Whole genome mapping of 5' RNA ends in bacteria by tagged sequencing : A comprehensive view in Enterococcus faecalis

Enterococcus faecalis is the third cause of nosocomial infections. To obtain the first comprehensive view of transcriptional organizations in this bacterium, we used a modified RNA-seq approach enabling to discriminate primary from processed 5'RNA ends. We also validated our approach by confirming known features in Escherichia coli. We mapped 559 transcription start sites and 352 processing sites in E. faecalis. A blind motif search retrieved canonical features of SigA- and SigN-dependent promoters preceding TSSs mapped. We discovered 95 novel putative regulatory RNAs, small- and antisense RNAs, and 72 transcriptional antisense organisations. Presented data constitute a significant insight into bacterial RNA landscapes and a step towards the inference of regulatory processes at transcriptional and post-transcriptional levels in a comprehensive manner

BC1-FMRP interaction is modulated by 2′-O-methylation: RNA-binding activity of the tudor domain and translational regulation at synapses

The brain cytoplasmic RNA, BC1, is a small non-coding RNA that is found in different RNP particles, some of which are involved in translational control. One component of BC1-containing RNP complexes is the fragile X mental retardation protein (FMRP) that is implicated in translational repression. Peptide mapping and computational simulations show that the tudor domain of FMRP makes specific contacts to BC1 RNA. Endogenous BC1 RNA is 2′-O-methylated in nucleotides that contact the FMRP interface, and methylation can affect this interaction. In the cell body BC1 2′-O-methylations are present in both the nucleus and the cytoplasm, but they are virtually absent at synapses where the FMRP–BC1–mRNA complex exerts its function. These results strongly suggest that subcellular region-specific modifications of BC1 affect the binding to FMRP and the interaction with its mRNA targets. We finally show that BC1 RNA has an important role in translation of certain mRNAs associated to FMRP. All together these findings provide further insights into the translational regulation by the FMRP–BC1 complex at synapses

Nucleotide post-transcriptional modifications of BC1 non-coding RNA during brain development: new insights into the BC1-FMRP complex

L’RNA BC1 (Brain Cytoplasmic 1 RNA) è un piccolo RNA non codificante (sncRNA) altamente espresso nel cervello dei roditori e appartenente alla famiglia degli elementi SINE (Short Interspersed Nuclear repetitive Elements). Nei neuroni l’RNA BC1 forma particelle ribonucleoproteiche (RNP), alcune delle quali reprimono la traduzione degli RNA messaggeri. Un componente chiave della RNP contenente BC1 è la proteina FMRP (Fragile X Mental Retardation Protein). Durante il mio lavoro di tesi è emerso che l’RNA BC1 è pseudouridinilato e metilato in vivo. E’ stato rilevante notare che uno dei due tipi di modificazioni (G46 e C47 2’-O-metilazioni) si verifica solo in una ben definita finestra temporale che corrisponde alla fine della sinaptogenesi. Esperimenti in vitro EMSA (Electrophoretic Mobility Shift Assay) sono stati condotti per individuare ogni possibile effetto di queste modificazioni post-trascrizionali (rispettivamente pseudouridine e 2’-O-metilazioni) sul legame tra l’RNA BC1 modificato e il dominio N-terminale (NT) di FMRP. È stato osservato che le modificazioni Gm46, Cm47 e Gm56 diminuiscono l’affinità di legame tra l’RNA BC1 e l’NT di FMRP. Uno studio traduzionale in vivo condotto su topi WT e FMR1 KO ha permesso di mostrare che nei topi FMR1KO l’efficienza traduzionale di tre mRNA target di FMRP (Map1B, -CaMKII e Arc) è deregolata solo nel cervello di topi di 3 settimane e non in quello di topi di 3 mesi. Noi proponiamo un modello nel quale lo stato delle modificazioni nucleotidiche post-trascrizionali del piccolo RNA non codificante BC1 potrebbe promuovere l’attività di chaperone degli RNA posseduta dalla proteina FMRP. In questo contesto, è possibile ipotizzare il ruolo di FMRP nel promuovere l’appaiamento e lo scambio dei filamenti di RNA tra BC1 e gli mRNA a cui esso è appaiato.The Brain Cytoplasmic RNA, BC1, is a small non-coding RNA (sncRNA) highly expressed in rodent brain and is part of the SINE elements (for short interspersed nuclear repetitive elements) family. In neurons, BC1 RNA forms different RNP particles, some of them repressing mRNA translation. One of the key components of the BC1-mRNP is the Fragile X Mental Retardation Protein. I show here that BC1 RNA is pseudouridinylated and 2’-O-methylated in vivo. Interestingly, 2’-O-methylations onG46 and C47 onlyoccurat a very defined temporal window,the end of synaptogenesis. In vitro EMSA experiments have been performed in order to detect any post-transcriptional modifications effect (pseudouridine and 2’-O-methylation respectively) on the binding between modified BC1 RNA and the FMRP N terminus. Interestingly, the Gm46, Cm47 and Gm56 modifications decrease the binding affinity between the BC1 RNA and the FMRP N terminus. An in vivo, translational study carried out in both WT and FMR1 KO mice, has allowed to demonstrate that in FMR1 KO mice the translational efficiency of the three FMRP mRNA targets: Map1B, -CaMKII and Arc was misregulated only in 3 weeks-old mouse brain, and not in 3 months-old mouse brain. A mechanistic model is proposed in which the post-transcriptional nucleotide modifications of BC1 ncRNA would promote the RNA chaperone activity of the FMRP protein. FMRP could promote the annealing and the exchange of both the BC1 and mRNA RNA strands binding

Molecular dynamics simulations show how the FMRP Ile304Asn mutation destabilizes the KH2 domain structure and affects its function

<div><p>Mutations or deletions of FMRP, involved in the regulation of mRNA metabolism in brain, lead to the Fragile X syndrome (FXS), the most frequent form of inherited intellectual disability. A severe manifestation of the disease has been associated with the Ile304Asn mutation, located on the KH2 domain of the protein. Several hypotheses have been proposed to explain the possible molecular mechanism responsible for the drastic effect of this mutation in humans. Here, we performed a molecular dynamics simulation and show that the Ile304Asn mutation destabilizes the hydrophobic core producing a partial unfolding of two α-helices and a displacement of a third one. The affected regions show increased residue flexibility and motion. Molecular docking analysis revealed strongly reduced binding to a model single-stranded nucleic acid in agreement with known data that the two partially unfolded helices form the RNA-binding surface. The third helix, which we show here to be also affected, is involved in the PAK1 protein interaction. These two functional binding sites on the KH2 domain do not overlap spatially, and therefore, they can simultaneously bind their targets. Since the Ile304Asn mutation affects both binding sites, this may justify the severe clinical manifestation observed in the patient in which both mRNA metabolism activity and cytoskeleton remodeling would be affected.</p></div

The catalytic activity of the translation termination factor methyltransferase Mtq2-Trm112 complex is required for large ribosomal subunit biogenesis

International audienceThe Mtq2-Trm112 methyltransferase modifies the eukaryotic translation termination factor eRF1 on the glutamine side chain of a universally conserved GGQ motif that is essential for release of newly synthesized peptides. Although this modification is found in the three domains of life, its exact role in eukaryotes remains unknown. As the deletion of MTQ2 leads to severe growth impairment in yeast, we have investigated its role further and tested its putative involvement in ribosome biogenesis. We found that Mtq2 is associated with nuclear 60S subunit precursors, and we demonstrate that its catalytic activity is required for nucleolar release of pre-60S and for efficient production of mature 5.8S and 25S rRNAs. Thus, we identify Mtq2 as a novel ribosome assembly factor important for large ribosomal subunit formation. We propose that Mtq2-Trm112 might modify eRF1 in the nucleus as part of a quality control mechanism aimed at proof-reading the peptidyl transferase center, where it will subsequently bind during translation termination

An Integrated Study on Analysis, Intelligence and Repair of Composite Structures with Damage(II)

本整合計劃將集合相關研究之教授，以分工合作方式研究受損複材結構(以樑、板為主)在受損(如脫層)後其結構之各種力學行為分析(層間應力、勁度、振頻與模態、挫曲負載與模態、強度)具偵測與控制前述力學行為功能之智慧結構的分析與設計、對受損複材結構加以修補之設計與分析，這些分工以各個子計劃分別進行；同時總計劃則進行協調合作討論、對各子計劃之工作和成果內容加以規範與整合，達成建立一套能用而有效之整合系統，可真正應用於針對複材結構受損後之各種力學行為分析，並對此行為之智慧監控、和修補之分析設計

Absence of the Fragile X Mental Retardation Protein results in defects of RNA editing of neuronal mRNAs in mouse

The fragile X syndrome (FXS), the most common form of inherited intellectual disability, is due to the absence of FMRP, a protein regulating RNA metabolism. Recently, an unexpected function of FMRP in modulating the activity of Adenosine Deaminase Acting on RNA (ADAR) enzymes has been reported both in Drosophila and Zebrafish. ADARs are RNA-binding proteins that increase transcriptional complexity through a post-transcriptional mechanism called RNA editing. In order to evaluate the ADAR2-FMRP interaction in mammals we analysed several RNA editing re-coding sites in the fmr1 knockout (KO) mice. Ex vivo and in vitro analysis revealed that absence of FMRP lead to an increase in the editing levels of brain specific mRNAs, indicating that FMRP might act as an inhibitor of editing activity. Proximity Ligation Assay (PLA) in mouse primary cortical neurons and in non-neuronal cells revealed that ADAR2 and FMRP co-localize in the nucleus. The ADAR2-FMRP co-localization was further observed by double-immunogold Electron Microscopy (EM) in the hippocampus. Moreover, ADAR2-FMRP interaction appeared to be RNA independent. Because changes in the editing pattern are associated with neuropsychiatric and neurodevelopmental disorders, we propose that the increased editing observed in the fmr1-KO mice might contribute to the FXS molecular phenotypes