Razvoj paketa coRdon u programskom jeziku R za analizu korištenja sinonimnih kodona i predviđanje ekspresije gena

Not all synonymous codons are used with equal frequency in prokaryotic genomes - this selective preference is termed codon usage (CU) bias and is an independent determinant of gene expression regulation at the level of translation. The synonymous codons corresponding to the most abundant cognate tRNA are considered optimal for translation. Codon usage bias can therefore be used to predict the relative expression levels of genes, by comparing CU bias of a specific gene to the CU bias of a set of genes known to be highly expressed. This approach can be efficiently used to predict highly expressed genes within a single genome but has also been demonstrated relevant at the level of entire microbial communities – metagenomes. This is possible because CU bias is shared among the microbial species in the same environment. By analysing CU bias of a metagenome, one can identify genes with high predicted expression across the given ecological niche, and identify enriched functions, i.e. the functional fingerprint of the analysed microbial community. Software tools to efficiently analyse and visualize metagenomics data in the context of translational optimisation and CU bias are scarce and this limits the ability to generate useful knowledge. As the part of this thesis, I have developed a Bioconductor-based R package for comprehensive analysis of CU and CU-based gene expressivity. The package also includes several methods for visualization of CU, and the CUbased functional analysis of annotated DNA sequences. The package is fast and flexible enough to handle even the largest metagenomics datasets.U genomima različitih prokariota svi sinonimni kodoni ne koriste se jednakom učestalošću - njihovo selektivno korištenje zaseban je mehanizam regulacije ekspresije gena na razini translacije. Sinonimni kodoni koje prepoznaju najbrojnije tRNA molekule u stanici smatraju se optimiziranima za translaciju. Stoga se selektivno korištenje sinonimnih kodona može koristiti za predviđanje relativne ekspresivnosti gena, uspoređujući obrazac korištenja kodona u određenom genu s kodonima koji se učestalo pojavljuju u skupu visoko eksprimiranih gena. Na ovaj način moguće je identificirati potencijalno jako eksprimirane gene na razini genoma, ali još važnije, i na razini čitavih metagenoma, jer je pokazano da je uzorak selektivnog korištenja kodona zajednički organizmima unutar iste mikrobne zajednice, bez obzira na njihovu filogenetsku udaljenost. Stoga je analizirajući korištenje kodona na razini metagenoma moguće predvidjeti koji geni su relativno visoko eksprimirani u određenoj mikrobnoj zajednici, kao i koje funkcije su zastupljene među tim genima - tzv. funkcijski otisak mikrobne zajednice. U sklopu ovog rada razvila sam paket u programskom jeziku R, baziran na sintaksi i smjernicama projekta Bioconductor, za kompletnu analizu korištenja sinonimnih kodona i ekspresivnosti gena. Uz to što omogućava računanje više različitih statistika koje kvantificiraju korištenje sinonimnih kodona, paket uključuje i nekoliko metoda za vizualizaciju korištenja sinonimnih kodona, omogućava funkcionalnu analizu anotiranih slijedova DNA te je dovoljno brz i fleksibilan za analizu velikih količina metagenomskih podataka

The structure and dynamics of the spliceosome

Eukariotski geni između kodirajućih sljedova, eksona, sadrže dugačke nekodirajuće introne. Geni se u cijelosti prepisuju u primarne transkripte mRNA, a introni se naknadno izrezuju djelovanjem multi-megadaltonskog ribonukleoproteinskog kompleksa za prekrajanje (eng. spliceosome). Ovaj kompleks sastavlja se postepeno u jezgri eukariotske stanice, na odgovarajućem pre-mRNA supstratu, a potom se aktivira kako bi mogao katalizirati reakcije izrezivanja introna i povezivanja eksona u zrelu molekulu mRNA. Najznačajnije karakteristike ovog kompleksa njegova su veličina (preko 3 MDa), brojnost komponenti (preko 200 proteina i 5 molekula RNA) te ribozimska i metaloenzimska priroda. Evolucijski očuvani elementi sekundarne strukture molekula RNA u aktivnom mjestu kompleksa imaju ključne katalitičke uloge, poput koordinacije metalnih iona, dok su proteini u ciklusu prekrajanja prvenstveno odgovorni za ispravno pozicioniranje i stabilizaciju ovih struktura. Još jedna značajna karakteristika je i izuzetna plastičnost kompleksa koja je dijelom posljedica velikog broja strukturno neuređenih proteina, a koja omogućuje uspješno prepoznavanje i izrezivanje introna iz najrazličitijih transkripata pre-mRNA. Izrezivanje introna mora biti izuzetno precizno ali i brzo, kako bi se u citoplazmu iz jezgre iznosile samo ispravno dorađene mRNA molekule. Stoga su stavljanje kompleksa, aktivacija i kataliza regulirani posttranslacijkskim modifikacijama i djelovanjem enzima koji uz utrošak ATP-a osiguravaju točnost ovih reakcija, a sve ključne reakcije ciklusa su reverzibilne. Prekrajanje je usto djelomično koordinirano s transkripcijom i drugim reakcijama metabolizma RNA. Nakon dovršetka reakcija prekrajanja, kompleks se rastavlja. Produkti reakcija, zrela mRNA i intron u obliku omče, jedan za drugim disociraju, kao u konačnici i komponente kompleksa koje se onda mogu uključiti u novi ciklus na drugom pre-mRNA supstratu.Nuclear pre-mRNA splicing is catalysed by the spliceosome, multi-megadalton ribonucleoprotein complex which is assembled in stepwise fashion and activated while bound to its substrate. Defining features of this complex are its size (over 3 MDa), myriad of components (more than 200 proteins and 5 RNAs), and the fact that it is a ribozyme, as well as a metaloenzyme. Evolutionary conserved elements in the secondary structure of RNAs carry out important catalytic functions, such as coordination of catalytic metal ions in the active site, while protein components are found to assist in formation of these secondary structures, as well as in stabilising their interactions. Another important characteristic of the spliceosome is its remarkable plasticity, which is in part due to a large number of intrinsically disordered proteins. This allows for introns from a wide range of vastly different pre-mRNA substrates to be successfully spliced. Splicing must be fast and precise at once, so as to assure that only the correctly spliced and modified RNAs are exported from nucleus to cytoplasm. To achieve this, spliceosome assembly, activation, and catalysis are highly regulated by different posttranslational modifications and through the action of the several enzymes that utilize ATP in order to maintain splicing reaction fidelity. In addition, all of the main reactions in the spliceosome cycle are reversible. Splicing is also, at least to some extent, coordinated with transcription and RNA metabolism which take place in the nucleus. Following the completion of splicing reactions, mature mRNA and intron in the form of lariat sequentially dissociate, as do the components of the spliceosome. These are then recycled in another spliceosome cycle

Razvoj paketa coRdon u programskom jeziku R za analizu korištenja sinonimnih kodona i predviđanje ekspresije gena

Not all synonymous codons are used with equal frequency in prokaryotic genomes - this selective preference is termed codon usage (CU) bias and is an independent determinant of gene expression regulation at the level of translation. The synonymous codons corresponding to the most abundant cognate tRNA are considered optimal for translation. Codon usage bias can therefore be used to predict the relative expression levels of genes, by comparing CU bias of a specific gene to the CU bias of a set of genes known to be highly expressed. This approach can be efficiently used to predict highly expressed genes within a single genome but has also been demonstrated relevant at the level of entire microbial communities – metagenomes. This is possible because CU bias is shared among the microbial species in the same environment. By analysing CU bias of a metagenome, one can identify genes with high predicted expression across the given ecological niche, and identify enriched functions, i.e. the functional fingerprint of the analysed microbial community. Software tools to efficiently analyse and visualize metagenomics data in the context of translational optimisation and CU bias are scarce and this limits the ability to generate useful knowledge. As the part of this thesis, I have developed a Bioconductor-based R package for comprehensive analysis of CU and CU-based gene expressivity. The package also includes several methods for visualization of CU, and the CUbased functional analysis of annotated DNA sequences. The package is fast and flexible enough to handle even the largest metagenomics datasets.U genomima različitih prokariota svi sinonimni kodoni ne koriste se jednakom učestalošću - njihovo selektivno korištenje zaseban je mehanizam regulacije ekspresije gena na razini translacije. Sinonimni kodoni koje prepoznaju najbrojnije tRNA molekule u stanici smatraju se optimiziranima za translaciju. Stoga se selektivno korištenje sinonimnih kodona može koristiti za predviđanje relativne ekspresivnosti gena, uspoređujući obrazac korištenja kodona u određenom genu s kodonima koji se učestalo pojavljuju u skupu visoko eksprimiranih gena. Na ovaj način moguće je identificirati potencijalno jako eksprimirane gene na razini genoma, ali još važnije, i na razini čitavih metagenoma, jer je pokazano da je uzorak selektivnog korištenja kodona zajednički organizmima unutar iste mikrobne zajednice, bez obzira na njihovu filogenetsku udaljenost. Stoga je analizirajući korištenje kodona na razini metagenoma moguće predvidjeti koji geni su relativno visoko eksprimirani u određenoj mikrobnoj zajednici, kao i koje funkcije su zastupljene među tim genima - tzv. funkcijski otisak mikrobne zajednice. U sklopu ovog rada razvila sam paket u programskom jeziku R, baziran na sintaksi i smjernicama projekta Bioconductor, za kompletnu analizu korištenja sinonimnih kodona i ekspresivnosti gena. Uz to što omogućava računanje više različitih statistika koje kvantificiraju korištenje sinonimnih kodona, paket uključuje i nekoliko metoda za vizualizaciju korištenja sinonimnih kodona, omogućava funkcionalnu analizu anotiranih slijedova DNA te je dovoljno brz i fleksibilan za analizu velikih količina metagenomskih podataka

Razvoj paketa coRdon u programskom jeziku R za analizu korištenja sinonimnih kodona i predviđanje ekspresije gena

Not all synonymous codons are used with equal frequency in prokaryotic genomes - this selective preference is termed codon usage (CU) bias and is an independent determinant of gene expression regulation at the level of translation. The synonymous codons corresponding to the most abundant cognate tRNA are considered optimal for translation. Codon usage bias can therefore be used to predict the relative expression levels of genes, by comparing CU bias of a specific gene to the CU bias of a set of genes known to be highly expressed. This approach can be efficiently used to predict highly expressed genes within a single genome but has also been demonstrated relevant at the level of entire microbial communities – metagenomes. This is possible because CU bias is shared among the microbial species in the same environment. By analysing CU bias of a metagenome, one can identify genes with high predicted expression across the given ecological niche, and identify enriched functions, i.e. the functional fingerprint of the analysed microbial community. Software tools to efficiently analyse and visualize metagenomics data in the context of translational optimisation and CU bias are scarce and this limits the ability to generate useful knowledge. As the part of this thesis, I have developed a Bioconductor-based R package for comprehensive analysis of CU and CU-based gene expressivity. The package also includes several methods for visualization of CU, and the CUbased functional analysis of annotated DNA sequences. The package is fast and flexible enough to handle even the largest metagenomics datasets.U genomima različitih prokariota svi sinonimni kodoni ne koriste se jednakom učestalošću - njihovo selektivno korištenje zaseban je mehanizam regulacije ekspresije gena na razini translacije. Sinonimni kodoni koje prepoznaju najbrojnije tRNA molekule u stanici smatraju se optimiziranima za translaciju. Stoga se selektivno korištenje sinonimnih kodona može koristiti za predviđanje relativne ekspresivnosti gena, uspoređujući obrazac korištenja kodona u određenom genu s kodonima koji se učestalo pojavljuju u skupu visoko eksprimiranih gena. Na ovaj način moguće je identificirati potencijalno jako eksprimirane gene na razini genoma, ali još važnije, i na razini čitavih metagenoma, jer je pokazano da je uzorak selektivnog korištenja kodona zajednički organizmima unutar iste mikrobne zajednice, bez obzira na njihovu filogenetsku udaljenost. Stoga je analizirajući korištenje kodona na razini metagenoma moguće predvidjeti koji geni su relativno visoko eksprimirani u određenoj mikrobnoj zajednici, kao i koje funkcije su zastupljene među tim genima - tzv. funkcijski otisak mikrobne zajednice. U sklopu ovog rada razvila sam paket u programskom jeziku R, baziran na sintaksi i smjernicama projekta Bioconductor, za kompletnu analizu korištenja sinonimnih kodona i ekspresivnosti gena. Uz to što omogućava računanje više različitih statistika koje kvantificiraju korištenje sinonimnih kodona, paket uključuje i nekoliko metoda za vizualizaciju korištenja sinonimnih kodona, omogućava funkcionalnu analizu anotiranih slijedova DNA te je dovoljno brz i fleksibilan za analizu velikih količina metagenomskih podataka

Modeling cell type evolution using single-cell and chromatin profiling approaches

Single-cell genomics methods have revolutionized the study of gene expression and regulation at the cell type level. Generating single-cell atlases for species at key phylogenetic positions of the animal tree of life (e.g.. early branching non-bilaterian animals) informs us not only about the biology of these understudied groups of organisms, but also enable comparative studies of cell type transcriptomic programs. This is the first step towards inferring the evolutionary trajectories of major animal cell types. In this context, the first part of my thesis describes the first single cell atlas of a stony coral, Stylophora pistillata, and the novel insights we gained from it. We also compared cell type transcriptomes of Stylophora to three other species in the Cnidaria phylum, and identified conserved expression programs and convergent mobilization of similar molecular pathways in potentially homologous cell types across species. However, because transcriptome similarities are confounded by pleiotropy and non-independence of gene expression programs, they do not always represent true cell type homologies. To infer more accurate cell type phylogenetic relationships we need to also compare their gene regulatory programs. The second part of my thesis focuses on detailed regulatory characterization of cell type programs in cnidarian Nematostella vectensis. We describe genome-wide regulatory logic of Nematostella, and characterize cell types by modular deployment of transcription factors (TFs) and gene regulatory networks (GRNs) in which they are involved. Together, the work presented in this thesis exemplifies the ways in which single cell approaches can be used to advance our understanding of cell type diversity, development, and evolution.Els mètodes de genòmica unicel·lular han revolucionat l'estudi de l'expressió gènica i la regulació a nivell de tipus cel·lular. La generació d'atles cel·lulars per a espècies en posicions filogenètiques clau de l'arbre de la vida animal (per exemple, animals basals, no bilaterals) ens informa no només sobre la biologia d'aquests grups d'organismes poc estudiats, sinó que també permet estudis comparatius dels tipus cel·lulars des d'una perspectiva molecular. Aquest és el primer pas per inferir les trajectòries evolutives dels principals tipus de cèl·lules animals. En aquest context, la primera part de la meva tesi descriu el primer atles cel·lular d'un corall dur, Stylophora pistillata, i el coneixement que en vam obtenir. També vam comparar els transcriptomes de tipus cel·lulars de Stylophora amb tres espècies més del fílum Cnidaria i vam identificar programes d'expressió conservats i mobilització convergent de vies moleculars similars en tipus de cèl·lules potencialment homòlogues entre espècies. Tanmateix, com que les similituds del transcriptoma es confonen per la pleiotropia i la no independència dels programes d'expressió gènica, no sempre representen necessàriament tipus cel·lulars homòlegs. Per inferir relacions filogenètiques dels tipus cel·lular, també hem de comparar els seus programes reguladors. La segona part de la meva tesi se centra en la caracterització reguladora detallada de programes de tipus cel·lular en l'anemona Nematostella vectensis (Cnidaria). Descrivim la lògica reguladora de tot el genoma de Nematostella i caracteritzem els tipus cel·lulars mitjançant el desplegament modular de factors de transcripció (TF) i xarxes reguladores de gens (GRN) en què estan implicats. En conjunt, el treball presentat en aquesta tesi exemplifica les maneres en què es poden utilitzar els enfocaments de genòmica de cèl·lula única per avançar en la nostra comprensió de la diversitat, el desenvolupament i l'evolució dels tipus cel·lulars animals.Programa de Doctorat en Biomedicin

Microbiota Alters Urinary Bladder Weight and Gene Expression.

We studied the effect of microbiota on the transcriptome and weight of the urinary bladder by comparing germ-free (GF) and specific pathogen-free (SPF) housed mice. In total, 97 genes were differently expressed (fold change > ±2; false discovery rate (FDR) p-value < 0.01) between the groups, including genes regulating circadian rhythm (Per1, Per2 and Per3), extracellular matrix (Spo1, Spon2), and neuromuscular synaptic transmission (Slc18a3, Slc5a7, Chrnb4, Chrna3, Snap25). The highest increase in expression was observed for immunoglobulin genes (Igkv1-122, Igkv4-68) of unknown function, but surprisingly the absence of microbiota did not change the expression of the genes responsible for recognizing microbes and their products. We found that urinary bladder weight was approximately 25% lighter in GF mice (p = 0.09 for males, p = 0.005 for females) and in mice treated with broad spectrum of antibiotics (p = 0.0002). In conclusion, our data indicate that microbiota is an important determinant of urinary bladder physiology controlling its gene expression and size

ACME dissociation: a versatile cell fixation-dissociation method for single-cell transcriptomics

Single-cell sequencing technologies are revolutionizing biology, but they are limited by the need to dissociate live samples. Here, we present ACME (ACetic-MEthanol), a dissociation approach for single-cell transcriptomics that simultaneously fixes cells. ACME-dissociated cells have high RNA integrity, can be cryopreserved multiple times, and are sortable and permeable. As a proof of principle, we provide single-cell transcriptomic data of different species, using both droplet-based and combinatorial barcoding single-cell methods. ACME uses affordable reagents, can be done in most laboratories and even in the field, and thus will accelerate our knowledge of cell types across the tree of life.This work was supported by an MRC grant (MR/S007849/1) and a Royal Society Grant (RGS\R1\191278) to JS. HG-C was supported by a Nigel Groome studentship from Oxford Brookes University. PA-C was supported by an EMBO Long Term Fellowship (ALTF-217-2018). JN was supported by funding from a BBSRC grant (BB/M011224/1) and the Osk. Huttunen Foundation (Doctoral grant

Stepwise emergence of the neuronal gene expression program in early animal evolution

The assembly of the neuronal and other major cell type programs occurred early in animal evolution. We can reconstruct this process by studying non-bilaterians like placozoans. These small disc-shaped animals not only have nine morphologically described cell types and no neurons but also show coordinated behaviors triggered by peptide-secreting cells. We investigated possible neuronal affinities of these peptidergic cells using phylogenetics, chromatin profiling, and comparative single-cell genomics in four placozoans. We found conserved cell type expression programs across placozoans, including populations of transdifferentiating and cycling cells, suggestive of active cell type homeostasis. We also uncovered fourteen peptidergic cell types expressing neuronal-associated components like the pre-synaptic scaffold that derive from progenitor cells with neurogenesis signatures. In contrast, earlier-branching animals like sponges and ctenophores lacked this conserved expression. Our findings indicate that key neuronal developmental and effector gene modules evolved before the advent of cnidarian/bilaterian neurons in the context of paracrine cell signaling.We thank Alex de Mendoza, Manuel Irimia, and Marta Iglesias for critical comments on the manuscript. We also thank Nikolaus Leisch for advice on placozoan fixation for in situ hybridization; Vikas Trivedi, Krisztina Arato, Kerim Anlas, and Maayan Schwarzkopf for help with in situ HCR; and Jase Gehring, Tara Chari, and Lior Pachter for counsel about the Clicktag (CT) protocol. Finally, we thank Niccolò Arecco for help with ChimeraX and Òscar Fornas and the CRG flow cytometry staff for help with FACS sorting and flow cytometry data analysis. Research in A.S.-P. group was supported by the European Research Council (ERC-StG 851647), the Spanish Ministry of Science and Innovation (PID2021-124757NB-I00), and AGAUR (2021-SGR2021-01219). We also acknowledge support of the Spanish Ministry of Science and Innovation to the EMBL partnership, the Centro de Excelencia Severo Ochoa, and the CERCA Programme (Generalitat de Catalunya). Research in B.S. group was supported by the Alexander von Humboldt Foundation and a TiHo President’s grant (ITZ-20-2). H.G.-V. was funded by the Max Planck Society through N. Dubilier and by the DFG (495424697). The CRG/UPF Proteomics Unit is part of the Spanish Infrastructure for Omics Technologies (ICTS OmicsTech). E.S. and C.C. acknowledge support from the Spanish Ministry of Science and Innovation (PID2020-115092GB-I00) and AGAUR (2021SGR01225). D.C and L.S acknowledge funding from the FWO (S000722N). X.G.-B. is supported by the European Union’s H2020 research and innovation program under Marie Skłodowska-Curie grant agreement 101031767. A.E. and C.N. are supported by FPI PhD fellowships from the Spanish Ministry of Science and Innovation

cGLRs are a diverse family of pattern recognition receptors in innate immunity

Cyclic GMP-AMP synthase (cGAS) is an enzyme in human cells that controls an immune response to cytosolic DNA. Upon binding DNA, cGAS synthesizes a nucleotide signal 2′3′-cGAMP that activates STING-dependent downstream immunity. Here, we discover that cGAS-like receptors (cGLRs) constitute a major family of pattern recognition receptors in innate immunity. Building on recent analysis in Drosophila, we identify &gt;3,000 cGLRs present in nearly all metazoan phyla. A forward biochemical screening of 150 animal cGLRs reveals a conserved mechanism of signaling including response to dsDNA and dsRNA ligands and synthesis of isomers of the nucleotide signals cGAMP, c-UMP-AMP, and c-di-AMP. Combining structural biology and in vivo analysis in coral and oyster animals, we explain how synthesis of distinct nucleotide signals enables cells to control discrete cGLR-STING signaling pathways. Our results reveal cGLRs as a widespread family of pattern recognition receptors and establish molecular rules that govern nucleotide signaling in animal immunity