CapTrap-Seq: A platform-agnostic and quantitative approach for high-fidelity full-length RNA transcript sequencing.

Long-read RNA sequencing is essential to produce accurate and exhaustive annotation of eukaryotic genomes. Despite advancements in throughput and accuracy, achieving reliable end-to-end identification of RNA transcripts remains a challenge for long-read sequencing methods. To address this limitation, we developed CapTrap-seq, a cDNA library preparation method, which combines the Cap-trapping strategy with oligo(dT) priming to detect 5'capped, full-length transcripts, together with the data processing pipeline LyRic. We benchmarked CapTrap-seq and other popular RNA-seq library preparation protocols in a number of human tissues using both ONT and PacBio sequencing. To assess the accuracy of the transcript models produced, we introduced a capping strategy for synthetic RNA spike-in sequences that mimics the natural 5'cap formation in RNA spike-in molecules. We found that the vast majority (up to 90%) of transcript models that LyRic derives from CapTrap-seq reads are full-length. This makes it possible to produce highly accurate annotations with minimal human intervention.

GENCODE: reference annotation for the human and mouse genomes in 2023.

GENCODE produces high quality gene and transcript annotation for the human and mouse genomes. All GENCODE annotation is supported by experimental data and serves as a reference for genome biology and clinical genomics. The GENCODE consortium generates targeted experimental data, develops bioinformatic tools and carries out analyses that, along with externally produced data and methods, support the identification and annotation of transcript structures and the determination of their function. Here, we present an update on the annotation of human and mouse genes, including developments in the tools, data, analyses and major collaborations which underpin this progress. For example, we report the creation of a set of non-canonical ORFs identified in GENCODE transcripts, the LRGASP collaboration to assess the use of long transcriptomic data to build transcript models, the progress in collaborations with RefSeq and UniProt to increase convergence in the annotation of human and mouse protein-coding genes, the propagation of GENCODE across the human pan-genome and the development of new tools to support annotation of regulatory features by GENCODE. Our annotation is accessible via Ensembl, the UCSC Genome Browser and https://www.gencodegenes.org.

Paired guide RNA CRISPR-Cas9 screening for protein-coding genes and lncRNAs involved in transdifferentiation of human B-cells to macrophages.

CRISPR-Cas9 screening libraries have arisen as a powerful tool to identify protein-coding (pc) and non-coding genes playing a role along different processes. In particular, the usage of a nuclease active Cas9 coupled to a single gRNA has proven to efficiently impair the expression of pc-genes by generating deleterious frameshifts. Here, we first demonstrate that targeting the same gene simultaneously with two guide RNAs (paired guide RNAs, pgRNAs) synergistically enhances the capacity of the CRISPR-Cas9 system to knock out pc-genes. We next design a library to target, in parallel, pc-genes and lncRNAs known to change expression during the transdifferentiation from pre-B cells to macrophages. We show that this system is able to identify known players in this process, and also predicts 26 potential novel ones, of which we select four (two pc-genes and two lncRNAs) for deeper characterization. Our results suggest that in the case of the candidate lncRNAs, their impact in transdifferentiation may be actually mediated by enhancer regions at the targeted loci, rather than by the lncRNA transcripts themselves. The CRISPR-Cas9 coupled to a pgRNAs system is, therefore, a suitable tool to simultaneously target pc-genes and lncRNAs for genomic perturbation assays.

MapToCleave: High-throughput profiling of microRNA biogenesis in living cells.

Previous large-scale studies have uncovered many features that determine the processing of microRNA (miRNA) precursors; however, they have been conducted in vitro. Here, we introduce MapToCleave, a method to simultaneously profile processing of thousands of distinct RNA structures in living cells. We find that miRNA precursors with a stable lower basal stem are more efficiently processed and also have higher expression in vivo in tissues from 20 animal species. We systematically compare the importance of known and novel sequence and structural features and test biogenesis of miRNA precursors from 10 animal and plant species in human cells. Lastly, we provide evidence that the GHG motif better predicts processing when defined as a structure rather than sequence motif, consistent with recent cryogenic electron microscopy (cryo-EM) studies. In summary, we apply a screening assay in living cells to reveal the importance of lower basal stem stability for miRNA processing and in vivo expression.

bsAS, an antisense long non-coding RNA, essential for correct wing development through regulation of blistered/DSRF isoform usage.

Natural Antisense Transcripts (NATs) are long non-coding RNAs (lncRNAs) that overlap coding genes in the opposite strand. NATs roles have been related to gene regulation through different mechanisms, including post-transcriptional RNA processing. With the aim to identify NATs with potential regulatory function during fly development, we generated RNA-Seq data in Drosophila developing tissues and found bsAS, one of the most highly expressed lncRNAs in the fly wing. bsAS is antisense to bs/DSRF, a gene involved in wing development and neural processes. bsAS plays a crucial role in the tissue specific regulation of the expression of the bs/DSRF isoforms. This regulation is essential for the correct determination of cell fate during Drosophila development, as bsAS knockouts show highly aberrant phenotypes. Regulation of bs isoform usage by bsAS is mediated by specific physical interactions between the promoters of these two genes, which suggests a regulatory mechanism involving the collision of RNA polymerases transcribing in opposite directions. Evolutionary analysis suggests that bsAS NAT emerged simultaneously to the long-short isoform structure of bs, preceding the emergence of wings in insects.

Scaffolds for Sustained Release of Ambroxol Hydrochloride, a Pharmacological Chaperone That Increases the Activity of Misfolded ß-Glucocerebrosidase.

Ambroxol is a pharmacological chaperone (PC) for Gaucher disease that increases lysosomal activity of misfolded ß-glucocerebrosidase (GCase) while displaying a safe toxicological profile. In this work, different poly(ε-caprolactone) (PCL)-based systems are developed to regulate the sustained release of small polar drugs in physiological environments. For this purpose, ambroxol is selected as test case since the encapsulation and release of PCs using polymeric scaffolds have not been explored yet. More specifically, ambroxol is successfully loaded in electrospun PCL microfibers, which are subsequently coated with additional PCL layers using dip-coating or spin-coating. The time needed to achieve 80% release of loaded ambroxol increases from ≈15 min for uncoated fibrous scaffolds to 3 days and 1 week for dip-coated and spin-coated systems, respectively. Furthermore, it is proven that the released drug maintains its bioactivity, protecting GCase against induced thermal denaturation.

Scalable Design of Paired CRISPR Guide RNAs for Genomic Deletion.

CRISPR-Cas9 technology can be used to engineer precise genomic deletions with pairs of single guide RNAs (sgRNAs). This approach has been widely adopted for diverse applications, from disease modelling of individual loci, to parallelized loss-of-function screens of thousands of regulatory elements. However, no solution has been presented for the unique bioinformatic design requirements of CRISPR deletion. We here present CRISPETa, a pipeline for flexible and scalable paired sgRNA design based on an empirical scoring model. Multiple sgRNA pairs are returned for each target, and any number of targets can be analyzed in parallel, making CRISPETa equally useful for focussed or high-throughput studies. Fast run-times are achieved using a pre-computed off-target database. sgRNA pair designs are output in a convenient format for visualisation and oligonucleotide ordering. We present pre-designed, high-coverage library designs for entire classes of protein-coding and non-coding elements in human, mouse, zebrafish, Drosophila melanogaster and Caenorhabditis elegans. In human cells, we reproducibly observe deletion efficiencies of ≥50% for CRISPETa designs targeting an enhancer and exonic fragment of the MALAT1 oncogene. In the latter case, deletion results in production of desired, truncated RNA. CRISPETa will be useful for researchers seeking to harness CRISPR for targeted genomic deletion, in a variety of model organisms, from single-target to high-throughput scales.

A Point Mutation in a lincRNA Upstream of GDNF Is Associated to a Canine Insensitivity to Pain: A Spontaneous Model for Human Sensory Neuropathies.

Human Hereditary Sensory Autonomic Neuropathies (HSANs) are characterized by insensitivity to pain, sometimes combined with self-mutilation. Strikingly, several sporting dog breeds are particularly affected by such neuropathies. Clinical signs appear in young puppies and consist of acral analgesia, with or without sudden intense licking, biting and severe self-mutilation of the feet, whereas proprioception, motor abilities and spinal reflexes remain intact. Through a Genome Wide Association Study (GWAS) with 24 affected and 30 unaffected sporting dogs using the Canine HD 170K SNP array (Illumina), we identified a 1.8 Mb homozygous locus on canine chromosome 4 (adj. p-val = 2.5x10-6). Targeted high-throughput sequencing of this locus in 4 affected and 4 unaffected dogs identified 478 variants. Only one variant perfectly segregated with the expected recessive inheritance in 300 sporting dogs of known clinical status, while it was never present in 900 unaffected dogs from 130 other breeds. This variant, located 90 kb upstream of the GDNF gene, a highly relevant neurotrophic factor candidate gene, lies in a long intergenic non-coding RNAs (lincRNA), GDNF-AS. Using human comparative genomic analysis, we observed that the canine variant maps onto an enhancer element. Quantitative RT-PCR of dorsal root ganglia RNAs of affected dogs showed a significant decrease of both GDNF mRNA and GDNF-AS expression levels (respectively 60% and 80%), as compared to unaffected dogs. We thus performed gel shift assays (EMSA) that reveal that the canine variant significantly alters the binding of regulatory elements. Altogether, these results allowed the identification in dogs of GDNF as a relevant candidate for human HSAN and insensitivity to pain, but also shed light on the regulation of GDNF transcription. Finally, such results allow proposing these sporting dog breeds as natural models for clinical trials with a double benefit for human and veterinary medicine.

DECKO: Single-oligo, dual-CRISPR deletion of genomic elements including long non-coding RNAs.

BACKGROUND: CRISPR genome-editing technology makes it possible to quickly and cheaply delete non-protein-coding regulatory elements. We present a vector system adapted for this purpose called DECKO (Double Excision CRISPR Knockout), which applies a simple two-step cloning to generate lentiviral vectors expressing two guide RNAs (gRNAs) simultaneously. The key feature of DECKO is its use of a single 165 bp starting oligonucleotide carrying the variable sequences of both gRNAs, making it fully scalable from single-locus studies to complex library cloning. RESULTS: We apply DECKO to deleting the promoters of one protein-coding gene and two oncogenic lncRNAs, UCA1 and the highly-expressed MALAT1, focus of many previous studies employing RNA interference approaches. DECKO successfully deleted genomic fragments ranging in size from 100 to 3000 bp in four human cell lines. Using a clone-derivation workflow lasting approximately 20 days, we obtained 9 homozygous and 17 heterozygous promoter knockouts in three human cell lines. Frequent target region inversions were observed. These clones have reductions in steady-state MALAT1 RNA levels of up to 98 % and display reduced proliferation rates. CONCLUSIONS: We present a dual CRISPR tool, DECKO, which is cloned using a single starting oligonucleotide, thereby affording simplicity and scalability to CRISPR knockout studies of non-coding genomic elements, including long non-coding RNAs.

CARMEN, a human super enhancer-associated long noncoding RNA controlling cardiac specification, differentiation and homeostasis.

Long noncoding RNAs (lncRNAs) are emerging as important regulators of developmental pathways. However, their roles in human cardiac precursor cell (CPC) remain unexplored. To characterize the long noncoding transcriptome during human CPC cardiac differentiation, we profiled the lncRNA transcriptome in CPCs isolated from the human fetal heart and identified 570 lncRNAs that were modulated during cardiac differentiation. Many of these were associated with active cardiac enhancer and super enhancers (SE) with their expression being correlated with proximal cardiac genes. One of the most upregulated lncRNAs was a SE-associated lncRNA that was named CARMEN, (CAR)diac (M)esoderm (E)nhancer-associated (N)oncoding RNA. CARMEN exhibits RNA-dependent enhancing activity and is upstream of the cardiac mesoderm-specifying gene regulatory network. Interestingly, CARMEN interacts with SUZ12 and EZH2, two components of the polycomb repressive complex 2 (PRC2). We demonstrate that CARMEN knockdown inhibits cardiac specification and differentiation in cardiac precursor cells independently of MIR-143 and -145 expression, two microRNAs located proximal to the enhancer sequences. Importantly, CARMEN expression was activated during pathological remodeling in the mouse and human hearts, and was necessary for maintaining cardiac identity in differentiated cardiomyocytes. This study demonstrates therefore that CARMEN is a crucial regulator of cardiac cell differentiation and homeostasis.

Evolution of selenophosphate synthetases: emergence and relocation of function through independent duplications and recurrent subfunctionalization.

Selenoproteins are proteins that incorporate selenocysteine (Sec), a nonstandard amino acid encoded by UGA, normally a stop codon. Sec synthesis requires the enzyme Selenophosphate synthetase (SPS or SelD), conserved in all prokaryotic and eukaryotic genomes encoding selenoproteins. Here, we study the evolutionary history of SPS genes, providing a map of selenoprotein function spanning the whole tree of life. SPS is itself a selenoprotein in many species, although functionally equivalent homologs that replace the Sec site with cysteine (Cys) are common. Many metazoans, however, possess SPS genes with substitutions other than Sec or Cys (collectively referred to as SPS1). Using complementation assays in fly mutants, we show that these genes share a common function, which appears to be distinct from the synthesis of selenophosphate carried out by the Sec- and Cys- SPS genes (termed SPS2), and unrelated to Sec synthesis. We show here that SPS1 genes originated through a number of independent gene duplications from an ancestral metazoan selenoprotein SPS2 gene that most likely already carried the SPS1 function. Thus, in SPS genes, parallel duplications and subsequent convergent subfunctionalization have resulted in the segregation to different loci of functions initially carried by a single gene. This evolutionary history constitutes a remarkable example of emergence and evolution of gene function, which we have been able to trace thanks to the singular features of SPS genes, wherein the amino acid at a single site determines unequivocally protein function and is intertwined to the evolutionary fate of the entire selenoproteome.

Structural basis for antiviral inhibition of the main protease, 3C, from human enterovirus 93.

Members of the Enterovirus genus of the Picornaviridae family are abundant, with common human pathogens that belong to the rhinovirus (HRV) and enterovirus (EV) species, including diverse echo-, coxsackie- and polioviruses. They cause a wide spectrum of clinical manifestations ranging from asymptomatic to severe diseases with neurological and/or cardiac manifestations. Pandemic outbreaks of EVs may be accompanied by meningitis and/or paralysis and can be fatal. However, no effective prophylaxis or antiviral treatment against most EVs is available. The EV RNA genome directs the synthesis of a single polyprotein that is autocatalytically processed into mature proteins at Gln↓Gly cleavage sites by the 3C protease (3C(pro)), which has narrow, conserved substrate specificity. These cleavages are essential for virus replication, making 3C(pro) an excellent target for antivirus drug development. In this study, we report the first determination of the crystal structure of 3C(pro) from an enterovirus B, EV-93, a recently identified pathogen, alone and in complex with the anti-HRV molecules compound 1 (AG7404) and rupintrivir (AG7088) at resolutions of 1.9, 1.3, and 1.5 Å, respectively. The EV-93 3C(pro) adopts a chymotrypsin-like fold with a canonically configured oxyanion hole and a substrate binding pocket similar to that of rhino-, coxsackie- and poliovirus 3C proteases. We show that compound 1 and rupintrivir are both active against EV-93 in infected cells and inhibit the proteolytic activity of EV-93 3C(pro) in vitro. These results provide a framework for further structure-guided optimization of the tested compounds to produce antiviral drugs against a broad range of EV species.

Picornavirus non-structural proteins as targets for new anti-virals with broad activity.

Picornaviridae is one of the largest viral families and is composed of 14 genera, six of which include human pathogens. The best known picornaviruses are enteroviruses (including polio, PV, and rhinoviruses), foot-and-mouth disease virus (FMDV), and hepatitis A virus (HAV). Although infections often are mild, certain strains may cause pandemic outbreaks accompanied with meningitis and/or paralysis. Vaccines are available for PV, HAV and FMDV. When the oral vaccines are given to immunocompromised individuals, they may be chronically infected, and remain secretors of vaccine-derived variants of virus for years. There is no effective prophylaxis available for these or other picornaviruses. So far, only the 3C protease from viruses in three genera has been fully characterized as an anti-viral target, whereas the mode of action of compounds targeting other non-structural proteins have remained largely unaddressed. Within the EU-supported FP6 project-VIZIER (Comparative Structural Genomics of Viral Enzymes Involved in Replication), the non-structural proteins were studied to identify conserved binding sites for broadly reactive anti-virals. The putative 2C helicase from echovirus-30 was shown to form ring-shaped hexamers typical for DNA-encoded SF3 helicases, and to possess ATPase activity. Hexamer formation of 2C from enterovirus 76 was in vitro shown to be dependent on the 44 N-terminal residues. Crystal structures of three enterovirus 3C proteases were solved and shown to be similar to those of other picornaviruses. A new binding site of VPg to the bottom of the thumb domain of CV-B3 3D polymerase was identified as a potential target. Broad anti-enterovirus compounds against 2C and 3A proteins were also identified, including thiazolobenzimidazoles (active against 2C) and TTP-8307 (targeting 3A). There is a need for more potent inhibitors against PV and other picornaviruses, which are potential silent reservoirs for re-emerging PV-like disease.

Structure and inhibition of herpesvirus DNA packaging terminase nuclease domain.

During viral replication, herpesviruses package their DNA into the procapsid by means of the terminase protein complex. In human cytomegalovirus (herpesvirus 5), the terminase is composed of subunits UL89 and UL56. UL89 cleaves the long DNA concatemers into unit-length genomes of appropriate length for encapsidation. We used ESPRIT, a high-throughput screening method, to identify a soluble purifiable fragment of UL89 from a library of 18,432 randomly truncated ul89 DNA constructs. The purified protein was crystallized and its three-dimensional structure was solved. This protein corresponds to the key nuclease domain of the terminase and shows an RNase H/integrase-like fold. We demonstrate that UL89-C has the capacity to process the DNA and that this function is dependent on Mn(2+) ions, two of which are located at the active site pocket. We also show that the nuclease function can be inactivated by raltegravir, a recently approved anti-AIDS drug that targets the HIV integrase.

Human mitochondrial mTERF wraps around DNA through a left-handed superhelical tandem repeat.

The regulation of mitochondrial DNA (mtDNA) processes is slowly being characterized at a structural level. We present here crystal structures of human mitochondrial regulator mTERF, a transcription termination factor also implicated in replication pausing, in complex with double-stranded DNA oligonucleotides containing the tRNA(Leu)(UUR) gene sequence. mTERF comprises nine left-handed helical tandem repeats that form a left-handed superhelix, the Zurdo domain.

Analysis of the stability and function of nucleoplasmin through cysteine mutants.

Xenopus laevis nucleoplasmin is a pentameric nuclear chaperone. The relation between the structure and the multifunctional aspects of the molecule has not yet been clearly established. In the course of analysing a C-terminally His-tagged recombinant version of the region equivalent to the trypsin resistant core (r-NP142) of the molecule, we found that this domain exhibited a substantially decreased oligomerization potential. To better understand the role of the three cysteines of nucleoplasmin on its pentameric functional structure, we have selectively mutated these residues to serine and generated three mutants (C15S, C35S, and C45S) both for the complete recombinant nucleoplasmin (r-NP) and the truncated r-NP142 non-tagged forms. We demonstrate that there are no disulphide bridges stabilizing either the monomer or the pentamer. Neither C15S nor C35S has any structural effects, while the mutation C45S abolishes the ability of r-NP142 to pentamerize. This structural impairment suggests that hydrophobic interactions of Cys 45 are critical for the stability of the protein. Our studies allow to analyse for the first time the structural and functional properties of nucleoplasmin in its monomeric form.

Mutation of the small acidic tract A1 drastically reduces nucleoplasmin activity.

Xenopus laevis nucleoplasmin is a molecular chaperone that mediates sperm decondensation and nucleosome assembly. Nucleoplasmin has three acidic tracts (A1, A2 and A3) and until recent years the long polyglutamic tract A2 was thought to be the binding site for basic proteins. However, the latest publications in this field show that neither A2 nor A3 is indispensable for histone and sperm-specific protein binding. In this work, we show that the mutation of only four acidic amino acid residues of the small A1 tract drastically reduces nucleoplasmin decondensing activity, pointing out this region as the potential binding site for sperm proteins.

Interaction of nucleoplasmin with core histones.

Nucleoplasmin is one of the most abundant proteins in Xenopus laevis oocytes, and it has been involved in the chromatin remodeling that takes place immediately after fertilization. This molecule has been shown to be responsible for the removal of the sperm-specific proteins and deposition of somatic histones onto the male pronuclear chromatin. To better understand the latter process, we have used sedimentation velocity, sedimentation equilibrium, and sucrose gradient fractionation analysis to show that the pentameric form of nucleoplasmin binds to a histone octamer equivalent consisting of equal amounts of the four core histones, H2A, H2B, H3, and H4, without any noticeable preference for any of these proteins. Removal of the histone N-terminal "tail" domains or the major C-terminal polyglutamic tracts of nucleoplasmin did not alter these binding properties. These results indicate that interactions other than those electrostatic in nature (likely hydrophobic) also play a critical role in the formation of the complex between the negatively charged nucleoplasmin and positively charged histones. Although the association of histones with nucleoplasmin may involve some ionic interactions, the interaction process is not electrostatically driven.

Nucleoplasmin interaction with protamines. Involvement of the polyglutamic tract.

Different recombinant forms of nucleoplasmin including truncations at the carboxyl-terminal end of the molecule (r-NP121, r-NP142) have been expressed and purified. All of them were found to oligomerize, forming pentameric complexes which, according to their hydrodynamic properties, can be modeled by oblate ellipsoids of constant width. In this model, the highly charged carboxyl ends appear to be arranged around a pentameric core along the plane defined by the major axes of the ellipsoid. Importantly, all the recombinant forms appear to be able to decondense protamine-containing sperm nuclei. However, although the stoichiometry with which protamines bind to these forms appears to be constant (2.5 mol of protamine/mol of nucleoplasmin pentamer), the efficiency with which they remove protamines from the sperm DNA decreases in the following order: o-NP > r-NP142 > or = r-NP >> r-NP121. Therefore, the main polyglutamic tract of nucleoplasmin (which is absent in r-NP121), while enhancing the efficiency of protamine removal, is not indispensable for sperm chromatin decondensation in the biological model we have used.