Two RNA-binding motifs in eIF3 direct HCV IRES-dependent translation.

The initiation of protein synthesis plays an essential regulatory role in human biology. At the center of the initiation pathway, the 13-subunit eukaryotic translation initiation factor 3 (eIF3) controls access of other initiation factors and mRNA to the ribosome by unknown mechanisms. Using electron microscopy (EM), bioinformatics and biochemical experiments, we identify two highly conserved RNA-binding motifs in eIF3 that direct translation initiation from the hepatitis C virus internal ribosome entry site (HCV IRES) RNA. Mutations in the RNA-binding motif of subunit eIF3a weaken eIF3 binding to the HCV IRES and the 40S ribosomal subunit, thereby suppressing eIF2-dependent recognition of the start codon. Mutations in the eIF3c RNA-binding motif also reduce 40S ribosomal subunit binding to eIF3, and inhibit eIF5B-dependent steps downstream of start codon recognition. These results provide the first connection between the structure of the central translation initiation factor eIF3 and recognition of the HCV genomic RNA start codon, molecular interactions that likely extend to the human transcriptome

Two RNA-binding motifs in eIF3 direct HCV IRES-dependent translation

The initiation of protein synthesis plays an essential regulatory role in human biology. At the center of the initiation pathway, the 13-subunit eukaryotic translation initiation factor 3 (eIF3) controls access of other initiation factors and mRNA to the ribosome by unknown mechanisms. Using electron microscopy (EM), bioinformatics and biochemical experiments, we identify two highly conserved RNA-binding motifs in eIF3 that direct translation initiation from the hepatitis C virus internal ribosome entry site (HCV IRES) RNA. Mutations in the RNA-binding motif of subunit eIF3a weaken eIF3 binding to the HCV IRES and the 40S ribosomal subunit, thereby suppressing eIF2-dependent recognition of the start codon. Mutations in the eIF3c RNA-binding motif also reduce 40S ribosomal subunit binding to eIF3, and inhibit eIF5B-dependent steps downstream of start codon recognition. These results provide the first connection between the structure of the central translation initiation factor eIF3 and recognition of the HCV genomic RNA start codon, molecular interactions that likely extend to the human transcriptome. © 2013 The Author(s)National Institutes of Health (NIH) [R56-AI095687 to J.H.D.C.; P50-GM102706 to J.A.D. and J.H.D.C.]; Spanish Ministry of Education through the Programa Nacional de Movilidad de Recursos Humanos del Plan Nacional de I-D+i 2008-2011 (to E.A.-P.). J.A.D. and E.N. are Howard Hughes Medical Institute Investigators. Funding for open access charge: NIH [P50-GM102706]Peer Reviewe

Plastic degradation by insect hexamerins: Near-atomic resolution structures of the polyethylene-degrading proteins from the wax worm saliva

14 p.-8 fig.Plastic waste management is a pressing ecological, social, and economic challenge. The saliva of the lepidopteran Galleria mellonella larvae is capable of oxidizing and depolymerizing polyethylene in hours at room temperature. Here, we analyze by cryo–electron microscopy (cryo-EM) G. mellonella’s saliva directly from the native source. The three-dimensional reconstructions reveal that the buccal secretion is mainly composed of four hexamerins belonging to the hemocyanin/phenoloxidase family, renamed Demetra, Cibeles, Ceres, and a previously unidentified factor termed Cora. Functional assays show that this factor, as its counterparts Demetra and Ceres, is also able to oxidize and degrade polyethylene. The cryo-EM data and the x-ray analysis from purified fractions show that they self-assemble primarily into three macromolecular complexes with striking structural differences that likely modulate their activity. Overall, these results establish the ground to further explore the hexamerins’ functionalities, their role in vivo, and their eventual biotechnological application.This work was funded by Roechling Stiftung to F.B., Consejo Superior de Investigaciones Cientificas (CSIC) to F.B., Ministerio de Ciencia e Innovación (grants PID2019-111215RB-100 and PDC2022-133955-I00) to T.T., Ministerio de Ciencia e Innovación (grant PID2019-111215RB-100) to T.T., the Generalitat de Catalunya (2017 SGR 1192) to M.S., the Spanish Ministry of Science, Innovation and Universities MCIN/AEI/10.13039/501100011033 ERDF “A way to make Europe” PID2021-129038NB-I00 to M.S., and MCIN/AEI/10.13039/501100011033 (grant PID2020-120275GB-I00) to E.A.-P.Peer reviewe

Characterization of SMG-9, an essential component of the nonsense-mediated mRNA decay SMG1C complex

SMG-9 is part of a protein kinase complex, SMG1C, which consists of the SMG-1 kinase, SMG-8 and SMG-9. SMG1C mediated phosphorylation of Upf1 triggers nonsense-mediated mRNA decay (NMD), a eukaryotic surveillance pathway that detects and targets for degradation mRNAs harboring premature translation termination codons. Here, we have characterized SMG-9, showing that it comprises an N-terminal 180 residue intrinsically disordered region (IDR) followed by a well-folded C-terminal domain. Both domains are required for SMG-1 binding and the integrity of the SMG1C complex, whereas the C-terminus is sufficient to interact with SMG-8. In addition, we have found that SMG-9 assembles in vivo into SMG-9:SMG-9 and, most likely, SMG-8:SMG-9 complexes that are not constituents of SMG1C. SMG-9 self-association is driven by interactions between the C-terminal domains and surprisingly, some SMG-9 oligomers are completely devoid of SMG-1 and SMG-8. We propose that SMG-9 has biological functions beyond SMG1C, as part of distinct SMG-9-containing complexes. Some of these complexes may function as intermediates potentially regulating SMG1C assembly, tuning the activity of SMG-1 with the NMD machinery. The structural malleability of IDRs could facilitate the transit of SMG-9 through several macromolecular complexes

Wax worm saliva and the enzymes therein are the key to polyethylene degradation by Galleria mellonella

11 p.-6 fig.Plastic degradation by biological systems with re-utilization of the by-products can be the future solution to the global threat of plastic waste accumulation. We report that the saliva of Galleria mellonella larvae (wax worms) is capable of oxidizing and depolymerizing polyethylene (PE), one of the most produced and sturdy polyolefin-derived plastics. This effect is achieved after a few hours’ exposure at room temperature and physiological conditions (neutral pH). The wax worm saliva can indeed overcome the bottleneck step in PE biodegradation, that is the initial oxidation step. Within the saliva, we identified two enzymes that can reproduce the same effect. This is the first report of enzymes with this capability, opening up the way to new ground-breaking solutions for plastic waste management through bio-recycling/up-cycling.Roechling Stiftung to FB Consejo Superior de Investigaciones Científicas (CSIC) to FB NATO Science for Peace and Security Programme (Grant SPS G5536) to TT Junta de Castilla y León, Consejería de Educación y Cultura y Fondo Social Europeo (Grant BU263P18) to TT Ministerio de Ciencia e Innovación (Grant PID2019-111215RB-100) to TT The Generalitat de Catalunya (2017 SGR 1192) to MS Ministerio de Ciencia e Innovación (Grant BFU2017-89143-P) to EA-PN

Análisis Estructural de proteínas reguladoras de GTPasas de la superfamilia Ras mediante microscopía electrónica

Leída en la Universidad Complutense de Madrid. Facultad de Ciencias Químicas el 11-21-2008; 120 págs.Todas las células perciben señales de su entorno que modulan procesos como su proliferación, diferenciación, migración o muerte. Estas señales pueden venir de moléculas solubles, de moléculas que conforman la matriz extracelular o a través del contac to directo con otras células, y son recibidas por receptores específicos celulares. La activación de estos receptores inicia multitud de rutas de señalización. Se ha realizado un gran esfuerzo en el estudio de estas rutas para ahondar en el conocimie nto de su función celular y porque su mal funcionamiento está ligado a numerosas enfermedades. Gracias a estos trabajos cada vez se tiene más claro que la transducción de la señal no ocurre de manera lineal, desde los receptores hasta sus respuestas celulares específicas, sino que existe una vasta interconexión entre distintas rutas de señalización. Muchas de las moléculas que forman parte de esta densa red son proteínas modulares que poseen una regulación compleja. En el desarrollo de esta tesi s se ha estudiado la estructura y regulación mediante microscopía electrónica de tres de estas proteínas, involucradas en las rutas de señalización de la superfamilia Ras. Se trata de: ¿ Vav3- Factor intercambiador de nucleótidos (GEF, Guanine nucle otide exchage factor) que activa a proteínas pertenecientes a la familia de GTPasas (GTP hidrolasas) Rho. ¿ Syk (Spleen tyrosine kinase)- Proteína tirosín quinasa responsable, entre otras cosas de la fosforilación y activación de Vav.Peer reviewe

With science, there is a future

2 p.-1 fig.Peer reviewe

Unraveling bacterial DNA replication initiation using cryo-EM

2 p.Peer reviewe

Desafíos y oportunidades en el futuro de la crio-microscopía electrónica

3 p.-2 fig.Peer reviewe

An Atypical AAA+ ATPase Assembly Controls Efficient Transposition through DNA Remodeling and Transposase Recruitment

SummaryTransposons are ubiquitous genetic elements that drive genome rearrangements, evolution, and the spread of infectious disease and drug-resistance. Many transposons, such as Mu, Tn7, and IS21, require regulatory AAA+ ATPases for function. We use X-ray crystallography and cryo-electron microscopy to show that the ATPase subunit of IS21, IstB, assembles into a clamshell-shaped decamer that sandwiches DNA between two helical pentamers of ATP-associated AAA+ domains, sharply bending the duplex into a 180° U-turn. Biochemical studies corroborate key features of the structure and further show that the IS21 transposase, IstA, recognizes the IstB•DNA complex and promotes its disassembly by stimulating ATP hydrolysis. Collectively, these studies reveal a distinct manner of higher-order assembly and client engagement by a AAA+ ATPase and suggest a mechanistic model where IstB binding and subsequent DNA bending primes a selected insertion site for efficient transposition