enamel microhardness changes under simulated cariogenic challenges are associated with genetic variations in these same genes. After creating an artificial caries lesion, associations could be seen between genetic variation in TUFT1 (p = 0.006) and TUIP11 (p = 0.0006) with enamel microhardness. Our results suggest that the influence of genetic variation of enamel formation genes may influence the dynamic interactions between the enamel surface and the oral cavity. © 2012 Shimizu et al

Widespread occurrence of non-canonical transcription termination by human RNA polymerase III

Human RNA polymerase (Pol) III-transcribed genes are thought to share a simple termination signal constituted by four or more consecutive thymidine residues in the coding DNA strand, just downstream of the RNA 3′-end sequence. We found that a large set of human tRNA genes (tDNAs) do not display any T≥4 stretch within 50 bp of 3′-flanking region. In vitro analysis of tDNAs with a distanced T≥4 revealed the existence of non-canonical terminators resembling degenerate T≥5 elements, which ensure significant termination but at the same time allow for the production of Pol III read-through pre-tRNAs with unusually long 3′ trailers. A panel of such non-canonical signals was found to direct transcription termination of unusual Pol III-synthesized viral pre-miRNA transcripts in gammaherpesvirus 68-infected cells. Genome-wide location analysis revealed that human Pol III tends to trespass into the 3′-flanking regions of tDNAs, as expected from extensive terminator read-through. The widespread occurrence of partial termination suggests that the Pol III primary transcriptome in mammals is unexpectedly enriched in 3′-trailer sequences with the potential to contribute novel functional ncRNAs

Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells

Bacterial type II CRISPR-Cas9 systems have been widely adapted for RNA-guided genome editing and transcription regulation in eukaryotic cells, yet their in vivo target specificity is poorly understood. Here we mapped genome-wide binding sites of a catalytically inactive Cas9 (dCas9) from Streptococcus pyogenes loaded with single guide RNAs (sgRNAs) in mouse embryonic stem cells (mESCs). Each of the four sgRNAs we tested targets dCas9 to between tens and thousands of genomic sites, frequently characterized by a 5-nucleotide seed region in the sgRNA and an NGG protospacer adjacent motif (PAM). Chromatin inaccessibility decreases dCas9 binding to other sites with matching seed sequences; thus 70% of off-target sites are associated with genes. Targeted sequencing of 295 dCas9 binding sites in mESCs transfected with catalytically active Cas9 identified only one site mutated above background levels. We propose a two-state model for Cas9 binding and cleavage, in which a seed match triggers binding but extensive pairing with target DNA is required for cleavage.National Institutes of Health (U.S.) (Grant RO1-GM34277)National Institutes of Health (U.S.) (Grant R01-CA133404)National Cancer Institute (U.S.) (Grant PO1-CA42063)National Cancer Institute (U.S.) (Cancer Center Support (Core) Grant P30-CA14051)National Institutes of Health (U.S.) (Director's Pioneer Award 1DP1-MH100706)Damon Runyon Cancer Research FoundationKinship Foundation. Searle Scholars ProgramSimons Foundatio

Retrospective Analysis of Six Years of Acute Flaccid Paralysis Surveillance and Polio Vaccine Coverage Reported by Italy, Serbia, Bosnia and Herzegovina, Montenegro, Bulgaria, Kosovo, Albania, North Macedonia, Malta, and Greece

Here we analyzed six years of acute flaccid paralysis (AFP) surveillance, from 2015 to 2020, of 10 countries linked to the WHO Regional Reference Laboratory, at the Istituto Superiore di Sanità, Italy. The analysis also comprises the polio vaccine coverage available (2015–2019) and enterovirus (EV) identification and typing data. Centralized Information System for Infectious Diseases and Laboratory Data Management System databases were used to obtain data on AFP indicators and laboratory performance and countries’ vaccine coverage from 2015 to 2019. EV isolation, identification, and typing were performed by each country according to WHO protocols. Overall, a general AFP underreporting was observed. Non-Polio Enterovirus (NPEV) typing showed a high heterogeneity: over the years, several genotypes of coxsackievirus and echovirus have been identified. The polio vaccine coverage, for the data available, differs among countries. This evaluation allows for the collection, for the first time, of data from the countries of the Balkan area regarding AFP surveillance and polio vaccine coverage. The need, for some countries, to enhance the surveillance systems and to promote the polio vaccine uptake, in order to maintain the polio-free status, is evident