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Open Access Publications from the University of California

Scholarly Works (5 results)

  • Thesis
  • Peer Reviewed
UC San Diego Electronic Theses and Dissertations (2020)

Ribonucleic acids (RNAs) serve as one of the main building blocks of organic life and with the advent of new understanding beyond an intermediary for protein coding, understanding it's function becomes imperative. Previous tools study RNA expression from cell populations such as tissue or cell lines, however, an expansive area of research has developed for the individual cells. The recognition of cell heterogeneity in normal and diseased tissues further highlighted the importance of accurate RNA detection in single cells. Single molecule RNA fluorescence in situ hybridization (smRNA-FISH) has revolutionized RNA detection and quantification by visualizing single RNA transcripts.

The work in this dissertation improves upon smRNA-FISH by developing quantum dot based fluorescent in situ technologies that uses hybridization of quantum dot-labeled single stranded DNA oligonucleotides to the RNA targets for visualization and quantification. In chapter 2, we demonstrate our proof-of-concept quantum dot single molecule RNA-FISH (qdot-smRNA-FISH) by comparing the sensitivity and specificity of qdot-smRNA-FISH against commercialized smRNA-FISH. In chapter 3, we describe qdot-smRNA-FISH for detection of isoforms (isoFISH), where different from smFISH that requires dozens of hybridization probes per gene, isoFISH requires only two hybridization probes per splicing variant. In chapter 4, we used the co-localization of multi-color quantum dot FISH (CoQ-FISH) to observe for the first time a fusion transcript formation of Eml4-Alk in non-small cell lung cancer biopsy samples from trans-splicing and not chromosomal rearrangement. In chapter 5, we develop cell surface qDot RNA-FISH (Surface-FISH), a method for visualization of RNAs on the cell surface. We identified RNA targets using RNA-seq on membrane coated nanoparticles from EL4 and applied Surface-FISH to observe regions of Malat1 and Neat1. In chapter 6, we developed Surface-PromFISH that uses randomized antisense probes to promiscuously label as many csRNAs as possible and made it compatible with co-staining of cell membrane proteins. We subjected the hybridization targets to RNA sequencing (Surface-FISHseq), which led to 51 putative PBMC csRNAs and we identified a potential function where antisense blocking of FNDC3B and CTSS lowers monocyte attachment.

    Cover page: Understanding diverse RNA function using quantum dot based fluorescence in situ hybridization
    • Article
    • Peer Reviewed
    UC San Diego Previously Published Works (2018)
    It remains challenging to identify all parts of the nuclear genome that are in proximity to nuclear speckles, due to physical separation between the nuclear speckle cores and chromatin. We hypothesized that noncoding RNAs including small nuclear RNA (snRNAs) and Malat1, which accumulate at the periphery of nuclear speckles (nsaRNA [nuclear speckle-associated RNA]), may extend to sufficient proximity to the genome. Leveraging a transcriptome-genome interaction assay (mapping of RNA-genome interactions [MARGI]), we identified clusters of nsaRNA-interacting genomic sequences (nsaPeaks). Posttranscriptional pre-mRNAs, which also accumulate to nuclear speckles, exhibited proximity to nsaPeaks but rarely to other genomic regions. Our combined DNA fluorescence in situ hybridization and immunofluorescence analysis in 182 single cells revealed a 3-fold increase in odds for nuclear speckles to localize near an nsaPeak than its neighboring genomic sequence. These data suggest a model that nsaRNAs are located in sufficient proximity to the nuclear genome and leave identifiable genomic footprints, thus revealing the parts of genome proximal to nuclear speckles.
      Cover page: RNAs as Proximity-Labeling Media for Identifying Nuclear Speckle Positions Relative to the Genome
      • Article
      • Peer Reviewed
      UC San Diego Previously Published Works (2016)
      The pervasive transcription of our genome presents a possibility of revealing new genomic functions by investigating RNA interactions. Current methods for mapping RNA-RNA interactions have to rely on an 'anchor' protein or RNA and often require molecular perturbations. Here we present the MARIO (Mapping RNA interactome in vivo) technology to massively reveal RNA-RNA interactions from unperturbed cells. We mapped tens of thousands of endogenous RNA-RNA interactions from mouse embryonic stem cells and brain. We validated seven interactions by RNA antisense purification and one interaction using single-molecule RNA-FISH. The experimentally derived RNA interactome is a scale-free network, which is not expected from currently perceived promiscuity in RNA-RNA interactions. Base pairing is observed at the interacting regions between long RNAs, including transposon transcripts, suggesting a class of regulatory sequences acting in trans. In addition, MARIO data reveal thousands of intra-molecule interactions, providing in vivo data on high-order RNA structures.
        Cover page: Mapping RNA–RNA interactome and RNA structure in vivo by MARIO
        • Article
        • Peer Reviewed
        UC San Diego Previously Published Works (2019)
        Fusion transcripts are used as biomarkers in companion diagnoses. Although more than 15,000 fusion RNAs have been identified from diverse cancer types, few common features have been reported. Here, we compared 16,410 fusion transcripts detected in cancer (from a published cohort of 9,966 tumor samples of 33 cancer types) with genome-wide RNA-DNA interactions mapped in two normal, noncancerous cell types [using iMARGI, an enhanced version of the mapping of RNA-genome interactions (MARGI) assay]. Among the top 10 most significant RNA-DNA interactions in normal cells, 5 colocalized with the gene pairs that formed fusion RNAs in cancer. Furthermore, throughout the genome, the frequency of a gene pair to exhibit RNA-DNA interactions is positively correlated with the probability of this gene pair to present documented fusion transcripts in cancer. To test whether RNA-DNA interactions in normal cells are predictive of fusion RNAs, we analyzed these in a validation cohort of 96 lung cancer samples using RNA sequencing (RNA-seq). Thirty-seven of 42 fusion transcripts in the validation cohort were found to exhibit RNA-DNA interactions in normal cells. Finally, by combining RNA-seq, single-molecule RNA FISH, and DNA FISH, we detected a cancer sample with EML4-ALK fusion RNA without forming the EML4-ALK fusion gene. Collectively, these data suggest an RNA-poise model, where spatial proximity of RNA and DNA could poise for the creation of fusion transcripts.
          • Article
          • Peer Reviewed
          UC San Diego Previously Published Works (2020)

          Background

          Compared to proteins, glycans, and lipids, much less is known about RNAs on the cell surface. We develop a series of technologies to test for any nuclear-encoded RNAs that are stably attached to the cell surface and exposed to the extracellular space, hereafter called membrane-associated extracellular RNAs (maxRNAs).

          Results

          We develop a technique called Surface-seq to selectively sequence maxRNAs and validate two Surface-seq identified maxRNAs by RNA fluorescence in situ hybridization. To test for cell-type specificity of maxRNA, we use antisense oligos to hybridize to single-stranded transcripts exposed on the surface of human peripheral blood mononuclear cells (PBMCs). Combining this strategy with imaging flow cytometry, single-cell RNA sequencing, and maxRNA sequencing, we identify monocytes as the major type of maxRNA+ PBMCs and prioritize 11 candidate maxRNAs for functional tests. Extracellular application of antisense oligos of FNDC3B and CTSS transcripts inhibits monocyte adhesion to vascular endothelial cells.

          Conclusions

          Collectively, these data highlight maxRNAs as functional components of the cell surface, suggesting an expanded role for RNA in cell-cell and cell-environment interactions.
            Cover page: Natural display of nuclear-encoded RNA on the cell surface and its impact on cell interaction
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