Dynamic interactions within sub-complexes of the H/ACA pseudouridylation guide RNP

H/ACA RNP complexes change uridines to pseudouridines in target non-coding RNAs in eukaryotes and archaea. H/ACA RNPs are comprised of a guide RNA and four essential proteins: Cbf5 (pseudouridine synthase), L7Ae, Gar1 and Nop10 in archaea. The guide RNA captures the target RNA via two antisense elements brought together to form a contiguous binding site within the pseudouridylation pocket (internal loop) of the guide RNA. Cbf5 and L7Ae interact independently with the guide RNA, and here we have examined the impacts of these proteins on the RNA in nucleotide protection assays. The results indicate that the interactions observed in a fully assembled H/ACA RNP are established in the sub-complexes, but also reveal a unique Cbf5–guide RNA interaction that is displaced by L7Ae. In addition, the results indicate that L7Ae binding at the kink (k)-turn of the guide RNA induces the formation of the upper stem, and thus also the pseudouridylation pocket. Our findings indicate that L7Ae is essential for formation of the substrate RNA binding site in the archaeal H/ACA RNP, and suggest that k-turn-binding proteins may remodel partner RNAs with important effects distant from the protein-binding site

Regulation of the RNA and DNA nuclease activities required for Pyrococcus furiosus Type III-B CRISPR-Cas immunity

Funding: National Institutes of Health (NIH) [R35GM118160 to M.P.T., R01GM097330 to S.B. and 1F31GM125365 toK.F.]; Biotechnology and Biological Sciences Research Council [REF: BB/S000313/1 to M.F.W.]. Funding for open access charge: NIH grant.Type III CRISPR-Cas prokaryotic immune systems provide anti-viral and anti-plasmid immunity via a dual mechanism of RNA and DNA destruction. Upon target RNA interaction, Type III crRNP effector complexes become activated to cleave both target RNA (via Cas7) and target DNA (via Cas10). Moreover, trans-acting endoribonucleases, Csx1 or Csm6, can promote the Type III immune response by destroying both invader and host RNAs. Here, we characterize how the RNase and DNase activities associated with Type III-B immunity in Pyrococcus furiosus (Pfu) are regulated by target RNA features and second messenger signaling events. In vivo mutational analyses reveal that either the DNase activity of Cas10 or the RNase activity of Csx1 can effectively direct successful anti-plasmid immunity. Biochemical analyses confirmed that the Cas10 Palm domains convert ATP into cyclic oligoadenylate (cOA) compounds that activate the ribonuclease activity of Pfu Csx1. Furthermore, we show that the HEPN domain of the adenosine-specific endoribonuclease, Pfu Csx1, degrades cOA signaling molecules to provide an auto-inhibitory off-switch of Csx1 activation. Activation of both the DNase and cOA generation activities require target RNA binding and recognition of distinct target RNA 3' protospacer flanking sequences. Our results highlight the complex regulatory mechanisms controlling Type III CRISPR immunity.Publisher PDFPeer reviewe

A journey down to hell: new thermostable protein-tags for biotechnology at high temperatures

The specific labelling of proteins in recent years has made use of self-labelling proteins, such as the SNAP-tag® and the Halotag®. These enzymes, by their nature or suitably engineered, have the ability to specifically react with their respective substrates, but covalently retaining a part of them in the catalytic site upon reaction. This led to the synthesis of substrates conjugated with, e.g., fluorophores (proposing them as alternatives to fluorescent proteins), but also with others chemical groups, for numerous biotechnological applications. Recently, a mutant of the OGT from Saccharolobus solfataricus (H5) very stable to high temperatures and in the presence of physical and chemical denaturing agents has been proposed as a thermostable SNAP-tag® for in vivo and in vitro harsh reaction conditions. Here, we show two new thermostable OGTs from Thermotoga neapolitana and Pyrococcus furiosus, which, respectively, display a higher catalytic activity and thermostability respect to H5, proposing them as alternatives for in vivo studies in these extreme model organisms

Irán y Arabia Saudí en Yemen

Este artículo analiza la implicación del régimen iraní en la guerra civil yemení como pretexto para la intervención saudí. Desde una perspectiva sociológica del poder, primero examinamos las motivaciones y los vínculos directos e indirectos entre los rebeldes hutíes y las élites iraníes. Luego usamos métodos cualitativos de investigación bibliográfica y análisis de fuentes periodísticas para examinar las razones de la decisión política de Arabia Saudí de intervenir en Yemen para extender el poder del príncipe bin Salmán y ampliar su apoyo popular. Podemos concluir que el papel de Irán en Yemen es insuficiente para entender a los hutíes como proxies. Por lo tanto, confirmamos que el príncipe saudí intensificó esta acusación para justificar la guerra y afirmar su posición dentro del sistema de poder interno

The Lsm2-8 complex determines nuclear localization of the spliceosomal U6 snRNA

Lsm proteins are ubiquitous, multifunctional proteins that are involved in the processing and/or turnover of many, if not all, RNAs in eukaryotes. They generally interact only transiently with their substrate RNAs, in keeping with their likely roles as RNA chaperones. The spliceosomal U6 snRNA is an exception, being stably associated with the Lsm2-8 complex. The U6 snRNA is generally considered to be intrinsically nuclear but the mechanism of its nuclear retention has not been demonstrated, although La protein has been implicated. We show here that the complete Lsm2-8 complex is required for nuclear accumulation of U6 snRNA in yeast. Therefore, just as Sm proteins effect nuclear localization of the other spliceosomal snRNPs, the Lsm proteins mediate U6 snRNP localization except that nuclear retention is the likely mechanism for the U6 snRNP. La protein, which binds only transiently to the nascent U6 transcript, has a smaller, apparently indirect, effect on U6 localization that is compatible with its proposed role as a chaperone in facilitating U6 snRNP assembly

Formation of the conserved pseudouridine at position 55 in archaeal tRNA

Pseudouridine (Ψ) located at position 55 in tRNA is a nearly universally conserved RNA modification found in all three domains of life. This modification is catalyzed by TruB in bacteria and by Pus4 in eukaryotes, but so far the Ψ55 synthase has not been identified in archaea. In this work, we report the ability of two distinct pseudouridine synthases from the hyperthermophilic archaeon Pyrococcus furiosus to specifically modify U55 in tRNA in vitro. These enzymes are (pfu)Cbf5, a protein known to play a role in RNA-guided modification of rRNA, and (pfu)PsuX, a previously uncharacterized enzyme that is not a member of the TruB/Pus4/Cbf5 family of pseudouridine synthases. (pfu)PsuX is hereafter renamed (pfu)Pus10. Both enzymes specifically modify tRNA U55 in vitro but exhibit differences in substrate recognition. In addition, we find that in a heterologous in vivo system, (pfu)Pus10 efficiently complements an Escherichia coli strain deficient in the bacterial Ψ55 synthase TruB. These results indicate that it is probable that (pfu)Cbf5 or (pfu)Pus10 (or both) is responsible for the introduction of pseudouridine at U55 in tRNAs in archaea. While we cannot unequivocally assign the function from our results, both possibilities represent unexpected functions of these proteins as discussed herein

Les reinterpretacions del confucianisme des del segle XIX fins l'actualitat

En aquest treball em proposo explicar les reinterpretacions més importants que ha sofert el confucianisme, enfocant-me principalment en el període comprès des del segle XIX fins a la nostra època. Però, primer explicaré breument alguns aspectes sobre Confuci i la visió que es tenia del confucianisme durant les dinasties Han i Song, que ens ajudaran a entendre el paper que va jugar el confucianisme durant la dinastia Qing i la tasca que van dur a terme estudiosos com Kang Youwei, així com les crítiques que aquests van rebre. Posteriorment parlaré sobre el Moviment del 4 de Maig i el rol que va tenir el confucianisme tant en aquest moment com a posteriori, fins arribar a l'època de la República Popular Xinesa i les mesures que va emprendre Mao Zedong vers el confucianisme. Finalment, explicaré les reinterpretacions més recents del confucianisme.En este trabajo me dispongo a exponer las reinterpretaciones más importantes que ha sufrido el confucianismo, enfocándome principalmente en el periodo comprendido desde el siglo XIX hasta nuestra época. Sin embargo, primero explicaré brevemente algunos aspectos sobre Confucio y la visión que se tenía del confucianismo durante las dinastías Han y Song, que nos ayudarán a entender el papel que jugó el confucianismo durante la dinastía Qing y la tarea que llevaron a cabo estudiosos como Kang Youwei, así como las críticas que estos recibieron. Posteriormente hablaré sobre el Movimiento del 4 de Mayo y el rol que tuvo el confucianismo tanto en este momento como a posteriori, hasta llegar a la época de la República Popular China y las medidas que emprendió Mao Zedong contra el confucianismo. Finalmente, explicaré las reinterpretaciones más recientes del confucianismo.In this work I will expose the major reinterpretations that has suffered Confucianism, focusing primarily on the period from the nineteenth century to the present day. But firs I will briefly explain some aspects of Confucius and which was the opinion about Confucianism during Han and Song dynasties, which will help us understand the role played by Confucianism during the Qing dynasty and the task carried out by scholars such as Kang Youwei and the critiques they received. I will talk later about the May 4th Movement and the role that Confucianism had both then and afterwards, up to the time of the People's Republic of China and the measures taken by Mao Zedong against Confucianism. Finally, I will explain the most recent reinterpretations of Confucianism

Structural basis for m7G-cap hypermethylation of small nuclear, small nucleolar and telomerase RNA by the dimethyltransferase TGS1

The 5′-cap of spliceosomal small nuclear RNAs, some small nucleolar RNAs and of telomerase RNA was found to be hypermethylated in vivo. The Trimethylguanosine Synthase 1 (TGS1) mediates this conversion of the 7-methylguanosine-cap to the 2,2,7-trimethylguanosine (m3G)-cap during maturation of the RNPs. For mammalian UsnRNAs the generated m2,2,7G-cap is one part of a bipartite import signal mediating the transport of the UsnRNP-core complex into the nucleus. In order to understand the structural organization of human TGS1 as well as substrate binding and recognition we solved the crystal structure of the active TGS1 methyltransferase domain containing both, the minimal substrate m7GTP and the reaction product S-adenosyl-l-homocysteine (AdoHcy). The methyltransferase of human TGS1 harbors the canonical class 1 methyltransferase fold as well as an unique N-terminal, α-helical domain of 40 amino acids, which is essential for m7G-cap binding and catalysis. The crystal structure of the substrate bound methyltransferase domain as well as mutagenesis studies provide insight into the catalytic mechanism of TGS1

ETS1, nucleolar and non-nucleolar TERT expression in nevus to melanoma progression.

TERT (telomerase reverse transcriptase) is the catalytic component of telomerase. TERT shows little expression in normal somatic cells but is commonly re-expressed in cancers, facilitating immortalization. Recently-discovered TERT promoter mutations create binding sites for ETS-family transcription factors to upregulate TERT. ETS1 is reported to be important for TERT upregulation in melanoma. However it is unclear when in melanoma progression TERT and ETS1 proteins are expressed. To elucidate this question, ETS1 and TERT immunohistochemistry were performed on a panel of benign (n=27) and dysplastic nevi (n=34), radial growth phase (n=29), vertical growth phase (n=25) and metastatic melanomas (n=27). Lesions were scored by percentage of positive cells. ETS1 was readily detectable in all lesions, but not in normal melanocytes. TERT was located in either the nucleolus, the nucleoplasm (non-nucleolar) or both. Non-nucleolar TERT increased in prevalence with progression, from 19% of benign nevi to 78% of metastases. It did not however correlate with cell proliferation (Ki-67 immunostaining), nor differ significantly in prevalence between primary melanomas with or without a TERT promoter mutation. These results demonstrate that ETS1 is expressed very early in melanoma progression, and interestingly only non-nucleolar TERT correlates clearly in prevalence with melanoma progression. It can be acquired at various stages and by mechanisms other than promoter mutations