Publications by authors named "Ryan J Marina"

7 Publications

  • Page 1 of 1

Robust single-cell discovery of RNA targets of RNA-binding proteins and ribosomes.

Nat Methods 2021 May 7;18(5):507-519. Epub 2021 May 7.

Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA.

RNA-binding proteins (RBPs) are critical regulators of gene expression and RNA processing that are required for gene function. Yet the dynamics of RBP regulation in single cells is unknown. To address this gap in understanding, we developed STAMP (Surveying Targets by APOBEC-Mediated Profiling), which efficiently detects RBP-RNA interactions. STAMP does not rely on ultraviolet cross-linking or immunoprecipitation and, when coupled with single-cell capture, can identify RBP-specific and cell-type-specific RNA-protein interactions for multiple RBPs and cell types in single, pooled experiments. Pairing STAMP with long-read sequencing yields RBP target sites in an isoform-specific manner. Finally, Ribo-STAMP leverages small ribosomal subunits to measure transcriptome-wide ribosome association in single cells. STAMP enables the study of RBP-RNA interactomes and translational landscapes with unprecedented cellular resolution.
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http://dx.doi.org/10.1038/s41592-021-01128-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8148648PMC
May 2021

Evaluation of Engineered CRISPR-Cas-Mediated Systems for Site-Specific RNA Editing.

Cell Rep 2020 11;33(5):108350

Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA 92093, USA; Stem Cell Program, University of California San Diego, La Jolla, CA 92093, USA; Institute for Genomic Medicine, University of California at San Diego, La Jolla, CA 92093, USA. Electronic address:

Site-directed RNA editing approaches offer great potential to correct genetic mutations in somatic cells while avoiding permanent off-target genomic edits. Nuclease-dead RNA-targeting CRISPR-Cas systems recruit functional effectors to RNA molecules in a programmable fashion. Here, we demonstrate a Streptococcus pyogenes Cas9-ADAR2 fusion system that uses a 3' modified guide RNA (gRNA) to enable adenosine-to-inosine (A-to-I) editing of specific bases on reporter and endogenously expressed mRNAs. Due to the sufficient nature of the 3' gRNA extension sequence, we observe that Cas9 gRNA spacer sequences are dispensable for directed RNA editing, revealing that Cas9 can act as an RNA-aptamer-binding protein. We demonstrate that Cas9-based A-to-I editing is comparable in on-target efficiency and off-target specificity with Cas13 RNA editing versions. This study provides a systematic benchmarking of RNA-targeting CRISPR-Cas designs for reversible nucleotide-level conversion at the transcriptome level.
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http://dx.doi.org/10.1016/j.celrep.2020.108350DOI Listing
November 2020

Elimination of Toxic Microsatellite Repeat Expansion RNA by RNA-Targeting Cas9.

Cell 2017 Aug 10;170(5):899-912.e10. Epub 2017 Aug 10.

Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, CA, USA; Stem Cell Program, University of California at San Diego, La Jolla, CA, USA; Institute for Genomic Medicine, University of California at San Diego, La Jolla, CA, USA; Molecular Engineering Laboratory, A(∗)STAR, Singapore, Singapore; Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore. Electronic address:

Microsatellite repeat expansions in DNA produce pathogenic RNA species that cause dominantly inherited diseases such as myotonic dystrophy type 1 and 2 (DM1/2), Huntington's disease, and C9orf72-linked amyotrophic lateral sclerosis (C9-ALS). Means to target these repetitive RNAs are required for diagnostic and therapeutic purposes. Here, we describe the development of a programmable CRISPR system capable of specifically visualizing and eliminating these toxic RNAs. We observe specific targeting and efficient elimination of microsatellite repeat expansion RNAs both when exogenously expressed and in patient cells. Importantly, RNA-targeting Cas9 (RCas9) reverses hallmark features of disease including elimination of RNA foci among all conditions studied (DM1, DM2, C9-ALS, polyglutamine diseases), reduction of polyglutamine protein products, relocalization of repeat-bound proteins to resemble healthy controls, and efficient reversal of DM1-associated splicing abnormalities in patient myotubes. Finally, we report a truncated RCas9 system compatible with adeno-associated viral packaging. This effort highlights the potential of RCas9 for human therapeutics.
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http://dx.doi.org/10.1016/j.cell.2017.07.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5873302PMC
August 2017

A tiling-deletion-based genetic screen for cis-regulatory element identification in mammalian cells.

Nat Methods 2017 Jun 17;14(6):629-635. Epub 2017 Apr 17.

Ludwig Institute for Cancer Research, La Jolla, California, USA.

Millions of cis-regulatory elements are predicted to be present in the human genome, but direct evidence for their biological function is scarce. Here we report a high-throughput method, cis-regulatory element scan by tiling-deletion and sequencing (CREST-seq), for the unbiased discovery and functional assessment of cis-regulatory sequences in the genome. We used it to interrogate the 2-Mb POU5F1 locus in human embryonic stem cells, and identified 45 cis-regulatory elements. A majority of these elements have active chromatin marks, DNase hypersensitivity, and occupancy by multiple transcription factors, which confirms the utility of chromatin signatures in cis-element mapping. Notably, 17 of them are previously annotated promoters of functionally unrelated genes, and like typical enhancers, they form extensive spatial contacts with the POU5F1 promoter. These results point to the commonality of enhancer-like promoters in the human genome.
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http://dx.doi.org/10.1038/nmeth.4264DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5490986PMC
June 2017

Epigenomics meets splicing through the TETs and CTCF.

Cell Cycle 2016 06 22;15(11):1397-9. Epub 2016 Apr 22.

b Center for Cancer Research, Laboratory of Receptor Biology and Gene Expression, National Cancer Institute , Bethesda , MD , USA.

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http://dx.doi.org/10.1080/15384101.2016.1171650DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4934062PMC
June 2016

TET-catalyzed oxidation of intragenic 5-methylcytosine regulates CTCF-dependent alternative splicing.

EMBO J 2016 Feb 28;35(3):335-55. Epub 2015 Dec 28.

Center for Cancer Research, Laboratory of Receptor Biology and Gene Expression, National Cancer Institute, Bethesda, MD, USA

Intragenic 5-methylcytosine and CTCF mediate opposing effects on pre-mRNA splicing: CTCF promotes inclusion of weak upstream exons through RNA polymerase II pausing, whereas 5-methylcytosine evicts CTCF, leading to exon exclusion. However, the mechanisms governing dynamic DNA methylation at CTCF-binding sites were unclear. Here, we reveal the methylcytosine dioxygenases TET1 and TET2 as active regulators of CTCF-mediated alternative splicing through conversion of 5-methylcytosine to its oxidation derivatives. 5-hydroxymethylcytosine and 5-carboxylcytosine are enriched at an intragenic CTCF-binding sites in the CD45 model gene and are associated with alternative exon inclusion. Reduced TET levels culminate in increased 5-methylcytosine, resulting in CTCF eviction and exon exclusion. In vitro analyses establish the oxidation derivatives are not sufficient to stimulate splicing, but efficiently promote CTCF association. We further show genomewide that reciprocal exchange of 5-hydroxymethylcytosine and 5-methylcytosine at downstream CTCF-binding sites is a general feature of alternative splicing in naïve and activated CD4(+) T cells. These findings significantly expand our current concept of the pre-mRNA "splicing code" to include dynamic intragenic DNA methylation catalyzed by the TET proteins.
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http://dx.doi.org/10.15252/embj.201593235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4741300PMC
February 2016

Diabetic Insult-Induced Redistribution of MicroRNA in Spatially Organized Mitochondria in Cardiac Muscle.

Circ Cardiovasc Genet 2015 Dec;8(6):747-8

From the Department of Cellular and Molecular Medicine, University of California, San Diego School of Medicine, La Jolla.

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http://dx.doi.org/10.1161/CIRCGENETICS.115.001258DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4683416PMC
December 2015