A Generalizable Strategy for Imaging pre-mRNA Levels in Living Subjects Using Spliceosome-Mediated RNA Trans-Splicing (original) (raw)
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Molecular imaging of gene expression in living subjects by spliceosome-mediated RNA trans-splicing
Spliceosome-mediated RNA trans-splicing (SMaRT) provides an effective means to reprogram mRNAs and the proteins they encode. SMaRT technology has a broad range of applications, including RNA repair and molecular imaging, each governed by the nature of the sequences delivered by the pre-trans-splicing molecule. Here, we show the ability of SMaRT to optically image the expression of an exogenous gene at the level of pre-mRNA splicing in cells and living animals. Because of the modular design of pre-trans-splicing molecules, there is great potential to employ SMaRT to image the expression of any arbitrary gene of interest in living subjects. In this report, we describe a model system that demonstrates the feasibility of imaging gene expression by trans-splicing in small animals. This represents a previously undescribed approach to molecular imaging of mRNA levels in living subjects. mRNA repair gene correction reporter
Pharmacology Research & Perspectives, 2015
The spliceosome has recently emerged as a new target for cancer chemotherapy and novel antitumor spliceosome targeted agents are under development. Here, we describe two types of novel pharmacodynamic assays that facilitate drug discovery and development of this intriguing class of innovative therapeutics; the first assay is useful for preclinical optimization of small-molecule agents that target the SF3B1 spliceosomal protein in animals, the second assay is an ex vivo validated, gel-based assay for the measurement of drug exposure in human leukocytes. The first assay utilizes a highly specific bioluminescent splicing reporter, based on the skipping of exons 4-11 of a Luc-MDM2 construct, which specifically yields active luciferase when treated with small-molecule spliceosome modulators. We demonstrate that this reporter can be used to monitor alternative splicing in whole cells in vitro. We describe here that cell lines carrying the reporter can be used in vivo for the efficient pharmacodynamic analysis of agents during drug optimization and development. We also demonstrate dose-and time-dependent on-target activity of sudemycin D6 (SD6), which leads to dramatic tumor regression. The second assay relies on the treatment of freshly drawn human blood with SD6 ex vivo treatment. Changes in alternative splicing are determined by RT-PCR using genes previously identified in in vitro experiments. The Luc-MDM2 alternative splicing bioluminescent reporter and the splicing changes observed in human leukocytes should allow for the more facile translation of novel splicing modulators into clinical application.
Intracellular Imaging with Genetically Encoded RNA-based Molecular Sensors
Nanomaterials, 2019
Genetically encodable sensors have been widely used in the detection of intracellular molecules ranging from metal ions and metabolites to nucleic acids and proteins. These biosensors are capable of monitoring in real-time the cellular levels, locations, and cell-to-cell variations of the target compounds in living systems. Traditionally, the majority of these sensors have been developed based on fluorescent proteins. As an exciting alternative, genetically encoded RNA-based molecular sensors (GERMS) have emerged over the past few years for the intracellular imaging and detection of various biological targets. In view of their ability for the general detection of a wide range of target analytes, and the modular and simple design principle, GERMS are becoming a popular choice for intracellular analysis. In this review, we summarize different design principles of GERMS based on various RNA recognition modules, transducer modules, and reporting systems. Some recent advances in the appl...
Single molecule–sensitive probes for imaging RNA in live cells
Nature Methods, 2009
To visualize native or non-engineered RNA in live cells with single-molecule sensitivity, we developed multiply labeled tetravalent RNA imaging probes (MTRIPs). When delivered with streptolysin O into living human epithelial cancer cells and primary chicken fibroblasts, MTRIPs allowed the accurate imaging of native mRNAs and a non-engineered viral RNA, of RNA co-localization with known RNA-binding proteins, and of RNA dynamics and interactions with stress granules.
A High-Throughput Assay to Identify Small-Molecule Modulators of Alternative Pre-mRNA Splicing
Journal of Biomolecular Screening, 2013
Alternative splicing (AS) is an efficient mechanism that involves the generation of transcriptome and protein diversity from a single gene. Defects in pre-messenger RNA (mRNA) splicing are an important cause of numerous diseases, including cancer. AS of pre-mRNA as a target for cancer therapy has not been well studied. We have reported previously that a splicing factor, polypyrimidine tract-binding protein (PTB), is overexpressed in ovarian tumors compared with matched normal controls, and knockdown of PTB expression by short-hairpin RNA impairs ovarian tumor cell growth, colony formation, and invasiveness. Given the complexity of PTB's molecular functions, a chemical method for controlling PTB activity might provide a therapeutic and experimental tool. However, no commercially available PTB inhibitors have yet been described. To expand our ability to find novel inhibitors, we developed a robust, fluorometric, cell-based high-throughput screening assay in 96-well plates that reports on the splicing activity of PTB. In an attempt to use the cells for large-scale chemical screens to identify PTB modulators, we established cell lines stably expressing the reporter gene. Our results suggest that this highthroughput assay could be used to identify small-molecule modulators of PTB activity. Based on these findings and the role that upregulated PTB has on cell proliferation and malignant properties of tumors, targeting PTB for inhibition with small molecules offers a promising strategy for cancer therapy.
Journal of Biological Chemistry, 2003
Alternative pre-messenger RNA splicing is a major contributor to proteomic diversity in higher eukaryotes and represents a key step in the control of protein function in a large variety of biological systems. As a means of artificially altering splice site choice, we have investigated the impact of positioning proteins in the vicinity of 5 splice sites. We find that a recombinant GST-MS2 protein interferes with 5 splice site use, most efficiently when it binds upstream of that site. To broaden the use of proteins as steric inhibitors of splicing, we have tested the activity of antisense oligonucleotides carrying binding sites for the heterogeneous nuclear ribonucleoprotein A1/A2 proteins. In a HeLa cell extract, tailed oligonucleotides complementary to exonic sequences elicit strong shifts in 5 splice site selection. In four different human cell lines, an interfering oligonucleotide carrying A1/A2 binding sites also shifted the alternative splicing of the Bcl-x pre-mRNA more efficiently than oligonucleotides acting through duplex formation only. The use of protein-binding oligonucleotides that interfere with U1 small nuclear ribonucleoprotein binding therefore represents a novel and powerful approach to control splice site selection in cells.
Single-Molecule Imaging of Transcriptionally Coupled and Uncoupled Splicing
Cell
It is believed that the introns are removed from pre-mRNAs during transcription, while the pre-mRNA is still tethered to the gene locus via RNA polymerase. However, during alternative splicing it is important that splicing be deferred until all of the exons and introns involved in the choice have been synthesized. We have developed an in situ RNA imaging method with singlemolecule sensitivity to define the intracellular sites of splicing. Using this approach, we found that the normally tight coupling between transcription and splicing is broken in situations where the intron's polypyrimidine tract is sequestered within strong secondary structures. We also found that in two cases of alternative splicing, in which certain exons are skipped due to the activity of the RNA binding proteins Sxl and PTB, splicing is uncoupled from transcription. This uncoupling occurs only on the perturbed introns, while the preceding and succeeding introns are removed cotranscriptionally.
Nanostructured Probes for In Vivo Gene Detection
Online, 2010
The ability to visualize in real-time the expression dynamics and localization of specific RNAs in vivo offers tremendous opportunities for biological and disease studies including cancer detection. However, quantitative methods such as real-time PCR and DNA microarrays rely on the use of cell lysates thus not able to obtain important spatial and temporal information. Fluorescence proteins and other reporter systems cannot image endogenous RNA in living cells. Fluorescence in situ hybridization (FISH) assays require washing to achieve specificity, therefore can only be used with fixed cells. Here we review the recent development of nanostructured probes for living cell RNA detection, and discuss the biological and engineering issues and challenges of quantifying gene expression in vivo. In particular, we describe methods that use oligonucleotide probes, combined with novel delivery strategies, to image the relative level, localization and dynamics of RNA in live cells. Examples of detecting endogenous mRNAs, as well as imaging their subcellular localization are given to illustrate the biological applications, and issues in probe design, delivery and target accessibility are discussed. The nanostructured probes promise to open new and exciting opportunities in sensitive gene detection for a wide range of biological and medical applications.