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The accuracy of crucial nuclear processes such as transcription, replication, and repair, depends on the local composition of chromatin and the regulatory proteins that reside there. Understanding these DNA-protein interactions at the level of specific genomic loci has remained challenging due to technical limitations. Here, we introduce a method termed "DNA O-MAP", which uses programmable peroxidase-conjugated oligonucleotide probes to biotinylate nearby proteins. We show that DNA O-MAP can be coupled with sample multiplexed quantitative proteomics and next-generation sequencing to quantify DNA-protein and DNA-DNA interactions at specific genomic loci., Competing Interests: Competing Interest Statement D.K.S. is a collaborator with Thermo Fisher Scientific, Genentech, Calico Labs, and AI Proteins. C.K.C., A.F.T., E.K., D.M.S., and B.J.B. have filed a patent application covering aspects of this work. B.J.B. is listed as an inventor on patent applications related to the SABER technology related to this work.

'Ribo-organisms' of the primordial RNA World would have needed ribozymes that catalyze RNA replication. McRae, Wan, Kristoffersen et al. recently revealed how these RNA replicases might have functioned by solving the structure of an artificial polymerase ribozyme. This work illustrates how complex RNA structures evolve, with implications for the origins of life., Competing Interests: Declaration of interests D.M.S. holds patents on methods for incorporating large RNA domains into CRISPR/Cas9 scaffolds, for in situ proximity-biotinylation tools for probing RNA subcellular localization, and for oligonucleotide-mediated, RNA-targeting proximity biotinylation tools to probe RNA interactions within intact cells. These patents are unrelated to the work covered in this manuscript., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

CRISPR-Display uses the S. pyogenes Cas9 protein to posttranscriptionally localize noncoding RNA (ncRNA) domains to any genomic site, by directly fusing these domains to the Cas9 sgRNA cofactor. This versatile technology enables numerous applications for interrogating natural chromatin-regulatory ncRNAs, or for utilizing artificial ncRNA and ribonucleoprotein (RNP) devices at individual chromatin loci. To achieve these, a successful CRISPR-Display experiment requires that chimeric sgRNA-ncRNA fusions are stably expressed and incorporated into Cas9 complexes, and that they retain their ncRNA "cargo" domains at the targeted genomic sites. Here, I describe a workflow for designing, building, and testing such chimeric sgRNA-ncRNA expression constructs. I detail strategies for choosing expression systems and sgRNA insertion topologies, for assaying the incorporation of sgRNA-ncRNA fusions into functional Cas9 complexes, and for surveying the activities of ncRNA domains at targeted genomic loci. This establishes an initial set of "best practices" for the design and implementation of CRISPR-Display experiments.

In the post-genomic era, thousands of putative noncoding regulatory regions have been identified, such as enhancers, promoters, long noncoding RNAs (lncRNAs), and a cadre of small peptides. These ever-growing catalogs require high-throughput assays to test their functionality at scale. Massively parallel reporter assays have greatly enhanced the understanding of noncoding DNA elements en masse Here, we present a massively parallel RNA assay (MPRNA) that can assay 10,000 or more RNA segments for RNA-based functionality. We applied MPRNA to identify RNA-based nuclear localization domains harbored in lncRNAs. We examined a pool of 11,969 oligos densely tiling 38 human lncRNAs that were fused to a cytosolic transcript. After cell fractionation and barcode sequencing, we identified 109 unique RNA regions that significantly enriched this cytosolic transcript in the nucleus including a cytosine-rich motif. These nuclear enrichment sequences are highly conserved and over-represented in global nuclear fractionation sequencing. Importantly, many of these regions were independently validated by single-molecule RNA fluorescence in situ hybridization. Overall, we demonstrate the utility of MPRNA for future investigation of RNA-based functionalities., (© 2018 The Authors. Published under the terms of the CC BY 4.0 license.)

Imaging and chromatin capture techniques have provided important insights into our understanding of nuclear organization. A limitation of these techniques is the inability to resolve allele-specific spatiotemporal properties of genomic loci in living cells. Here, we describe an allele-specific CRISPR live-cell DNA imaging technique (SNP-CLING) to provide the first comprehensive insights into allelic positioning across space and time in mouse embryonic stem cells and fibroblasts. With 3D imaging, we studied alleles on different chromosomes in relation to one another and relative to nuclear substructures such as the nucleolus. We find that alleles maintain similar positions relative to each other and the nucleolus; however, loci occupy unique positions. To monitor spatiotemporal dynamics by SNP-CLING, we performed 4D imaging and determined that alleles are either stably positioned or fluctuating during cell state transitions, such as apoptosis. SNP-CLING is a universally applicable technique that enables the dissection of allele-specific spatiotemporal genome organization in live cells.

The spatial organization of RNA within cells is a crucial factor influencing a wide range of biological functions throughout all kingdoms of life. However, a general understanding of RNA localization has been hindered by a lack of simple, high-throughput methods for mapping the transcriptomes of subcellular compartments. Here, we develop such a method, termed APEX-RIP, which combines peroxidase-catalyzed, spatially restricted in situ protein biotinylation with RNA-protein chemical crosslinking. We demonstrate that, using a single protocol, APEX-RIP can isolate RNAs from a variety of subcellular compartments, including the mitochondrial matrix, nucleus, cytosol, and endoplasmic reticulum (ER), with specificity and sensitivity that rival or exceed those of conventional approaches. We further identify candidate RNAs localized to mitochondria-ER junctions and nuclear lamina, two compartments that are recalcitrant to classical biochemical purification. Since APEX-RIP is simple, versatile, and does not require special instrumentation, we envision its broad application in a variety of biological contexts.

While long intergenic noncoding RNAs (lincRNAs) and mRNAs share similar biogenesis pathways, these transcript classes differ in many regards. LincRNAs are less evolutionarily conserved, less abundant, and more tissue-specific, suggesting that their pre- and post-transcriptional regulation is different from that of mRNAs. Here, we perform an in-depth characterization of the features that contribute to lincRNA regulation in multiple human cell lines. We find that lincRNA promoters are depleted of transcription factor (TF) binding sites, yet enriched for some specific factors such as GATA and FOS relative to mRNA promoters. Surprisingly, we find that H3K9me3-a histone modification typically associated with transcriptional repression-is more enriched at the promoters of active lincRNA loci than at those of active mRNAs. Moreover, H3K9me3-marked lincRNA genes are more tissue-specific. The most discriminant differences between lincRNAs and mRNAs involve splicing. LincRNAs are less efficiently spliced, which cannot be explained by differences in U1 binding or the density of exonic splicing enhancers but may be partially attributed to lower U2AF65 binding and weaker splicing-related motifs. Conversely, the stability of lincRNAs and mRNAs is similar, differing only with regard to the location of stabilizing protein binding sites. Finally, we find that certain transcriptional properties are correlated with higher evolutionary conservation in both DNA and RNA motifs and are enriched in lincRNAs that have been functionally characterized., (© 2017 Melé et al. Published by Cold Spring Harbor Laboratory Press.)

Noncoding RNAs play diverse roles throughout biology and exhibit broad functional capacity. To investigate and harness these capabilities, we developed clustered regularly interspaced short palindromic repeats (CRISPR)-Display (CRISP-Disp), a targeted localization method that uses Cas9 to deploy large RNA cargos to DNA loci. We demonstrate that functional RNA domains up to at least 4.8 kb long can be inserted in CRISPR guide RNA at multiple points, allowing the construction of Cas9 complexes with protein-binding cassettes, artificial aptamers, pools of random sequences and natural long noncoding RNAs. A unique feature of CRISP-Disp is the multiplexing of distinct functions at multiple targets, limited only by the availability of functional RNA motifs. We anticipate the use of CRISP-Disp for ectopically targeting functional RNAs and ribonucleoprotein (RNP) complexes to genomic loci.

Early life presumably required polymerase ribozymes capable of replicating RNA. Known polymerase ribozymes best approximating such replicases use as their catalytic engine an RNA-ligase ribozyme originally selected from random RNA sequences. Here we report 3.15-Å crystal structures of this ligase trapped in catalytically viable preligation states, with the 3'-hydroxyl nucleophile positioned for in-line attack on the 5'-triphosphate. Guided by metal- and solvent-mediated interactions, the 5'-triphosphate hooks into the major groove of the adjoining RNA duplex in an unanticipated conformation. Two phosphates and the nucleophile jointly coordinate an active-site metal ion. Atomic mutagenesis experiments demonstrate that active-site nucleobase and hydroxyl groups also participate directly in catalysis, collectively playing a role that in proteinaceous polymerases is performed by a second metal ion. Thus artificial ribozymes can use complex catalytic strategies that differ markedly from those of analogous biological enzymes.

RNA crystallization and phasing represent major bottlenecks in RNA structure determination. Seeking to exploit antibody fragments as RNA crystallization chaperones, we have used an arginine-enriched synthetic Fab library displayed on phage to obtain Fabs against the class I ligase ribozyme. We solved the structure of a Fab-ligase complex at 3.1-Å resolution using molecular replacement with Fab coordinates, confirming the ribozyme architecture and revealing the chaperone's role in RNA recognition and crystal contacts. The epitope resides in the GAAACAC sequence that caps the P5 helix, and this sequence retains high-affinity Fab binding within the context of other structured RNAs. This portable epitope provides a new RNA crystallization chaperone system that easily can be screened in parallel to the U1A RNA-binding protein, with the advantages of a smaller loop and Fabs' high molecular weight, large surface area and phasing power.

The class I ligase was among the first ribozymes to have been isolated from random sequences and represents the catalytic core of several RNA-directed RNA polymerase ribozymes. The ligase is also notable for its catalytic efficiency and structural complexity. Here, we report an improved version of this ribozyme, arising from selection that targeted the kinetics of the chemical step. Compared with the parent ribozyme, the improved ligase achieves a modest increase in rate enhancement under the selective conditions and shows a sharp reduction in [Mg(2+)] dependence. Analysis of the sequences and kinetics of successful clones suggests which mutations play the greatest part in these improvements. Moreover, backbone and nucleobase interference maps of the parent and improved ligase ribozymes complement the newly solved crystal structure of the improved ligase to identify the functionally significant interactions underlying the catalytic ability and structural complexity of the ligase ribozyme.

Primordial organisms of the putative RNA world would have required polymerase ribozymes able to replicate RNA. Known ribozymes with polymerase activity best approximating that needed for RNA replication contain at their catalytic core the class I RNA ligase, an artificial ribozyme with a catalytic rate among the fastest of known ribozymes. Here we present the 3.0 angstrom crystal structure of this ligase. The architecture resembles a tripod, its three legs converging near the ligation junction. Interacting with this tripod scaffold through a series of 10 minor-groove interactions (including two A-minor triads) is the unpaired segment that contributes to and organizes the active site. A cytosine nucleobase and two backbone phosphates abut the ligation junction; their location suggests a model for catalysis resembling that of proteinaceous polymerases.