High-Spatial-Resolution Multi-Omics Sequencing via Deterministic Barcoding in Tissue.

We present deterministic barcoding in tissue for spatial omics sequencing (DBiT-seq) for co-mapping of mRNAs and proteins in a formaldehyde-fixed tissue slide via next-generation sequencing (NGS). Parallel microfluidic channels were used to deliver DNA barcodes to the surface of a tissue slide, and crossflow of two sets of barcodes, A1-50 and B1-50, followed by ligation in situ, yielded a 2D mosaic of tissue pixels, each containing a unique full barcode AB. Application to mouse embryos revealed major tissue types in early organogenesis as well as fine features like microvasculature in a brain and pigmented epithelium in an eye field. Gene expression profiles in 10-µm pixels conformed into the clusters of single-cell transcriptomes, allowing for rapid identification of cell types and spatial distributions. DBiT-seq can be adopted by researchers with no experience in microfluidics and may find applications in a range of fields including developmental biology, cancer biology, neuroscience, and clinical pathology.

Phase Separation of FUS Is Suppressed by Its Nuclear Import Receptor and Arginine Methylation.

Cytoplasmic FUS aggregates are a pathological hallmark in a subset of patients with frontotemporal dementia (FTD) or amyotrophic lateral sclerosis (ALS). A key step that is disrupted in these patients is nuclear import of FUS mediated by the import receptor Transportin/Karyopherin-ß2. In ALS-FUS patients, this is caused by mutations in the nuclear localization signal (NLS) of FUS that weaken Transportin binding. In FTD-FUS patients, Transportin is aggregated, and post-translational arginine methylation, which regulates the FUS-Transportin interaction, is lost. Here, we show that Transportin and arginine methylation have a crucial function beyond nuclear import-namely to suppress RGG/RG-driven phase separation and stress granule association of FUS. ALS-associated FUS-NLS mutations weaken the chaperone activity of Transportin and loss of FUS arginine methylation, as seen in FTD-FUS, promote phase separation, and stress granule partitioning of FUS. Our findings reveal two regulatory mechanisms of liquid-phase homeostasis that are disrupted in FUS-associated neurodegeneration.

Template and target-site recognition by human LINE-1 in retrotransposition.

The long interspersed element-1 (LINE-1, hereafter L1) retrotransposon has generated nearly one-third of the human genome and serves as an active source of genetic diversity and human disease1. L1 spreads through a mechanism termed target-primed reverse transcription, in which the encoded enzyme (ORF2p) nicks the target DNA to prime reverse transcription of its own or non-self RNAs2. Here we purified full-length L1 ORF2p and biochemically reconstituted robust target-primed reverse transcription with template RNA and target-site DNA. We report cryo-electron microscopy structures of the complete human L1 ORF2p bound to structured template RNAs and initiating cDNA synthesis. The template polyadenosine tract is recognized in a sequence-specific manner by five distinct domains. Among them, an RNA-binding domain bends the template backbone to allow engagement of an RNA hairpin stem with the L1 ORF2p C-terminal segment. Moreover, structure and biochemical reconstitutions demonstrate an unexpected target-site requirement: L1 ORF2p relies on upstream single-stranded DNA to position the adjacent duplex in the endonuclease active site for nicking of the longer DNA strand, with a single nick generating a staggered DNA break. Our research provides insights into the mechanism of ongoing transposition in the human genome and informs the engineering of retrotransposon proteins for gene therapy.

Second-Strand Synthesis-Based Massively Parallel scRNA-Seq Reveals Cellular States and Molecular Features of Human Inflammatory Skin Pathologies.

High-throughput single-cell RNA-sequencing (scRNA-seq) methodologies enable characterization of complex biological samples by increasing the number of cells that can be profiled contemporaneously. Nevertheless, these approaches recover less information per cell than low-throughput strategies. To accurately report the expression of key phenotypic features of cells, scRNA-seq platforms are needed that are both high fidelity and high throughput. To address this need, we created Seq-Well S3 ("Second-Strand Synthesis"), a massively parallel scRNA-seq protocol that uses a randomly primed second-strand synthesis to recover complementary DNA (cDNA) molecules that were successfully reverse transcribed but to which a second oligonucleotide handle, necessary for subsequent whole transcriptome amplification, was not appended due to inefficient template switching. Seq-Well S3 increased the efficiency of transcript capture and gene detection compared with that of previous iterations by up to 10- and 5-fold, respectively. We used Seq-Well S3 to chart the transcriptional landscape of five human inflammatory skin diseases, thus providing a resource for the further study of human skin inflammation.

Mega-scale experimental analysis of protein folding stability in biology and design.

Advances in DNA sequencing and machine learning are providing insights into protein sequences and structures on an enormous scale1. However, the energetics driving folding are invisible in these structures and remain largely unknown2. The hidden thermodynamics of folding can drive disease3,4, shape protein evolution5-7 and guide protein engineering8-10, and new approaches are needed to reveal these thermodynamics for every sequence and structure. Here we present cDNA display proteolysis, a method for measuring thermodynamic folding stability for up to 900,000 protein domains in a one-week experiment. From 1.8 million measurements in total, we curated a set of around 776,000 high-quality folding stabilities covering all single amino acid variants and selected double mutants of 331 natural and 148 de novo designed protein domains 40-72 amino acids in length. Using this extensive dataset, we quantified (1) environmental factors influencing amino acid fitness, (2) thermodynamic couplings (including unexpected interactions) between protein sites, and (3) the global divergence between evolutionary amino acid usage and protein folding stability. We also examined how our approach could identify stability determinants in designed proteins and evaluate design methods. The cDNA display proteolysis method is fast, accurate and uniquely scalable, and promises to reveal the quantitative rules for how amino acid sequences encode folding stability.

Genetic Characterization of Three Distinct Mechanisms Supporting RNA-Driven DNA Repair and Modification Reveals Major Role of DNA Polymerase ζ.

DNA double-stranded breaks (DSBs) are dangerous lesions threatening genomic stability. Fidelity of DSB repair is best achieved by recombination with a homologous template sequence. In yeast, transcript RNA was shown to template DSB repair of DNA. However, molecular pathways of RNA-driven repair processes remain obscure. Utilizing assays of RNA-DNA recombination with and without an induced DSB in yeast DNA, we characterize three forms of RNA-mediated genomic modifications: RNA- and cDNA-templated DSB repair (R-TDR and c-TDR) using an RNA transcript or a DNA copy of the RNA transcript for DSB repair, respectively, and a new mechanism of RNA-templated DNA modification (R-TDM) induced by spontaneous or mutagen-induced breaks. While c-TDR requires reverse transcriptase, translesion DNA polymerase ζ (Pol ζ) plays a major role in R-TDR, and it is essential for R-TDM. This study characterizes mechanisms of RNA-DNA recombination, uncovering a role of Pol ζ in transferring genetic information from transcript RNA to DNA.

Sequence-specific activation of the DNA sensor cGAS by Y-form DNA structures as found in primary HIV-1 cDNA.

Cytosolic DNA that emerges during infection with a retrovirus or DNA virus triggers antiviral type I interferon responses. So far, only double-stranded DNA (dsDNA) over 40 base pairs (bp) in length has been considered immunostimulatory. Here we found that unpaired DNA nucleotides flanking short base-paired DNA stretches, as in stem-loop structures of single-stranded DNA (ssDNA) derived from human immunodeficiency virus type 1 (HIV-1), activated the type I interferon-inducing DNA sensor cGAS in a sequence-dependent manner. DNA structures containing unpaired guanosines flanking short (12- to 20-bp) dsDNA (Y-form DNA) were highly stimulatory and specifically enhanced the enzymatic activity of cGAS. Furthermore, we found that primary HIV-1 reverse transcripts represented the predominant viral cytosolic DNA species during early infection of macrophages and that these ssDNAs were highly immunostimulatory. Collectively, our study identifies unpaired guanosines in Y-form DNA as a highly active, minimal cGAS recognition motif that enables detection of HIV-1 ssDNA.

Structure of the human Mediator-RNA polymerase II pre-initiation complex.

Mediator is a conserved coactivator complex that enables the regulated initiation of transcription at eukaryotic genes1-3. Mediator is recruited by transcriptional activators and binds the pre-initiation complex (PIC) to stimulate the phosphorylation of RNA polymerase II (Pol II) and promoter escape1-6. Here we prepare a recombinant version of human Mediator, reconstitute a 50-subunit Mediator-PIC complex and determine the structure of the complex by cryo-electron microscopy. The head module of Mediator contacts the stalk of Pol II and the general transcription factors TFIIB and TFIIE, resembling the Mediator-PIC interactions observed in the corresponding complex in yeast7-9. The metazoan subunits MED27-MED30 associate with exposed regions in MED14 and MED17 to form the proximal part of the Mediator tail module that binds activators. Mediator positions the flexibly linked cyclin-dependent kinase (CDK)-activating kinase of the general transcription factor TFIIH near the linker to the C-terminal repeat domain of Pol II. The Mediator shoulder domain holds the CDK-activating kinase subunit CDK7, whereas the hook domain contacts a CDK7 element that flanks the kinase active site. The shoulder and hook domains reside in the Mediator head and middle modules, respectively, which can move relative to each other and may induce an active conformation of the CDK7 kinase to allosterically stimulate phosphorylation of the C-terminal domain.

Nano3P-seq: transcriptome-wide analysis of gene expression and tail dynamics using end-capture nanopore cDNA sequencing.

RNA polyadenylation plays a central role in RNA maturation, fate, and stability. In response to developmental cues, polyA tail lengths can vary, affecting the translation efficiency and stability of mRNAs. Here we develop Nanopore 3' end-capture sequencing (Nano3P-seq), a method that relies on nanopore cDNA sequencing to simultaneously quantify RNA abundance, tail composition, and tail length dynamics at per-read resolution. By employing a template-switching-based sequencing protocol, Nano3P-seq can sequence RNA molecule from its 3' end, regardless of its polyadenylation status, without the need for PCR amplification or ligation of RNA adapters. We demonstrate that Nano3P-seq provides quantitative estimates of RNA abundance and tail lengths, and captures a wide diversity of RNA biotypes. We find that, in addition to mRNA and long non-coding RNA, polyA tails can be identified in 16S mitochondrial ribosomal RNA in both mouse and zebrafish models. Moreover, we show that mRNA tail lengths are dynamically regulated during vertebrate embryogenesis at an isoform-specific level, correlating with mRNA decay. Finally, we demonstrate the ability of Nano3P-seq in capturing non-A bases within polyA tails of various lengths, and reveal their distribution during vertebrate embryogenesis. Overall, Nano3P-seq is a simple and robust method for accurately estimating transcript levels, tail lengths, and tail composition heterogeneity in individual reads, with minimal library preparation biases, both in the coding and non-coding transcriptome.

Enhanced ac4C detection in RNA via chemical reduction and cDNA synthesis with modified dNTPs.

The functional analysis of epitranscriptomic modifications in RNA is constrained by a lack of methods that accurately capture their locations and levels. We previously demonstrated that the RNA modification N4-acetylcytidine (ac4C) can be mapped at base resolution through sodium borohydride reduction to tetrahydroacetylcytidine (tetrahydro-ac4C), followed by cDNA synthesis to misincorporate adenosine opposite reduced ac4C sites, culminating in C:T mismatches at acetylated cytidines (RedaC:T). However, this process is relatively inefficient, resulting in <20% C:T mismatches at a fully modified ac4C site in 18S rRNA. Considering that ac4C locations in other substrates including mRNA are unlikely to reach full penetrance, this method is not ideal for comprehensive mapping. Here, we introduce "RetraC:T" (reduction to tetrahydro-ac4C and reverse transcription with amino-dATP to induce C:T mismatches) as a method with enhanced ability to detect ac4C in cellular RNA. In brief, RNA is reduced through NaBH4 or the closely related reagent sodium cyanoborohydride (NaCNBH3) followed by cDNA synthesis in the presence of a modified DNA nucleotide, 2-amino-dATP, that preferentially binds to tetrahydro-ac4C. Incorporation of the modified dNTP substantially improved C:T mismatch rates, reaching stoichiometric detection of ac4C in 18S rRNA. Importantly, 2-amino-dATP did not result in truncated cDNA products nor increase mismatches at other locations. Thus, modified dNTPs are introduced as a new addition to the toolbox for detecting ac4C at base resolution.

The enemy within: endogenous retroelements and autoimmune disease.

Inappropriate or chronic detection of self nucleic acids by the innate immune system underlies many human autoimmune diseases. We discuss here an unexpected source of endogenous immunostimulatory nucleic acids: the reverse-transcribed cDNA of endogenous retroelements. The interplay between innate immune sensing and clearance of retroelement cDNA has important implications for the understanding of immune responses to infectious retroviruses such as human immunodeficiency virus (HIV). Furthermore, the detection of cDNA by the innate immune system reveals an evolutionary tradeoff: selection for a vigorous, sensitive response to infectious retroviruses may predispose the inappropriate detection of endogenous retroelements. We propose that this tradeoff has placed unique constraints on the sensitivity of the DNA-activated antiviral response, with implications for the interactions of DNA viruses and retroviruses with their hosts. Finally, we discuss how better understanding of the intersection of retroelement biology and innate immunity can guide the way to novel therapies for specific autoimmune diseases.

Distinct and overlapping RNA determinants for binding and target-primed reverse transcription by Bombyx mori R2 retrotransposon protein.

Eukaryotic retrotransposons encode a reverse transcriptase that binds RNA to template DNA synthesis. The ancestral non-long terminal repeat (non-LTR) retrotransposons encode a protein that performs target-primed reverse transcription (TPRT), in which the nicked genomic target site initiates complementary DNA (cDNA) synthesis directly into the genome. The best understood model system for biochemical studies of TPRT is the R2 protein from the silk moth Bombyx mori. The R2 protein selectively binds the 3' untranslated region of its encoding RNA as template for DNA insertion to its target site in 28S ribosomal DNA. Here, binding and TPRT assays define RNA contributions to RNA-protein interaction, template use for TPRT and the fidelity of template positioning for TPRT cDNA synthesis. We quantify both sequence and structure contributions to protein-RNA interaction. RNA determinants of binding affinity overlap but are not equivalent to RNA features required for TPRT and its fidelity of template positioning for full-length TPRT cDNA synthesis. Additionally, we show that a previously implicated RNA-binding protein surface of R2 protein makes RNA binding affinity dependent on the presence of two stem-loops. Our findings inform evolutionary relationships across R2 retrotransposon RNAs and are a step toward understanding the mechanism and template specificity of non-LTR retrotransposon mobility.

De novo variants in FRMD5 are associated with developmental delay, intellectual disability, ataxia, and abnormalities of eye movement.

Proteins containing the FERM (four-point-one, ezrin, radixin, and moesin) domain link the plasma membrane with cytoskeletal structures at specific cellular locations and have been implicated in the localization of cell-membrane-associated proteins and/or phosphoinositides. FERM domain-containing protein 5 (FRMD5) localizes at cell adherens junctions and stabilizes cell-cell contacts. To date, variants in FRMD5 have not been associated with a Mendelian disease in OMIM. Here, we describe eight probands with rare heterozygous missense variants in FRMD5 who present with developmental delay, intellectual disability, ataxia, seizures, and abnormalities of eye movement. The variants are de novo in all for whom parental testing was available (six out of eight probands), and human genetic datasets suggest that FRMD5 is intolerant to loss of function (LoF). We found that the fly ortholog of FRMD5, CG5022 (dFrmd), is expressed in the larval and adult central nervous systems where it is present in neurons but not in glia. dFrmd LoF mutant flies are viable but are extremely sensitive to heat shock, which induces severe seizures. The mutants also exhibit defective responses to light. The human FRMD5 reference (Ref) cDNA rescues the fly dFrmd LoF phenotypes. In contrast, all the FRMD5 variants tested in this study (c.340T>C, c.1051A>G, c.1053C>G, c.1054T>C, c.1045A>C, and c.1637A>G) behave as partial LoF variants. In addition, our results indicate that two variants that were tested have dominant-negative effects. In summary, the evidence supports that the observed variants in FRMD5 cause neurological symptoms in humans.

The recurrent de novo c.2011C>T missense variant in MTSS2 causes syndromic intellectual disability.

MTSS2, also known as MTSS1L, binds to plasma membranes and modulates their bending. MTSS2 is highly expressed in the central nervous system (CNS) and appears to be involved in activity-dependent synaptic plasticity. Variants in MTSS2 have not yet been associated with a human phenotype in OMIM. Here we report five individuals with the same heterozygous de novo variant in MTSS2 (GenBank: NM_138383.2: c.2011C>T [p.Arg671Trp]) identified by exome sequencing. The individuals present with global developmental delay, mild intellectual disability, ophthalmological anomalies, microcephaly or relative microcephaly, and shared mild facial dysmorphisms. Immunoblots of fibroblasts from two affected individuals revealed that the variant does not significantly alter MTSS2 levels. We modeled the variant in Drosophila and showed that the fly ortholog missing-in-metastasis (mim) was widely expressed in most neurons and a subset of glia of the CNS. Loss of mim led to a reduction in lifespan, impaired locomotor behavior, and reduced synaptic transmission in adult flies. Expression of the human MTSS2 reference cDNA rescued the mim loss-of-function (LoF) phenotypes, whereas the c.2011C>T variant had decreased rescue ability compared to the reference, suggesting it is a partial LoF allele. However, elevated expression of the variant, but not the reference MTSS2 cDNA, led to similar defects as observed by mim LoF, suggesting that the variant is toxic and may act as a dominant-negative allele when expressed in flies. In summary, our findings support that mim is important for appropriate neural function, and that the MTSS2 c.2011C>T variant causes a syndromic form of intellectual disability.

Loss-of-function variants in TIAM1 are associated with developmental delay, intellectual disability, and seizures.

TIAM Rac1-associated GEF 1 (TIAM1) regulates RAC1 signaling pathways that affect the control of neuronal morphogenesis and neurite outgrowth by modulating the actin cytoskeletal network. To date, TIAM1 has not been associated with a Mendelian disorder. Here, we describe five individuals with bi-allelic TIAM1 missense variants who have developmental delay, intellectual disability, speech delay, and seizures. Bioinformatic analyses demonstrate that these variants are rare and likely pathogenic. We found that the Drosophila ortholog of TIAM1, still life (sif), is expressed in larval and adult central nervous system (CNS) and is mainly expressed in a subset of neurons, but not in glia. Loss of sif reduces the survival rate, and the surviving adults exhibit climbing defects, are prone to severe seizures, and have a short lifespan. The TIAM1 reference (Ref) cDNA partially rescues the sif loss-of-function (LoF) phenotypes. We also assessed the function associated with three TIAM1 variants carried by two of the probands and compared them to the TIAM1 Ref cDNA function in vivo. TIAM1 p.Arg23Cys has reduced rescue ability when compared to TIAM1 Ref, suggesting that it is a partial LoF variant. In ectopic expression studies, both wild-type sif and TIAM1 Ref are toxic, whereas the three variants (p.Leu862Phe, p.Arg23Cys, and p.Gly328Val) show reduced toxicity, suggesting that they are partial LoF variants. In summary, we provide evidence that sif is important for appropriate neural function and that TIAM1 variants observed in the probands are disruptive, thus implicating loss of TIAM1 in neurological phenotypes in humans.

Sequencing of individual barcoded cDNAs using Pacific Biosciences and Oxford Nanopore Technologies reveals platform-specific error patterns.

Long-read transcriptomics require understanding error sources inherent to technologies. Current approaches cannot compare methods for an individual RNA molecule. Here, we present a novel platform-comparison method that combines barcoding strategies and long-read sequencing to sequence cDNA copies representing an individual RNA molecule on both Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT). We compare these long-read pairs in terms of sequence content and isoform patterns. Although individual read pairs show high similarity, we find differences in (1) aligned length, (2) transcription start site (TSS), (3) polyadenylation site (poly(A)-site) assignment, and (4) exon-intron structures. Overall, 25% of read pairs disagree on either TSS, poly(A)-site, or splice site. Intron-chain disagreement typically arises from alignment errors of microexons and complicated splice sites. Our single-molecule technology comparison reveals that inconsistencies are often caused by sequencing error-induced inaccurate ONT alignments, especially to downstream GUNNGU donor motifs. However, annotation-disagreeing upstream shifts in NAGNAG acceptors in ONT are often confirmed by PacBio and are thus likely real. In both barcoded and nonbarcoded ONT reads, we find that intron number and proximity of GU/AGs better predict inconsistencies with the annotation than read quality alone. We summarize these findings in an annotation-based algorithm for spliced alignment correction that improves subsequent transcript construction with ONT reads.

Profiling RNA at chromatin targets in situ by antibody-targeted tagmentation.

Whereas techniques to map chromatin-bound proteins are well developed, mapping chromatin-associated RNAs remains a challenge. Here, we describe Reverse Transcribe and Tagment (RT&Tag), in which RNAs associated with a chromatin epitope are targeted by an antibody followed by a protein A-Tn5 transposome. Localized reverse transcription generates RNA/cDNA hybrids that are subsequently tagmented by Tn5 transposases for downstream sequencing. We demonstrate the utility of RT&Tag in Drosophila cells for capturing the noncoding RNA roX2 with the dosage compensation complex and maturing transcripts associated with silencing histone modifications. We also show that RT&Tag can detect N6-methyladenosine-modified mRNAs, and show that genes producing methylated transcripts are characterized by extensive promoter pausing of RNA polymerase II. The high efficiency of in situ antibody tethering and tagmentation makes RT&Tag especially suitable for rapid low-cost profiling of chromatin-associated RNAs.

Light-Seq: light-directed in situ barcoding of biomolecules in fixed cells and tissues for spatially indexed sequencing.

We present Light-Seq, an approach for multiplexed spatial indexing of intact biological samples using light-directed DNA barcoding in fixed cells and tissues followed by ex situ sequencing. Light-Seq combines spatially targeted, rapid photocrosslinking of DNA barcodes onto complementary DNAs in situ with a one-step DNA stitching reaction to create pooled, spatially indexed sequencing libraries. This light-directed barcoding enables in situ selection of multiple cell populations in intact fixed tissue samples for full-transcriptome sequencing based on location, morphology or protein stains, without cellular dissociation. Applying Light-Seq to mouse retinal sections, we recovered thousands of differentially enriched transcripts from three cellular layers and discovered biomarkers for a very rare neuronal subtype, dopaminergic amacrine cells, from only four to eight individual cells per section. Light-Seq provides an accessible workflow to combine in situ imaging and protein staining with next generation sequencing of the same cells, leaving the sample intact for further analysis post-sequencing.

Nanopore-based RNA sequencing deciphers the formation, processing, and modification steps of rRNA intermediates in archaea.

Ribosomal RNA (rRNA) maturation in archaea is a complex multistep process that requires well-defined endo- and exoribonuclease activities to generate fully mature linear rRNAs. However, technical challenges prevented detailed mapping of rRNA processing steps and a systematic analysis of rRNA maturation pathways across the tree of life. In this study, we used long-read (PCR)-cDNA and direct RNA nanopore-based sequencing to study rRNA maturation in three archaeal model organisms, namely the Euryarchaea Haloferax volcanii and Pyrococcus furiosus and the Crenarchaeon Sulfolobus acidocaldarius Compared to standard short-read protocols, nanopore sequencing facilitates simultaneous readout of 5'- and 3'-positions, which is required for the classification of rRNA processing intermediates. More specifically, we (i) accurately detect and describe rRNA maturation stages by analysis of terminal read positions of cDNA reads and thereupon (ii) explore the stage-dependent installation of the KsgA-mediated dimethylations in H. volcanii using base-calling and signal characteristics of direct RNA reads. Due to the single-molecule sequencing capacity of nanopore sequencing, we could detect hitherto unknown intermediates with high confidence, revealing details about the maturation of archaea-specific circular rRNA intermediates. Taken together, our study delineates common principles and unique features of rRNA processing in euryarchaeal and crenarchaeal representatives, thereby significantly expanding our understanding of rRNA maturation pathways in archaea.

Artifacts and biases of the reverse transcription reaction in RNA sequencing.

RNA sequencing has spurred a significant number of research areas in recent years. Most protocols rely on synthesizing a more stable complementary DNA (cDNA) copy of the RNA molecule during the reverse transcription reaction. The resulting cDNA pool is often wrongfully assumed to be quantitatively and molecularly similar to the original RNA input. Sadly, biases and artifacts confound the resulting cDNA mixture. These issues are often overlooked or ignored in the literature by those that rely on the reverse transcription process. In this review, we confront the reader with intra- and intersample biases and artifacts caused by the reverse transcription reaction during RNA sequencing experiments. To fight the reader's despair, we also provide solutions to most issues and inform on good RNA sequencing practices. We hope the reader can use this review to their advantage, thereby contributing to scientifically sound RNA studies.