Poster Session 8: RNP Biogenesis & Modification 20:00 - 21:00 Friday, 29th May, 2020 Poster

39 Arginine-to-glycine mutants of HIV Rev arginine-rich motif with altered specificity

Ingrid Ghattas1, Kazuma Takaoka2, Taiichi Sakamoto2, Kazuo Harada3, Colin Smith1 1American University of Beirut, Beirut, Lebanon. 2Chiba Institute of Technology, Narashino, Chiba, Japan. 3Tokyo Gakugei University, Koganei, Tokyo, Japan

Abstract

Arginine-rich motifs (ARMs) binding small, structured RNAs occur in important viral regulatory processes, including well-studied examples with structural models, such as the human immunodeficiency virus (HIV) Rev- RRE interaction that mediates the nuclear export of incompletely spliced viral transcripts. The Rev ARM-IIB interaction relies on its α-helical ARM and four amino acids, Arg35, Arg39, Asn40, and Arg44, that make contacts to five guanine bases in the internal loop formed by non-canonical purine-purine base pairs G47:A73 and G48:G71. Many functional variants of the Rev ARM-IIB interaction have been discovered, yet how easily it can evolve new specificities is poorly explored. Previously, a double mutant of Rev ARM, R35G-N40V, which uses a distinct, unknown strategy to recognize IIB, was discovered and characterized, prompting further exploration. Recently, several Arg-to-Gly mutants (R35G, R38G, and R41G) were discovered that bind mutant RRE G50A;C69A yet do not bind wild-type RRE IIB. This constellation of Arg-to-Gly mutants with altered specificity was intriguing, especially in light of R35G-N40V employing a recognition strategy distinct from the wild-type interaction. Here, we characterize Rev R35G, R38G, and R41G by mutational profiling, gel-shifts, isothermal titration calorimetry, and site-specific mutations. The amino acid usage of the mutants are not clearly distinct from wild-type Rev, suggesting recognition strategies similar to the wild-type interaction, and only R38G displays high affinity in vitro. Mutational footprinting was undertaken to reveal which RRE residues are important for R35G, R38G, R41G, and R35G-N40V. A reporter system recapitulating the dimerization of Rev ARM on RRE II ABC shows that R35G, R38G, and R41G function in the dimer context, but that R35G-N40V does not. We speculate Rev R38G-RRE G50A;C69A has a structure similar to the wild-type interaction and that entropic destabilization imparted by the glycine mutation is compensated by the RNA mutations.

Presenting author email [email protected]

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RNPs: Biogenesis, Structure & Function 68 RNA Binding to ATP Synthase: A journey into the unknown

Aindrila Chatterjee, Thileepan Sekaran, Thomas Schwarzl, Matthias W Hentze European Molecular Biology Laboratory (EMBL), Heidelberg, Germany

Abstract

FO-F1 ATP synthase forms the last component of mitochondrial respiratory chain, generating ATP from ADP and inorganic phosphate. Highly sensitive to metabolic cues, it has decisive roles in cell differentiation, immune response, aging and pathogenic remodeling of tissues. Recent surveys of RNA-bound proteomes have revealed a surprising enrichment of catalytic (F1) components of ATP synthase as putative RNA binders. This association is highly conserved across different eukaryotic phyla including plants, yeast, flies, mice and humans.

Our study focuses on exploring this intriguing finding and identifying the functional role of the ATP synthase F1 module (F1-ATPase) - RNA interaction in mammalian cells. Divided into four connected modules, we have first biochemically verified the specific RNA-interacting components of F1-ATPase. Second, using enhanced CLIP (crosslinking and immunoprecipitation) and deep sequencing, we have identified the RNA(s) associated with F1-ATPase. This information will be used to design RNA and/or protein binding-deficient mutants of the corresponding components. Finally, based on curated experimental data and established molecular toolkits, we are designing functional assays to test the significance of F1-ATPase-RNA association. I will present our strategy and current data. Taken together, these experiments will define the premise for developing a working hypothesis on the biology of RNA-ATP synthase interaction. This study explores a novel avenue of research into the regulatory potential of RNAs on ATP synthase and vice versa, with the far reaching goal of gaining an insight into the complex biology of coupling bioenergetic and transcriptional fluxes in a cell.

Presenting author email [email protected]

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RNPs: Biogenesis, Structure & Function 71 Network Theory Reveals Fundamental Principles of Spliceosome Assembly and Structure

Clarisse van der Feltz, Harpreet Kaur, Yichen Sun, Aaron Hoskins U. Wisconsin-Madison, Madison, WI, USA

Abstract

Cryo-EM has revolutionized structural biology of the spliceosome. Dozens of yeast and human spliceosome structures are now available with each depicting thousands of molecular interactions. This revolution has presented a challenge in the field for quantitatively describing each structure as well as evaluating the changes between different structures. We have cataloged every protein/protein, protein/RNA, and RNA/RNA interaction in available cryo-EM structures of yeast spliceosomes. We have then applied computational methods based in network theory to calculate structure-defined spliceosome modules and connectivity; the eigenvector and betweenness centralities for each splicing factor; and changes to modularity, centrality, and connectivity as the spliceosome assembles, becomes activated for catalysis, and performs the splicing reaction. Our results re-define the textbook, snRNP-centric view of spliceosome assembly and catalysis by showing how a high degree of structural integration allows splicing factors to move between structural modules. The connectivity of the spliceosome is highly dynamic with some factors always playing a central role (e.g., Prp8) and others becoming critical only at specific stages (e.g., U2 snRNA, Ecm2, Prp45). Elongated, non-globular spliceosome proteins often exhibit high centrality and contribute significantly to spliceosome network connectivity. Finally, we have carried out an in-depth network analysis of the Prp8 protein and U6 snRNA at amino acid or nucleotide-level resolution, respectively. These results provide a detailed, quantitative view of structural connectivity to U6 and Prp8 and allow us to bridge computational analysis of spliceosome structures to splicing phenotypes observed with U6 or Prp8 mutations in yeast. In sum, our results reveal how quantitative, mathematical models of large macromolecular machines can yield new insights into their assembly, dynamics, and structure.

Presenting author email [email protected]

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RNPs: Biogenesis, Structure & Function 123 The interplay of Lin28a and YB-1 proteins impacting the mRNA translation regulation

Anastasiia Samsonova1, Krystel El Hage1, Bénédicte Desforges1, Vandana Joshi1, Marie-Jeanne Clement1, Guillaume Lambert1, Pierrick Craveur2, Loïc Hamon1, David Pastré1 1SABNP, Univ Evry, INSERM U1204, University Paris-Saclay, Evry, France. 2Synsight, a/s IncubAlliance, Orsay, France

Abstract

YB-1 and Lin28a are two RNA-binding proteins (RBPs) that contain a single cold-shock domain, a conserved RNA-binding beta barrel structure. Both of these proteins bind directly to mRNAs in cytoplasm to regulate translation. YB-1 and Lin28a are also considered as cancer molecular markers involved into translational control of oncogenes. Sequence and structure similarity of these two RBPs raises the question whether they could act together causing the pathology progression in humans. Well-known role of Lin28a in pluripotent cells is inhibition the pri-let-7 miRNA maturation, but it is important to indicate that expression of Lin28a is taking place even while the let-7 pathway is not yet activated during cell differentiation. Here, using biochemical and biophysical methods, we showed YB-1 and Lin28a ability to bind single stranded nucleic acids in similar cooperative manner. Moreover, due to high structure similarity of Lin28a and YB-1 cold- shock domains, these proteins are able to form a mixed polymer upon mRNA binding. Our NMR experiments revealed some amino acid residues of Lin28a that are affected upon proteins multimerization, and further, using the method developed in our laboratory, we observed that mutation of these residues disturbs the proteins mixing pattern. Using cell biology approaches, we showed the co-localization of YB-1 and Lin28 in cytoplasm, and also we got the evidence of YB-1-dependend Lin28a function in differentiating cells. Overall, these results bring us to the better understanding of proteins network in cells and their activity with impact on translation regulation.

Presenting author email [email protected]

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RNPs: Biogenesis, Structure & Function 182 Structure and RNA binding properties of Lsm1-7 and Lsm2-8 complexes

Eric Montemayor, Johanna Virta, Samuel Hayes, Yuichiro Nomura, David Brow, Samuel Butcher University of Wisconsin, Madison, WI, USA

Abstract

Eukaryotes possess highly conserved Lsm proteins that assemble into heteroheptameric complexes that bind RNA and control a diverse range of biological processes. The cytoplasmic Lsm1-7 complex plays a major role in mRNA decay by binding to mRNA and recruiting the decapping machinery to facilitate degradation. The highly related Lsm 2-8 complex is nuclear, binds the 3′ end of U6 snRNA, and is also involved in nuclear mRNA decay and telomerase biogenesis. Here, we establish the molecular basis for Lsm-RNA recognition and specificity and present four structures at high-resolution (1.8-2.3 Å) of S. pombe Lsm1-7 and Lsm2-8 complexes bound to RNAs. The structures of Lsm2-8 bound to unmodified and post-transcriptionally modified poly-uridine RNA reveal how it binds RNA with increased affinity for the unique 2′,3′ cyclic phosphate end of U6, which forms an unusual structure that is recognized by highly conserved residues in Lsm3 and the C-terminus of Lsm8. In contrast, the Lsm1-7 complex has a strong preference to bind polyuridine tracts followed by a 3′ terminal purine (UUUUR-3′-OH) as revealed by extensive binding affinity measurements against a panel of RNA sequences. In the course of these investigations we discovered that the C-terminal helical region of Lsm1 is a gate that does not contribute to affinity, but rather serves to increase binding specificity. The structures of the Lsm1-7-RNA complexes reveal the presence of an unexpected and atypical purine binding pocket in Lsm5, explaining why Lsm5 has a divergent binding pocket sequence. These structures establish the molecular basis for RNA interactions with the Lsm1-7 and Lsm2-8 complexes, which we expect to be broadly general across eukaryotic biology. This research further reveals for the first time how the modular and highly related Lsm complexes can assemble into quaternary structures with unique RNA binding properties.

Presenting author email [email protected]

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RNPs: Biogenesis, Structure & Function 255 Influence of hypoxia on the RNA binding proteins complexes and translation in leukemia cells

Magdalena Wolczyk, Marta Jakubik, Laura Turos, Milena Wiech, Zofia Dabrowska, Katarzyna Piwocka, Paulina Podszywalow-Bartnicka Nencki Institute of Experimental Biology, Warsaw, Poland

Abstract

Leukemia cells migrate along different environments, from hypoxic in the bone marrow to oxygen-reach in the blood circulation (so called normoxia), activation of stress response pathways might modulate sensitivity of leukemia cells to therapy. We have observed that activation of mild stress response induces treatment- resistance of leukemia cells [Cell Cycle, 2012]. We have also found that expression of oncogene in CML leukemia cells induces stress granules formation by T-cell intracellular antigen-1 (Tia) proteins and leads to TIAR-dependent down-regulation of BRCA1 protein synthesis [Cell Cycle, 2014; BBA, 2017]. Herein, we would like to present our results concerning response of the RNA binding proteins (RBPs) to adaptation of leukemia cells to low oxygen level. Using various leukemia cell lines (K562, Lama-84, BV173, HL-60 and Nalm-6) as well as primary CML cells cultured in normoxia or in the hypoxia workstation (1.5% O2, 5% CO2) we observed that exposition to hypoxia activates distinct signaling pathways during acute phase (first hours) than in the chronic phase (over 72h), what affects nuclear/cytoplasmic shuttling of the RBPs. TMT-mass spectrometry analysis of protein composition of cytoplasmic RBPs complexes of normoxia versus hypoxia-adopted cells revealed that oxygen conditions affect the activity of RBPs in the cytoplasm, along with changes in monosome/polysome profile of the cells. Our results point to translational control as important component of leukemia cells adaptation to hypoxia. This work was supported by research grants from the National Science Centre: UMO- 2014/15/D/NZ3/05187 and UMO-2018/30/M/NZ3/00274 to P.P.-B and UMO-2016/23/N/NZ3/02232 to M.W.

Presenting author email [email protected]

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RNPs: Biogenesis, Structure & Function 256 The structure and function of held out wings, an essential RBP during spermatogenesis in Drosophila melanogaster

Michaela Agapiou1, Tayah Hopes1, Amanda Bretman2, Thomas Edwards3,1, Julie Aspden1 1School of Molecular and Cellular Biology, University of Leeds, Leeds, United Kingdom. 2School of Biology, University of Leeds, Leeds, United Kingdom. 3Astbury Centre, University of Leeds, Leeds, United Kingdom

Abstract

Held out wings (HOW) is an RNA-binding protein essential for spermatogenesis in D. melanogaster. It is part of the signal transduction and activation of RNA (STAR) family of proteins, which contain a single maxi-KH domain. HOW’s orthologues include quaking in mammals and GLD-1 in C. elegans, both important post- transcriptional regulators of RNAs in processes such as gametogenesis and myelination. Similar to quaking there are multiple protein isoforms of HOW, the two established ones are HOW(L) and HOW(S). The longer isoform, HOW(L), is nuclear and important for regulating the mitotic cell divisions before meiosis. The shorter HOW(S) isoform is cytoplasmic, and has not been well studied in the context of spermatogenesis.

To identify and characterise the RNAs important in HOW(S)’s function, we have carried out RIP-seq from HOW(S)-expressing cells in the testes, the spermatogonia in the 1-2 cell cysts. This identified 115 new potential HOW(S) targets using differential transcript enrichment analyses. These transcripts are enriched for the GO term ‘positive regulation of signal transduction’, coinciding with HOW belonging to the STAR family of proteins. The previously published HOW response element (HRE), based upon its interaction with the 3’UTR of stripe, is ACUAA. With a much larger number of HOW target 3’-UTRs, we have found an RNA motif, that closely matches the HRE but extends it to a hexamer: (A/G/U)CUAAC. This was found in 49% of these targets’ 3’-UTRs. Further, we identified a novel motif within 5’-UTRs, GCG(A/U/C)G, in 65% of these transcripts.

Fluorescence anisotropy experiments have revealed that recombinant HOW RNA-binding domain preferentially binds polyU over polyA. Next, we will determine the affinity of HOW for the RNA motifs we have identified in both 3’-UTR and 5’-UTRs. Work is also underway to determine the structure of HOW bound to an RNA motif by X-ray crystallography. This will provide molecular detail into the binding of HOW to the RNA targets we have identified, providing insight into the importance of HOW-RNA interactions during D. melanogaster spermatogenesis.

Presenting author email [email protected]

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RNPs: Biogenesis, Structure & Function 433 Single-molecule observation of the effects of Hero proteins on TDP-43 conformation

Andy Y. W. Lam1,2, Kotaro Tsuboyama1,3, Hisashi Tadakuma1,4, Yukihide Tomari1,2 1Institute for Quantitative Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan. 2Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan. 3Department of Pharmacology, Northwestern University, Chicago, Illinois, USA. 4School of Life Science and Technology, ShanghaiTech University, Shanghai, China

Abstract

Many RNA binding proteins (RBPs) contain low-complexity Prion-like domains (PrLDs) that contribute to their functionality and allow them to undergo liquid-liquid phase transitions. However, these intrinsically disordered domains make RBPs prone to misfolding, forming pathological aggregates in various neurodegenerative diseases. For example, TAR DNA-binding protein 43 (TDP-43) is found aggregated in about 45% of Frontotemporal lobar degeneration (FTLD) and 97% of Amyotrophic lateral sclerosis (ALS) cases. While chaperones are thought to counteract aggregation by reversing misfolding, our lab has recently identified a widespread family of Heat-resistant obscure (Hero) proteins that also suppress aggregation of TDP-43. Unlike canonical chaperones, Hero proteins are completely unstructured, highly hydrophilic and electrostatically charged, suggesting an enigmatic mechanism of action. However, due to the difficulty of studying conformationally diverse proteins, the mechanisms of misfolding, aggregation, and their prevention remain elusive. Single-molecule Förster resonance energy transfer (smFRET) is one of the few methods able to observe conformational states within molecular ensembles. Here we use genetic code expansion in HEK293T cells to produce full-length TDP-43 with unnatural amino acids for the site-specific introduction of fluorescent dyes. Utilizing smFRET we probe the conformational states of the TDP-43 PrLD under the influence of Hero proteins. We found that while TDP-43 populates a broad distribution of intermediate FRET, Hero proteins shift this to distinct very low and high FRET conformational states. Our results are consistent with a model where Hero proteins prevent TDP-43 from sampling aggregation-prone states within the dynamic conformational ensemble.

Presenting author email [email protected]

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RNPs: Biogenesis, Structure & Function 436 TSSC4 is a novel U5 snRNP-specific chaperon

Klara Klimesova1, Jitka Vojackova1, Celine Verheggen2, Edouard Bertrand2, David Stanek1 1Institute of Molecular Genetics, Prague, Czech Republic. 2Institut de Génétique Moléculaire de Montpellier, Montpellier, France

Abstract

The spliceosome is formed from pre-assembled small nuclear ribonucleoproteins particles (snRNPs), each containing a small nuclear RNA and a specific set of proteins. Three of these snRNPs enter the splicing reaction as a pre-formed U4/U6•U5 tri-snRNP. During splicing the tri-snRNP undergoes extensive rearrangement, individual snRNPs are released and recycled before entering a next splicing reaction. Using SILAC, we have identified a protein called TSSC4 to interact with U5-specific proteins PRPF8 and EFTUD2. We further show that TSSC4 is a new U5 snRNP specific protein, which also associates with components of the NTC complex. We identified TSSC4 regions important for interaction with U5 snRNP and NTC. TSSC4 knockdown in HeLa cells impairs snRNP biogenesis and consistently with this result we observe increased accumulation of snRNP components in Cajal bodies, including a mono U5-specific protein CD2BP2. Together we conclude that TSSC4 as a novel U5 snRNP specific factor important snRNP biogenesis and speculate that TSSC4 also acts during recycling phase of the spliceosomal cycle.

Presenting author email [email protected]

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RNPs: Biogenesis, Structure & Function 572 smFRET studies of the DEAH-box helicase DHX36 with RNA G-quadruplexes

Michael Banco1, Tapas Paul2, Sua Myong2, Adrian Ferré-D'Amaré1 1National Institutes of Health, Bethesda, MD, USA. 2Johns Hopkins, Baltimore, MD, USA

Abstract

G-quadruplexes (G4s) are stable nonconical nucleic acid structures that are suggested to regulate numerous essential biological processes, which include genome stability, transcription, and translation. These nucleic acid structures are formed by multiple tracts of guanosines self-assembling into stacked planar quartets coordinated by a metal ion. Several helicases have been shown to exhibit G4 unwinding activity, but the molecular mechanism of resolving G4 structures by these proteins still remains unclear. DHX36 is an essential G4-specific helicase that possesses high affinity to parallel DNA/RNA G4s. Previously, we determined multiple crystal structures of DHX36, including one complexed with the Myc promoter G4, that provided insights into the unwinding mechanism of the helicase (Chen et. al., Nature 558:465, 2018). These structures indicated DHX36 undergoes an ATP-independent conformational change upon binding to a G4, which consequently pulls the strand by one nucleotide and rearranges the bound G4 into an unstable state. Furthermore, previous smFRET studies demonstrated DHX36 inducing an ATP-independent unfolded state and exhibited repetitive unfolding in an ATP-dependent manner for a RNA G4 (Tippana et. al., Nat Commun 10:1855, 2019). Here, we utilized smFRET methods to demonstrate DHX36 inducing a similar destabilized structure upon binding and unfolds in a repetitive ATP-dependent manner for a RNA G4 formed in the human telomerase RNA component (hTERC). Interestingly, our smFRET measurements of the hTERC RNA G4 demonstrated slower ATP-dependent repetitive unfolding and dissociation of DHX36 from the G4 in comparison to previously published RNA G4s. Additionally, we provide further evidence that the observed FRET fluctuations are a result of DHX36 rearranging the G4 structure. This work was supported, in part, by the Intramural Program of the National Heart, Lung, and Blood Institute, NIH.

Presenting author email mike.banco@nih,gov

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RNPs: Biogenesis, Structure & Function 619 Analysis of mRNP Complexes by Fluorescence Correlation and Cross- Correlation Spectroscopy

Àngels Mateu-Regué1, Jan Christiansen2, Christian Hellriegel3, Finn Cilius Nielsen1 1Center for Genomic Medicine, Rigshospitalet, Copenhagen, Denmark. 2Department of Biology, University of Copenhagen, Copenhagen, Denmark. 3Carl Zeiss RMS / Harvard Center for Biological Imaging, Cambridge, MA 02138, USA

Abstract

Understanding the mRNA life cycle requires analysis of the dynamic macromolecular composition and stoichiometry of mRNPs. Fluorescence correlation and cross-correlation spectroscopy (FCS and FCCS) are appealing technologies to study mRNP complexes because they readily may provide information about the molecular composition, stoichiometry and dynamics of the particles. We developed protocols for analysis of live cells and cellular lysates, and demonstrate the feasibility of analysing common cytoplasmic mRNPs composed of core factor YBX1, IMPs (IMPs or IGF2BPs) and their interactions with other RNA binding proteins such as PABPC1, ELAVL2 (HuB), STAU1 and FMRP. FCS is fast and reproducible and data recorded in live cells is largely reconciled in lysates. FCCS corroborated previously reported RNA dependent interactions between the factors and provided an estimate of the relative overlap between the factors in the mRNPs. We concluded that FCS and FCCS provide a new and useful approach for the quantitative and dynamic analysis of mRNP macromolecular complexes that may complement current biochemical approaches.

Presenting author email [email protected]

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RNPs: Biogenesis, Structure & Function 655 Mapping the role of hRNase P individual protein subunits via CRISPR/Cas9

Ilias Skeparnias, Athanasios-Nasir Shaukat, Constantinos Stathopoulos Department of Biochemistry, School of Medicine, University of Patras, Patras, Greece

Abstract

RNase P is an essential ribonucleoprotein responsible for the maturation of the 5’ leader of precursor tRNAs, 5S rRNA and specific long and small non-coding RNAs. Human nuclear RNase P consists of one catalytic RNA (H1) and 10 protein subunits (Rpps) some of which have known alternatives roles during transcription. In the present study, the viability of HeLa cells after ablation of either RPP21 or RPP29 using CRISPR/Cas9 was examined. Both proteins have been previously shown to play role on DNA damage and serve either as scaffold for recruitment of HDR proteins or non-coding RNAs to DSB sites, after depletion of their expression using siRNAs. The recent structure of the hRNase P complex revealed that RPP21 stacks between RPP29 and RPP38 and that both RPP21 and RPP29 are important for the local architecture of the wrist module of the holoenzyme. Disruption of either RPP21 or RPP29 genes resulted in undetectable expression, but did not affect cell viability. In the case of RPP21 knockout, observed morphological alterations in the edited cells were accompanied by significantly lower growth rates, indicating possible deregulation of their proliferation rates. Interestingly, and albeit their slow growth, RPP21 knockout cells survival suggests a metabolic rewiring that seems to be unaffected by the apparent RNase P deficiency. Expression of important transcription and translation factors was examined through qRT-PCR and western blot analysis and revealed significant alterations that could explain the observed phenotype. Overall, our data coincide with previous reports and support the notion that some of the protein subunits of RNase P, although important for the overall architecture of the holoenzyme, can be dispensable for cell viability, raising questions on the protein content of RNase P, under various conditions, including stress. Moreover, our observations highlight the possible alternative roles of Rpps beyond their participation in RNase P complex formation.

Presenting author email [email protected]

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RNPs: Biogenesis, Structure & Function 656 A novel and essential nematode protein links spliced leader trans-splicing to NAD-utilising pathways and RNA 3’ end formation and degradation

Rosie Spencer, Rotimi Fasimoye, Haithem ElMassoudi, Eva Soto-Martin, Bernadette Connolly, Jonathan Pettitt, Berdnt Müller University of Aberdeen, Aberdeen, United Kingdom

Abstract

Spliced leader trans-splicing is an essential RNA processing step, related to cis-splicing, that forms the 5’ end of mRNAs in many different eukaryotes. In nematodes it is known that the majority of spliced leader trans- splicing events are dependent upon the SL1 snRNA, but the proteins allowing this RNA to function in trans- splicing are poorly understood. To gain insight into these protein components, we have developed a novel reporter gene assay to monitor SL1 trans-splicing (Philippe et al., NAR 45, 8474-8483). We have used this assay to confirm that selected candidate proteins are indeed involved in SL1 trans-splicing. Our recent unpublished data expands and refines our understanding of the proteins involved in SL1 trans-splicing: we have analysed factors co-immunoprecipitating with the SL1-specific protein SNA-1, providing a comprehensive view of SL1 snRNP components, and giving us insight into the interaction of the SL1 snRNP with the spliceosome. Importantly, we have identified SNA-3, a new, highly conserved nematode-specific protein involved in SL1 trans-splicing. SNA-3 contains NADAR domains, which have been linked to NAD/ADP-ribose metabolism. While little is known about domain function, plant and bacterial proteins containing NADAR domains have been shown to have N-glycosidase activity. Further exploration of the factors interacting with SNA-3 implicates it in RNA 3’ end formation and degradation.

Presenting author email [email protected]

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RNPs: Biogenesis, Structure & Function 786 Determinants of RNA recognition by the FinO-like domain of the E. coli ProQ protein

Ewa Stein1, Joanna Kwiatkowska1, Maciej Basczok1, Chandra M. Gravel2, Katherine E. Berry2, Mikołaj Olejniczak1 1Institute of Molecular Biology and Biotechnology, Poznań, Poland. 2Mount Holyoke College, South Hadley, MA, USA

Abstract

The regulation of bacterial adaptation to changing environmental conditions is partly dependent on small regulatory RNAs. The function of regulatory RNAs is often dependent on RNA binding proteins, such as the chaperone protein Hfq. Recent studies revealed that another protein, named ProQ, is a global RNA binding protein involved in the interactions with numerous small RNAs in E. coli and S. enterica. The ProQ protein consists of the N-terminal FinO-like domain, which is involved in RNA binding, a positively charged linker, and the C-terminal Tudor domain. Recent studies of RNA binding profiles of ProQ and Hfq in E. coli and S. enterica showed that these proteins bind distinct, although partly overlapping, pools of RNA ligands in bacterial cells. To better understand how the ProQ protein recognizes RNA ligands, we compared the binding of six different RNA molecules to full-length ProQ protein and the isolated FinO-like domain (ProQ NTD). The data showed that the ProQ NTD specifically recognized Rho-independent terminators in its RNA ligands. The analysis of RNA mutants showed that the binding of the ProQ NTD was dependent on the base-pairing in the lower part of the Rho-independent terminator hairpin preceding the 3' oligoU tail of at least four uridines of length. Additionally, our data showed that the A-rich motif upstream of the terminator serves as a negative determinant of binding to the Hfq protein, both in in vitro binding assays and when monitored using bacterial three-hybrid assay in E. coli cells. In summary, our data suggest that the specific binding of ProQ to RNA ligands in E. coli depends both on RNA sequence and structure elements recognized by ProQ and on those that prevent the binding of Hfq.

Presenting author email [email protected]

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RNPs: Biogenesis, Structure & Function 812 Mechanism of mRNA Association Underlying Heteromeric hERG Channel Assembly

Jennifer Knickelbine, Yusi Cui, Fang Liu, Lingjun Li, Gail Robertson University of Wisconsin-Madison, Madison, WI, USA

Abstract

The expression of cardiac ion channels must be precisely controlled to produce the regular beating of the heart and protect from arrhythmia. One of the most important channels in the heart is the IKr (Kv11.1) voltage-gated potassium channel, encoded by the human ether-à-go-go-related gene (hERG), which controls ventricular repolarization. These channels are tetramers composed of 2 types of subunits, termed 1a and 1b, and heteromeric channels conduct much more current than homomers. Thus, aberrant assembly and function of the channel can give rise to impaired repolarization, Long QT syndrome, and sudden cardiac death. Previous studies showed that hERG 1a and 1b mRNAs co-purify with nascent hERG 1a protein from lysates of co-transfected HEK cells, cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs), and native myocardium. This association does not require translation of 1b protein, and knockdown of either transcript reduces the levels of both transcripts, suggesting that the association likely occurs at the mRNA level. This project aims to determine the mechanism by which hERG mRNAs associate to facilitate co-translational, heteromeric assembly. We used several in vitro techniques (RNA dot blot, electrophoretic mobility shift assay (EMSA), and immunoprecipitation (IP) with mixed lysates from separately transfected HEK cells) to look for direct interactions between 1a and 1b mRNAs, and found that they do not associate under these conditions. We also used affinity capture mass spectrometry to identify RNA-binding proteins (RBPs) associated with the mRNAs in co-transfected HEK cells. We detected 133 proteins that specifically co-immunoprecipitate with hERG, 54 of which have RNA-binding gene ontology, and 13 which significantly enrich with hERG under 3 IP conditions. We are validating these interactions in living cells using immunofluorescence (IF), single molecule fluorescence in situ hybridization (smFISH) and other approaches. This work will establish a mechanism by which 1a and 1b mRNAs associate to produce heteromeric hERG channels and could identify new targets underlying cardiac arrhythmias.

Presenting author email [email protected]

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RNPs: Biogenesis, Structure & Function 844 A Neuronal Atlas of RNA-Binding Protein Expression and Localization at Single-Cell Resolution

John Laver1, Eviatar Yemini2, Oliver Hobert2, Mihail Sarov3, John Calarco1 1Department of Cell and Systems Biology, University of Toronto, Toronto, Canada. 2Department of Biological Sciences, Howard Hughes Medical Institute, Columbia University, New York, USA. 3Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany

Abstract

The nervous system is composed of many different types of neurons, each unique with regard to their functions and morphologies. The specification, development, and function of neurons and neuron subtypes require precise post-transcriptional control of gene expression, through regulation of mRNA splicing, localization, translation, and stability. These processes are regulated by trans-acting factors, such as RNA- binding proteins (RBPs) and small RNAs, hundreds of which are encoded in eukaryotic genomes. To gain a systems-level understanding of post-transcriptional regulation in the nervous system, we aim to define a comprehensive atlas of neuronal RBP expression in a whole organism, the nematode Caenorhabditis elegans, with single-cell resolution. To assess neuron-specific RBP expression and subcellular localization, we are using a microscopy-based approach, visualizing RBPs in C. elegans by expressing GFP-tagged RBP transgenes driven by endogenous regulatory elements. An initial survey of 40 RBPs has revealed a diversity of expression patterns: one-third of the RBPs exhibit expression restricted to particular subsets of neurons, and at the subcellular level, approximately equal proportions of the RBPs localize to either the cytoplasm, the nucleus, or both, with some RBPs displaying different subcellular localization patterns in different neurons. We have now begun to define the expression of each RBP with single-neuron precision, using NeuroPAL, a multi-colour neuronal identification strain in which individual neurons are marked with up to four different fluorescent proteins. A complete understanding of where and when each RBP is expressed in the nervous system will yield important insights into the RBP regulatory networks controlling neuronal development and function.

Presenting author email [email protected]

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RNPs: Biogenesis, Structure & Function 941 A 4-base pair core-enclosing helix in yeast telomerase RNA is essential for activity and for binding to the TERT catalytic protein subunit

Melissa Mefford1, Evan Hass1, David Zappulla1,2 1Johns Hopkins University, Baltimore, MD, USA. 2Lehigh University, Bethlehem, PA, USA

Abstract

Telomerase countervails the chromosome end-replication problem, preventing cellular senescence. In humans, an increase in telomerase is associated with 90% of cancers, whereas a decrease underlies premature aging and telomere syndromes. At its core, the telomerase RNP is composed of a dedicated reverse transcriptase (TERT) and a lncRNA. The majority of the 1157-nt Saccharomyces cerevisiae telomerase RNA, TLC1, is evolving extremely rapidly, but the catalytic core secondary structure is conserved, with aspects even shared with humans. In vivo, TLC1 can be pared down by 67% to just conserved regions, and these “Mini-T” RNAs maintain stable, shortened telomeres. Mini-T’s contain the catalytic core (with its template, template-boundary helix, pseudoknot/base-triples, and core-enclosing helix), as well as binding sites for Est1, Ku, and Sm7, at the ends of arms that radiate from the core. Telomerase RNA is likely to contain multiple critical binding sites for TERT, one of which we hypothesize is the core-enclosing helix (CEH). To test this, we used circularly permuted Mini-T to avoid disrupting the Area of Required Connectivity in the core, allowing us to specifically examine how CEH structure relates to telomerase activity and TERT binding. In such contexts, we consistently observed that a 4-bp CEH is necessary for telomerase to be active in vitro and maintain yeast telomeres in vivo, whereas ΔCEH, 1-bp, and 2-bp alleles were catalytically dead and senesced. We then used a new CRISPR•dCas9-based “CARRY two-hybrid” assay to assess binding of our circularly permuted Mini-T RNAs to TERT. The results of these tests showed that the 4-bp CEH RNA binds to TERT, but the shorter-CEH constructs do not, consistent with the telomerase activity and in-vivo complementation results. We conclude that a major reason why the core-enclosing helix is essential in yeast telomerase RNA is because it is needed to bind TERT to form the active core enzyme. Although the nucleotides comprising the CEH 4-bp stem are nearly invariant among Saccharomyces species, our results obtained using sequence-randomized and truncated-CEH alleles strongly suggest that CEH-TERT binding at the core of the telomerase RNP is dictated more by secondary than primary structure.

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RNPs: Biogenesis, Structure & Function 22 H/ACA box redundancy and conservation in the human fungal pathogen C. albicans

Kathryn Rodgers, Ethan Pickerill, Douglas Bernstein Ball State University, Muncie, IN, USA

Abstract

RNA can be modified in over 100 ways and absence of these modifications has a variety of impacts on cell health. One of the most prevalent RNA modifications is pseudouridylation. Pseudouridylation is catalyzed by pseudouridine synthases that cleave the C-N glyosidic bond and reattach the uracil at the C1 position to the ribose. rRNA pseudouridylation in Archaebacteria and Eukaryotes require H/ACA box snoRNA to guide the pseudouridine synthase to the target residue. H/ACA box snoRNAs form a hairpin secondary structure and two antisense regions located on an internal loop uses base pairing to target the pseudouridine synthases to their target residues. Here we characterize H/ACA box snoRNAs of the human fungal pathogen C. albicans. Our analysis suggests C. albicans H/ACA box snoRNAs are more poorly conserved among Candida species than CD Box snoRNAs or their S. cerevisiae counterparts. Furthermore, while C. albicans encodes 27 H/ACA box snoRNAs, multiple snoRNAs are predicted to target the same rRNA residue. We individually knocked out such redundant snoRNAs and identified which snoRNA/s are required to perform specific rRNA modifcations. Our data suggests pseudouridylation machinery varies among budding yeast lineages.

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Ribosome Biogenesis & Modification 37 Puf6 and Loc1 can bind RPL43 mRNA and control the production of this ribosomal protein

Ning-Hsiang Hsu, Le-Yun Yueh, Chih-Yi Chu, Kai-Yin Lo National Taiwan University, Taipei City, Taiwan

Abstract

The level of ribosome biogenesis is highly connected with cell growth rate. Many ribosomal proteins have extra-ribosomal functions. Overexpression or insufficient supply of ribosomal proteins may impair the cellular growth. Therefore, the supply of ribosomal proteins is tightly controlled in response to rRNA synthesis and environmental stimuli. Puf6 and Loc1 are RNA binding proteins. In our previous study, Puf6 and Loc1 are also identified as the dedicated chaperones of the ribosomal protein, Rpl43. They associate with Rpl43 and are critical to maintain the protein level and proper loading of Rpl43. In this study, Puf6 and Loc1 were shown to interact with RPL43 mRNA. The production of Rpl43 was controlled by its intron and 3’UTR and these elements were also regulated by Puf6 and Loc1. While the levels of mature RPL43 mRNAs were not decreased in puf6Δ and loc1Δ, the translation of RPL43 mRNA dropped in these two mutants. Interestingly, the ternary complex, Rpl43, Puf6 and Loc1, showed lower affinity with mRNA but higher affinity with rRNA. The results from this study suggest that the dedicated chaperones of Rpl43 not only control the protein level from stability but also regulate the production and incorporation of this ribosomal protein.

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Ribosome Biogenesis & Modification 89 The function of translation initiation factor eIF4G in ribosome biogenesis is connected with Brix family proteins in Saccharomyces cerevisiae

Yun-Ting Tseng, Yu-Cheng Sung, Kai-Yin Lo Department of Agricultural Chemistry, National Taiwan University, Taipei, Taiwan

Abstract eIF4G, an important eukaryotic translation initiation factor, recruits 40S ribosomal subunit to mRNA and start protein translation. It is a scaffold protein, bridging eIF4E (Cap-binding protein), eIF4A (RNA helicase), PABP (poly-A binding protein) and mRNA. In S. cerevisiae, it was reported that deletion of eIF4G1 caused defects in 60S biogenesis. However, no study has shown the molecular mechanism of eIF4G in 60S biogenesis. In addition to translation, ribosome biogenesis is also a key pathway to regulate protein levels. More than 200 trans-acting (assembly) factors are required for rRNA processing, assembly of rRNAs and proteins, stabilization of ribosomal proteins, and checking the accuracy in ribosome assembly. In this study, both eIF4G1 (Tif4631) and eIF4G2 (Tif4632) in yeast were shown to involve in 60S ribosome biogenesis. Deletion of eIF4G disturbed biogenesis pathway of 60S but not 40S. The direct protein-protein interaction was observed between eIF4G1 and Brix family proteins, including Ssf1/2, Brx1, Rpf1 and Rpf2. Besides, strong genetic interactions were observed between TIF4631 and Brix family. In eIF4GΔ, Brix family proteins changed the cellular distribution from nucleolus to nucleus and increased the retention with 60S. Ssf1, Brx1, and Rpf1 are assembly factors involved in PET (polypeptide exit tunnel) maturation. Our data indicate that eIF4G is also involved in facilitating PET maturation in 60S ribosome biogenesis. This is the first study to dissect the molecular role and importance of eIF4G in 60S biogenesis.

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Ribosome Biogenesis & Modification 181 Building and maintaining the mammalian nucleolus: insights uncovered by a phenotypic, genome-wide RNAi screen

Lisa Ogawa McLean1, Yulia Surovtseva2, Laura Abriola2, Susan Baserga1,3 1Yale University, New Haven, CT, USA. 2Yale Center for Molecular Discovery, West Haven, CT, USA. 3Yale School of Medicine, New Haven, CT, USA

Abstract

The nucleolus is a large and dynamic phase-separated organelle in the nucleus that originates from transcription of pre-ribosomal RNA (rRNA) from the tandemly repeated ribosomal DNA loci. In the mammalian genome there are 10 of these nucleolar organizing regions, yet cell lines differ in the observed average number of nucleoli per nucleus (Farley et al., 2015), and the mechanisms that govern these differences in nucleolar number remain unclear. In the human breast epithelial MCF10A cell line, the mean number of nucleoli per nucleus is 3; however, siRNA depletion of factors required for ribosome biogenesis cause a decrease in this number to 1 (Freed et al., 2012). A genome-wide siRNA screen of 18,017 genes using this one nucleolus phenotype as a readout revealed known as well as previously unknown proteins required for making ribosomes (Farley-Barnes et al., 2018). Intriguingly, this same screening campaign also uncovered factors that cause an increase in nucleolar number to 5 or more (n=113; unpublished), and bioinformatic analyses reveal that these hits are associated with mitosis and DNA replication, not ribosome biogenesis. Small molecule inhibition of the cell cycle using several commercially available drugs validates a role for DNA replication and mitosis in maintaining normal nucleolar number. Additionally, functional biochemical assays on a subset of hits (n=14) uncovers proteins required for pre-rRNA transcription (n=10/14) and global protein synthesis (n=13/14). In mammalian cells, nucleoli dissolve and re-form each mitosis, which includes the coalescence of individual nucleolar organizing regions into a single nucleolus. Taken together, we hypothesize that our phenotypic siRNA screen using increased nucleolar number as a readout has revealed a unique subset of proteins required to build the nucleolus in mitosis and maintain its structure and function during the cell cycle.

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Ribosome Biogenesis & Modification 278 Bud23 promotes the progression of the Small Subunit Processome to the pre-40S ribosome

Joshua Black, Richa Sardana, Ezzeddine Elmir, Arlen Johnson University of Texas at Austin, Austin, TX, USA

Abstract

Ribosomes are the molecular machines that synthesize proteins. Each ribosome is composed of a large and a small subunit which, respectively, carry out the essential functions of polypeptide synthesis and mRNA decoding. Ribosome production is tightly linked to cellular growth as cells must produce enough ribosomes to meet their protein needs. However, ribosome assembly is an energetically and metabolically expensive pathway that must be balanced with other cellular energy needs and regulated accordingly. The earliest metastable assembly intermediate of the eukaryotic ribosomal small subunit (SSU or 40S) is the SSU Processome, a large complex of protein and RNA factors that is thought to represent an early checkpoint in assembly pathway. Progression of the SSU Processome to the pre-40S particle requires the removal of the U3 snoRNA and biogenesis factors as well as cleavage of the ribosomal RNA. While the factors that drive these events are largely known, how progression of the SSU Processome is regulated is not understood. The methyltransferase Bud23 has a role during the progression of the SSU Processome, but its function, beyond methylation of G1575 in the 3’ basal subdomain of 18S rRNA, has not characterized. Here, we have carried out a comprehensive genetic screen in Saccharomyces cerevisiae to understand Bud23 function. We identified 67 bud23Δ-suppressing mutations in genes encoding the SSU Processome factors Dhr1, Imp4, Utp2 (Nop14), Bms1 and the SSU protein Rps28A. These factors form a physical interaction network that links the 3’ basal subdomain to the U3 snoRNA and many of the suppressing mutations weaken protein-protein and protein-RNA interactions. Importantly, this network links Bud23 to the RNA helicase Dhr1 and the GTPase Bms1. We previously showed that Dhr1 is required for unwinding the U3 snoRNA, and Bms1 is thought to drive conformational changes to promote rRNA cleavage. Moreover, particles isolated from cells lacking Bud23 accumulated late SSU Processome factors and inhibited cleavage at sites A1 and A2. We propose a model in which the Bud23-triggered dissociation of factors in the 3’ basal domain facilitates progression of the SSU Processome.

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Ribosome Biogenesis & Modification 289 Localization of yeast RNA exosome active subunit Rrp44 hints pre-rRNA processing as its main function in yeast

Ellen Okuda1, Fernando Gonzales-Zubiate2, Olivier Gadal3, Carla Oliveira1 1University of Sao Paulo, Sao Paulo, SP, Brazil. 2Yachay Tech University, San Miguel de Urcuquí, Ecuador. 3Université de Toulouse, Toulouse, France

Abstract

The yeast RNA exosome is essential for pre-ribosomal RNA processing, which starts co-transcriptionally in the nucleolus. Despite being considered as a nuclear and cytoplasmic protein, we show that the exosome active subunit Rrp44/Dis3 localizes mainly to the nucleus, and is concentrated in the nucleolus, where the early pre- rRNA processing reactions take place. We identified putative nuclear localization signals in Rrp44 sequence and the karyopherins that might recognize those sequences. In addition, we show that two core exosome subunits also localize to the nucleus and are concentrated in the nucleolus. Our results shed more light on the localization of the yeast exosome and have implications regarding the main function of this RNase complex.

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Ribosome Biogenesis & Modification 396 The snoRNP assembly factor Bcd1 regulates cellular translation by controlling the ribosomal RNA 2’-O-methylation pattern

Sohail Khoshnevis1, R. Elizabeth Dreggors1, Virginie Marchand2, Yuri Motorin3, Homa Ghalei1 1Emory University, Atlanta, GA, USA. 2CNRS-Lorraine University-INSERM, Vandoeuvre-les-Nancy, France. 3CNRS-Lorraine University, Vandoeuvre-les-Nancy, France

Abstract

Precise modification and processing of ribosomal (r)RNAs is required for the production of ribosomes that are capable of efficient and accurate translation of cellular proteins. Small nucleolar ribonucleoproteins (snoRNPs) guide the modification and processing of rRNAs and are thus critical for all eukaryotic cells. Bcd1, an essential nuclear zinc finger protein, is an early regulator for biogenesis of box C/D snoRNPs and controls the steady- state levels of box C/D snoRNAs through a largely unknown mechanism. We previously showed that a conserved motif in Bcd1 is required for the interactions of the protein with box C/D snoRNAs and the core snoRNP protein, Snu13 and that both of these interactions are critical for the assembly of snoRNPs and ribosome biogenesis. Here we define the binding site of Bcd1 on precursor box C/D snoRNAs. Further, we show that failure to recruit Bcd1 results in the production of hypo 2’-O-methylated ribosomes with distinct translational properties. Taken together, our results reveal a key step in the maturation pathway of box C/D snoRNAs, which is critical for maintaining the fidelity of the pool of cellular ribosomes.

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Ribosome Biogenesis & Modification 408 Modulation of rRNA processing pathways conserves energetic reserves and maintains nucleolar structure during stress

Witold Szaflarski1, Mateusz Sowiński1, Marta Leśniczak1, Sandeep Ojha2, Sulochan Malla3, Anaïs Aulas4, Pavel Ivanov5,6, Shawn Lyons7 1Department of Histology and Embryology, Poznan University of Medical Sciences, Poznan, Poland. 2Department of Biochemistry, Boston University School of Medicine of Biochemistry, Boston, MA, USA. 3Department of Biochemistry, Boston University School of MedicineDepartment of Biochemistry, Boston University School of Medicine, Boston, MA, USA. 4Centre de Recherche en Cancérologie, Marseille, France. 5Department of Medicine, Harvard Medical School, Boston, MA, USA. 6Division of Rheumatology and Clinical Immunology, Brigham and Women's Hospital, Boston, MA, USA. 7Department of Biochemistry, Boston University School of Medicine, Boston, MA, USA

Abstract

Production of ribosomes is a major energetic task of a growing cell owing to the intricacy of these massive marcomolecular machines. Each human ribosome contains 80 ribosomal proteins and 4 non-coding RNAs. The majority of the assembly steps of pre-ribosomes take place in a non-membranous liquid-liquid phase separated (LLPS) organelle called the nucleolus. A major driver of nucleolar structure is pre-rRNA as a result of an abundance of transcription. However, in response to stress, pro-growth functions are rapidly attenuated. Global translation and, in particular, ribosomal protein translation are rapidly inhibited by the integrated stress response (ISR). However, how these processes are coordinately regulated with rRNA transcription are not known. Here, we show rather than inhibiting rRNA transcription, which would disrupt nucleolar structure, cells inhibit the first step of rRNA processing, thereby preserving nucleolar structure. We show that this is not a result of translation inhibition or dependent upon the ISR, but an independent stress response pathway. Both inter- and intranucleolar dynamics are diminished; however, this in not co-incident with the formation of functional amyloids as has been shown for other stresses (e.g. acidosis). Failure to coordinately regulate ribosomal protein translation and rRNA production results in nucleolar fragmentation. Our study unveils a novel stress response pathway that conserves energy, preserves LLPS of the nucleolus and prevents further stress by regulation of rRNA processing.

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Ribosome Biogenesis & Modification 457 Analyses of the important functional domains of translation initiation factor eIF4G in ribosome biogenesis in Saccharomyces cerevisiae

Yu-Cheng Sung, Yun-Ting Tseng, Kai-Yin Lo Department of Agricultural Chemistry, National Taiwan University, Taipei, Taiwan

Abstract eIF4G is an essential translation initiation factor and is important in controlling cell growth, development, and lifespan. Apart from its role in translation, eIF4G has also been shown to involve in degradation and splicing of mRNAs in the nucleus. In this study, eIF4G was shown to have additional functional role in 60S ribosome biogenesis. Under depletion of eIF4G, defects in rRNA processing, export and assembly of nascent ribosomal subunits could be detected. Several important domains have been identified in eIF4G, like PAB1, 4E, RS (Arginine-Serine rich), to interacts with Pab1 (poly-A binding protein), eIF4E, and RNA, respectively. The individual domain was mutated in eIF4G and examined which domain was important in 60S biogenesis and interaction with the transacting factors, including Ssf1/2, Brx1, Rpf1, Rpf2, and Imp4. These factors have a conserved Brix domain and are all involved in ribosome biogenesis. The potential interaction domain of Brix family proteins with eIF4G was also analyzed. In eIF4G mutant, the loading of Brix proteins was incorrect and further altered the compositions of the nascent ribosomal subunits. The results in this study showed how eIF4G associated with 60S subunits potentially and its potential functional role of in ribosome biogenesis.

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Ribosome Biogenesis & Modification 499 A point mutation in motif V of DEAD-box RNA helicase Dbp4 confers both cold- and temperature-sensitivity

Sophie Sleiman1, Kevin J.Mc Naught2, Andrew D. Klocko2, Eric U. Selker2, François Dragon1 1Département des sciences biologiques, Université du Québec à Montréal and Centre d'excellence en recherche sur les maladies orphelines, Montréal, Québec, Canada. 2Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403, USA

Abstract

Neurospora crassa mutant crib-1 (cytoplasmic ribosome biosynthesis) was identified nearly 50 years ago as a cold-sensitive mutant defective in production of the small subunit of cytoplasmic ribosomes. Soon thereafter, the mutant was shown to be defective in processing of the rRNA precursor, especially when grown in the cold, resulting in a severe reduction of 18S rRNA. More recently, whole-genome sequencing and complementation studies revealed that crib-1 carries a single point mutation affecting motif V of the DEAD-box RNA helicase DBP-4. The yeast ortholog Dbp4 is essential for SSU processome formation and 18S rRNA processing, and associates with U3 and U14 snoRNAs. Northern hybridization analyses showed that pre-rRNA processing in the crib-1 mutant was delayed and led to the accumulation of an aberrant 23S precursor. Introducing the same point mutation in yeast Dbp4 conferred slow growth at the permissive temperature (30°C). In contrast to N. crassa, the yeast mutant displayed both cold- and temperature-sensitivity, with a more severe phenotype in the cold. Sucrose density gradients indicated that yeast ribosome biogenesis is altered at both restrictive temperatures, and northern hybridization analyses confirmed the presence of the aberrant 23S pre-rRNA. Sucrose density gradients with extracts from the N. crassa crib-1 mutant revealed that sedimentation of U14 snoRNA was altered as with depletion of yeast Dbp4

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Ribosome Biogenesis & Modification 554 Biochemical characterization of circularly permuted GTPase Lsg1 and its role in eukaryotic ribosome biogenesis

Sharmishtha Musalgaonkar, Arlen Johnson University of Texas at Austin, Austin, TX, USA

Abstract

In eukaryotic cells, ribosome biosynthesis and maturation is a complex process that involves about 200 factors and ribosome export across the nuclear membrane. Eukaryotic ribosome biogenesis starts in the nucleolus, progresses in the nucleoplasm and final steps of maturation take place in the cytoplasm. Here we have biochemically characterized a circularly permuted GTPase called Lsg1, that is highly conserved amongst all eukaryotes and required for the release of nuclear export adaptor protein Nmd3 from a nascent 60S subunit in the cytoplasm. We have previously shown that the activation of Lsg1 GTPase center requires its interaction with both 60S and Nmd3. To understand finer details of last steps of 60S maturation, here we have used mutations in conserved motifs of Lsg1 that disrupt important events like GTP binding/hydrolysis or interaction with Nmd3 and 60S subunits. We demonstrate that mutations in GTPase domain of Lsg1, disrupt its interaction with helix 69 of LSU also affect GTP binding and hydrolysis.

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Ribosome Biogenesis & Modification 631 Ribosome engineering reveals the importance of 5S rRNA autonomy for ribosome assembly

Shijie Huang1, Nikolay Aleksashin1, Anna Loveland2, Dorota Klepacki1, Kaspar Reier3, Amira Kefi1, Teresa Szal1, Jaanus Remme3, Luc Jaeger4, Nora Vázquez-Laslop1, Andrei Korostelev2, Alexander Mankin1 1University of Illinois at Chicago, Chicago, IL, USA. 2University of Massachusetts Medical School, Worcester, MA, USA. 3University of Tartu, Riia, Tartu, Estonia. 4University of California, Santa Barbara, CA, USA

Abstract

The 120-nt long 5S rRNA, is an indispensable component of cytoplasmic ribosomes in all living organisms. The functions of 5S rRNA and the reasons for its evolutionary preservation as an independent molecule remain unclear. Here we used ribosome engineering to investigate whether maintaining 5S rRNA as an independent molecule is critical for ribosome function and cell survival. By fusing circularly permutated 5S rRNA (cp5S) with 23S rRNA and deleting all wild type 5S rRNA genes, we generated a bacterial strain completely devoid of free 5S rRNA. Viability of the engineered cells demonstrates that autonomous 5S rRNA is not required for cell growth and is unlikely to have essential functions outside the ribosome. The fully-assembled ribosomes carrying 23S-cp5S hybrid rRNA and lacking free 5S rRNA are highly active in translation. However, the engineered cells accumulate aberrant 50S subunits that are unable to form stable 70S ribosomes. Cryo-EM analysis revealed a dramatically malformed peptidyl transferase center in the misassembled 50S subunits. The results of our experiments argue that the key evolutionary force preserving the autonomous nature of the smallest rRNA is its role in ribosome biogenesis.

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Ribosome Biogenesis & Modification 633 Unexpectedly complex mitoribosomes in Andalucia godoyi, a protist with the most bacteria-like mitochondrial genome

Matus Valach1, José Angel Gonzalez Alcazar1, Matt Sarrasin1, B. Franz Lang1, Michael W. Gray2, Gertraud Burger1 1Université de Montréal, Montreal, QC, Canada. 2Dalhousie University, Halifax, NS, Canada

Abstract

A ubiquitous feature of the mitochondrion is its translation machinery, essential for synthesis of mitochondrion-encoded proteins. Compared to ribosomes of bacteria from which mitochondria evolved, mitoribosomes display a huge diversity of deviant structural and compositional features. To complement studies that have focused mainly on highly derived organisms such as animals, fungi, and kinetoplastids, we investigated mitoribosomal proteins in jakobids, a lineage of unicellular protists from the Discoba superphylum and having the most bacteria-like mitochondrial genomes. Our in silico analyses of the genomes and transcriptomes from four jakobid genera uncovered homologs of nearly all mitoribosomal proteins present in at least two other major eukaryotic lineages. To confirm that the identified candidates are indeed structural components of the jakobid mitoribosome, we isolated the small mitoribosomal subunit of Andalucia godoyi and examined its makeup by mass spectrometry. In this species, we infer 30 and 44 mitoribosomal proteins of the small and large subunit, respectively, which is a substantial increase over the number of ribosomal proteins in bacteria (21 and 33, respectively) and on par with the observed mitoribosome complexity in fast-evolving lineages such as yeasts and animals. It has been proposed that mitochondrial ribosomal RNAs (rRNAs) evolve by rapid expansions and truncations of their structural elements, and that the resulting anatomical instabilities were compensated by the recruitment of pre-existing proteins to the mitoribosome. However, this view is at odds with what we found in Andalucia, where ancestral, bacteria-like mitochondrial rRNAs coexist with the extra set of ribosomal proteins typical for mitochondria. Therefore, we posit that numerous accessory proteins had already been accreted to the mitoribosome in the last common ancestor of eukaryotes and that this pre-adaptation subsequently allowed structural fluctuation in the rRNAs and further acquisition of supernumerary lineage- specific components.

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Ribosome Biogenesis & Modification 676 Investigating the N- and C-terminal Extensions of the Yeast DEAD-box Protein Dbp6

Payton DeMarzo, Mlana Lore, Crystal Young-Erdos Eckerd College, St. Petersburg, FL, USA

Abstract

DEAD-box proteins are RNA-dependent ATPases that are involved in all aspects of RNA metabolism. Also known as DEAD-box helicases, these enzymes bind RNA and ATP and unwind RNA duplexes via a highly conserved helicase core. Despite being highly conserved in both sequence and structure, each DEAD-box protein has a unique intracellular function and is therefore essential. The N- and C-terminal extensions that flank this core differ in both length and composition between DEAD-box proteins and have been shown to contribute to RNA specificity and binding, thereby conferring the diverse cellular roles of DEAD-box proteins. To gain insight into this enzymatic specificity, we are investigating the significance of the 192 amino acid N-terminal and 63 amino acid C-terminal extensions of Dbp6, a DEAD-box protein required for yeast ribosome assembly. To find the minimum functional length of the flanking regions, we have systematically designed truncation constructs and observed their effects on the growth of yeast cells in which endogenous Dbp6 has been depleted. Our results indicate that the deletion of up to 168 amino acids at the N-terminus (ΔN168) or 27 amino acids at the C- terminus (ΔC27) give either wild-type level or slow growth. Removal of five more residues at the N-terminus or two more at the C-terminus results in a lethal phenotype. Interestingly, even though Dbp6 ΔN143 and Dbp6 ΔC21 each result in near wild-type level growth, the double truncation construct Dbp6 ΔN143ΔC21 results in no growth. Western blot analysis confirmed that truncated proteins were indeed present, supporting the hypothesis that the additional amino acids removed may be required for a unique biochemical function of Dbp6. We are currently cloning the truncations of Dbp6 into bacterial expression vectors in order to over- express and purify the truncated proteins. Future experiments include measuring the ATP and RNA binding and RNA duplex unwinding activities of truncated Dbp6 in vitro. Since the human ortholog of Dbp6 has been implicated in lung cancer progression, we hope that these insights into the molecular workings of Dbp6 can contribute to our broader understanding of DEAD-box proteins and their uses as potential cancer therapeutic targets.

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Ribosome Biogenesis & Modification 694 The 47S pre-rRNA processing cascade of articular chondrocytes is impaired in an in vitro model of osteoarthritis

A. Chabronova, G. G.H. van den Akker, E.G.J. Ripmeester, B.A.C. Housmans, A. Cremers, D. Surtel, M.M.J. Caron, L.W. van Rhijn, T.J.M. Welting Laboratory for Experimental Orthopedics, Department of Orthopedic Surgery, MUMC+, Maastricht, Netherlands

Abstract

INTRODUCTION: Osteoarthritis (OA) is the most common degenerative joint disorder, characterized by progressive loss and destruction of articular cartilage. Chondrocytes are the only cells present in cartilage with a primary function of producing and maintaining the cartilage extracellular matrix (ECM). This requires continuous translation of ECM-related mRNAs by ribosomes. In the human system, three ribosomal RNAs, 18S, 5.8S, and 28S, arise from a single 47S pre-rRNA transcript. A limited number of small nucleolar RNAs (snoRNAs), such as RMRP are known to guide endoribonucleolytic cleavages of the 47S pre-rRNA transcript. Here, we hypothesized that 47S pre-rRNA processing is impaired in osteoarthritic articular chondrocytes and contributes to OA progression.

METHODS & RESULTS: To test our hypothesis, we set-up an OA-mimicking in vitro model using human primary articular chondrocytes exposed to osteoarthritic synovial fluid (OA SF) for 1 to 14 days. The dynamics of the pre-rRNA processing cascade was evaluated using custom-made qPCR primers covering selected cleavage sites (01, 2 and 4a) within the 47S transcript. Upon OA SF treatment, we observed accelerated cleavage at site 01 within 5’ ETS (external transcribed spacer) region, and site 2 within ITS1 (internal transcribed spacer 1). Cleavage at site 2 separates pre-18S and pre-5.8S-28S intermediates and it is guided by snoRNA RMRP, expression of which was increased upon OA-SF treatment. On the contrary, cleavage at site 4a within ITS2 region was significantly hindered, thus leading to the accumulation of unprocessed pre-5.8S and pre-28S intermediates. DISCUSSION & CONCLUSIONS: Using our in vitro OA model we found evidence for altered pre-rRNA processing dynamics, suggesting that 47S processing is vulnerable to changes in the chondrocyte’s extracellular environment. The observed accelerated processing towards mature 18S rRNA, and hindered processing towards mature 5.8S and 28S rRNAs can potentially affect ribosome biogenesis and thus compromise the chondrocyte’s translational capacity. Future experiments are focusing on the validation of these findings by northern blotting and detailed unbiased mapping of the 47S processing cascade using RNA sequencing technology.

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Ribosome Biogenesis & Modification 746 Impaired chondrocyte U3 snoRNA expression in osteoarthritis impacts the chondrocyte protein translation apparatus

Ellen G.J. Ripmeester1, Marjolein M.J. Caron1, Guus G.H. Akker, van den1, Don A.M. Surtel1, Andy Cremers1, Panagiotis Balaskas2, Philip Dyer2, Bas A.C. Housmans1, Alzbeta Chabronova1, Aibek Smagul2, Y. Fang2, Lodewijk W. Rhijn, van1, Mandy J. Peffers2, Tim J.M. Welting1 1Maastricht University, Maastricht, Netherlands. 2University of Liverpool, Liverpool, United Kingdom

Abstract

INTRODUCTION: Osteoarthritis (OA) is the most prevalent degenerative joint disease, which presents with a disturbed chondrocyte homeostasis and is accompanied with a catabolic signature. Although pathways controlling ribosome activity have been described to regulate chondrocyte homeostasis, ribosome biogenesis in osteoarthritis is unexplored. We hypothesized that U3 small nucleolar RNA (snoRNA), a key factor in ribosomal RNA (rRNA) maturation, is critical for chondrocyte protein translation capacity in osteoarthritis. METHODS: U3 snoRNA expression analysis was performed on osteoarthritic and non-osteoarthritic cartilage and in an experimental murine OA model. Expression of rRNAs and chondrocyte phenotypic markers were measured using RT-qPCR. U3 snoRNA expression was reduced by transfection of an anti-sense oligonucleotide, and induced by introduction of a U3 snoRNA mini-gene. Overall protein translational capacity was determined using puromycylation assays. Differential proteomes after U3 snoRNA knockdown in SW1353 cells were established by protein mass spectrometry. U3 snoRNA promoter activity was determined by bioluminescent techniques. RESULTS: Microarray and RT-qPCR analyses demonstrated reduced expression of U3 snoRNA in OA cartilage, compared to non-OA, with similar results obtained from murine OA joints. OA synovial fluid reduced U3 expression via inhibition of U3 gene promoter activity. Altering U3 snoRNA expression changed the expression of OA-relevant chondrocyte phenotypical genes. OA chondrocytes showed reduced protein translation compared to non-OA chondrocytes. This was recapitulated following reduction of U3 snoRNA expression, resulting in reduced rRNA levels and translational capacity. Reciprocally, induced expression of U3 snoRNA was accompanied by increasing rRNA levels and protein translation. Whole proteome analysis revealed an impact of reduced U3 snoRNA expression on protein translational processes. Finally, cellular U3 snoRNA levels were increased by BMP-7 treatment via upregulation of U3 promoter activity, leading to higher rRNA expression levels and protein translation. DISCUSSION & CONCLUSION: We demonstrate that U3 snoRNA expression, a critical non-coding RNA needed for ribosome biogenesis, is reduced in OA chondrocytes. This has consequences for chondrocyte rRNA levels and protein translational capacity. U3 snoRNA expression might be a therapeutic target for OA treatment and our data indeed show that BMP-7, a chondrocyte anabolic morphogen, is capable of inducing chondrocyte U3 levels, and improving chondrocyte protein translation capacity.

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Ribosome Biogenesis & Modification 835 Exit the ribosome, enter the nucleus! Nuclear import of ribosomal proteins

Jutta Hafner, Maximilian Mack, Ingrid Roessler, Brigitte Pertschy University of Graz, Graz, Austria

Abstract

The ribosome, an outstanding molecular machine, synthesizes all cellular proteins and is assembled as well as matured in a complex process involving rRNA, r-proteins and more than 200 ribosome biogenesis factors. The spatial separation of r-protein and pre-rRNA synthesis necessitates the transport of r-proteins from the cytoplasm to their rRNA assembly site into the nucleus. For instance, in the eukaryotic model organism Saccharomyces cerevisiae, every minute, over 150,000 newly synthesized r-proteins need to be imported into the nucleus. Therefore, the efficient delivery of r-proteins is an essential task in all eukaryotic cells and an immense burden on the nuclear import machinery. Import cargoes are usually recognized by nuclear import receptors (importins) via their diverse, often basic, nuclear import signals (NLS). Besides, importin binding is not only important for nuclear import itself, but also for the protection of basic cargoes from aggregation. So far, 10 different human and yeast importins, with various, but frequently overlapping cargo specificities, have been described. To gain an overview of the import pathway of ribosomal proteins in eukaryotes, we systematically analyzed published data of physical interaction-studies in human and yeast. Additionally, we performed in- silico predictions of potential NLS sequences in all r-proteins. Results of our analyses will be presented on this poster.

Presenting author email [email protected]

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Ribosome Biogenesis & Modification 886 Nucleolar stress induction by platinum(II) compounds

Emily Sutton, Christine McDevitt, Jack Prochnau, Matthew Yglesias, Austin Mroz, Min Chieh Yang, Rachael Cunninhgam, Christopher Hendon, Victoria DeRose University of Oregon, Eugene, OR, USA

Abstract

Although platinum(II) can crosslink both DNA and RNA, most research into the mode of action of platinum(II) chemotherapeutic agents has focused on the DNA damage response. Recently, studies have revealed surprising links between a subset of these drugs and the nucleolus, which is the site of ribosomal RNA (rRNA) synthesis and ribosome biogenesis. Unlike cisplatin and carboplatin, oxaliplatin is linked to nucleolar stress- induced cell death pathways at low doses in mammalian tissue culture. Of these three FDA-approved compounds, only oxaliplatin robustly induces redistribution of nucleophosmin (NPM1) at low doses, which is a hallmark of the nucleolar stress response. Given the diverse potential ligands of Pt(II), we sought to identify the structural features that may be responsible for inducing this stress response. Using a compound library, we tested various structural factors and have found that while changes in ligand ring size and aromaticity can be tolerated, ring orientation is a critical determinant of nucleolar stress induction. This study has provided significant insight into the unique specificity of ligand requirements necessary for stress induction, however the biological mechanism by which oxaliplatin-like Pt(II) compounds induce nucleolar stress and cause NPM1 redistribution is still not well understood. To that end, we have begun to assess biological processes that may be specifically disrupted by oxaliplatin leading up to NPM1 redistribution, including rRNA synthesis, rRNA processing and nucleolar DNA damage. These current studies are engaged in identifying the targets and causes of this uniquely observed nucleolar stress response.

Presenting author email [email protected]

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Ribosome Biogenesis & Modification 889 Structural basis of the Erm-mediated resistance to macrolide antibiotics.

Maxim Svetlov1, Egor Syroegin1, Elena Aleksandrova1, Steven Gregory2, Alexander Mankin1, Yury Polikanov1 1University of Illinois at Chicago, Chicago, Illinois, USA. 2University of Rhode Island, Kingston, Rhode Island, USA

Abstract

Macrolides represent one of the most successful classes of clinically important antibiotics. They inhibit bacterial growth by binding to the 70S ribosome and interfering with protein biosynthesis. The main mechanism of resistance to macrolides widely employed by pathogens is dimethylation of the 23S rRNA nucleotide A2058 in the drug binding site which is catalyzed by the Erm-type rRNA methyltransferases. This modification also confers resistance to other classes of ribosome targeting antibiotics, including lincosamides and streptogramins B. Here, we present the first crystal structure of the Erm-dimethylated 70S ribosome at 2.4Å resolution and describe changes in the drug-binding site on the ribosome leading to macrolide resistance. Our structural analysis uncovers the molecular basis for the Erm-mediated drug resistance and should facilitate the development of new macrolide antibiotics active against the Erm-expressing pathogens.

Presenting author email [email protected]

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Ribosome Biogenesis & Modification 908 Time-resolved DMS footprinting in E. coli reveals delayed folding of peripheral rRNA helices in newly made ribosomes

Yumeng Hao, Ryan Hulscher, Boris Zinshteyn, Sarah Woodson Johns Hopkins University, Baltimore, MD, USA

Abstract

During active growth, E. coli cells synthesize more than 3,000 ribosomes per minute, underscoring the need for accurate and efficient ribosome biogenesis. Previous studies of bacterial ribosome biogenesis stalled assembly by depriving the cell of essential ribosomal proteins and assembly factors. This approach can re- route the assembly pathways, however, and may not reveal how native rRNAs fold in the cell over time. To understand how ribosomes assemble during normal growth, we metabolically labeled nascent transcripts with 4-thiouridine (4SU) and used dimethyl sulfate (DMS) footprinting to monitor pre-rRNA structural changes in real time in E. coli cells (4U-DMS-Seq). The slow folding patterns in the late ribosome folding intermediates correlated with changes in tertiary packing, refolding of protein binding sites, structural rearrangements and hypomethylation at the decoding sites. However, the active sites are protected, likely due to the interactions with assembly factors in cell. Our method reveals structural hallmarks of late assembly intermediates, and how rRNAs interact with ribosomal proteins and assembly factors. 4U-DMS-Seq can be generalized to study ribosome biogenesis in other organisms and examine the assembly of other large RNA-protein complexes.

Presenting author email [email protected]

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Ribosome Biogenesis & Modification 943 Casein Kinase 2 Catalytic Subunits a1/a2 are bona fide Members of the SSU Processome’s Utp-C Subcomplex and may Regulate Ribosome Biosynthesis

James Cluf1,2, Elise Poole1,2, J. Michael Charette1,2 1Brandon University, Brandon, Manitoba, Canada. 2Children's Hospital Research Institute of Manitoba, Winnipeg, Manitoba, Canada

Abstract

The Small Subunit Processome (SSU Processome) is a large ribonucleoprotein complex responsible for the assembly of the SSU of the ribosome. It consists of five sub-complexes, one of which (the UTP-C subunit) is believed to contain the protein kinase/casein kinase CK2 complex. It consists of the catalytic CKα/a1 and CKα/a2 and the regulatory CKβ/b1 and CKβ/b2 subunits. CK2 is a ubiquitous and constitutively active serine/threonine kinase implicated in many cellular processes. In addition to its putative membership in the SSU processome, CK2 is also known to regulate the activity of RNA Polymerase I and III and to stoichiometrically coordinate ribosomal protein production with ribosome assembly. Our objective is to validate the membership of CK2 in the SSU processome and to determine the contribution of CK2 proteins to the regulation of ribosome assembly. In a yeast model system, we are determining the role of CK2 in the regulation of ribosome assembly by genetically depleting cells of individual and pairs of CK2 subunits. As growth is directly correlated to ribosome assembly, growth curves were used as a surrogate for ribosome assembly. The growth of wildtype cells was compared to that of cells genetically depleted of individual and pairs of CK2 components. Membership of CK2 in the SSU processome was confirmed by co-IP of each of the individual CK2 proteins with the known SSU processome components Kre33 and U3 snoRNA. Pre-rRNA processing defects will be confirmed by northern analysis. Growth curves reveal that genetic depletion of individual catalytic subunits CKα/a1 and CKα/a2 results in a reduction in growth while simultaneous depletion of both catalytic subunits is lethal, as seen by an arrest in growth. Interestingly, catalytically dead CKα/a1 and CKα/a2 mutants are dominant negative. Co-IPs of catalytic and regulatory subunits with Kre33 have confirmed membership of CK2 within SSU Processome. Thus, we validated the membership of the CK2 in the SSU processome and show that the proteins regulate cell growth, likely through a dysregulation of ribosome assembly.

Presenting author email [email protected]

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Ribosome Biogenesis & Modification 955 METTL17 is is critical for mouse embryonic development

Kyrylo Krasnykov, Radha Raman Pandey, Elena Delfino, Kuan-Ming Chen, David Homolka, Ramesh Pillai University of Geneva, Geneva, Switzerland

Abstract

Embryonic development of mammals represents a complex and dynamic process of coordinated cell division and differentiation events. Myriads of known enzymes and signaling molecules identified as markers and regulators of embryonic transitions are biologically/biochemically active. METTL17 is a mitochondrial protein apparently participating in the assembly of the small subunit of the mitochondrial ribosome. As a pivotal part of the small subunit of the mitochondrial ribosome, METTL17 should be required in embryonic stages particularly dependent of the properly functioning mitochondria network. Indeed, Mettl17-/- leads to a developmental arrest of mouse embryos at the E8.5 stage corresponding to the early organogenesis in which mitochondria network remodeling comes into play to make further developmental transitions. Bulk RNAseq identified several Mettl17-/- dysregulated genes, however was not efficient for their functional annotation. To specifically study cell types most affected by Mettl17-/-, we used scRNA-seq technology. Our study identified that all the supportive cell progenitors involved in the anterior-posterior patterning of mouse embryos were strongly underrepresented in E7.5 mouse embryos upon Mettl17-/-. Additionally, the transcriptional changes of the mouse embryonic stem cells caused by Mettl17-/- disrupted supportive developmental trajectory, therefore explaining a complete embryonic arrest at the E8.5 stage characterized as a phase of somitogenesis and embryonic axes patterning.

Presenting author email [email protected]

Topic category

Ribosome Biogenesis & Modification