A universal approach to investigate circRNA protein coding function.
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ABSTRACT:
SUBMITTER: Mo D
PROVIDER: S-EPMC6690939 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Protein-encoding circular RNAs (circRNAs) are newly identified RNA molecules characterized by intense interaction with translating ribosome. Emerging evidence has implicated physiological and pathological significance of these non-canonical RNAs, yet a large body of them remains unidentified. Due to limited tools at hand, we developed CircProPlus, an automated computational pipeline for de novo detection of translated circRNAs. In comparison to previously established CircPro, CircProPlus adjusts the overall workflow and integrates more robust implements for achieving easier accessibility, higher flexibility and productivity. In present study, we tested the performance of CircProPlus when using different circRNA-detecting implements (i.e., CIRI2, CirComPara2) in the evaluation of coding ability of circRNAs. Results showed that CirComPara2, a state-of-the-art algorithm, consistently outperformed CIRI2 when coupled with CircProPlus in testing real data collected from different RNA libraries and species, which highlighted its potency in data mining of circRNAs with protein-coding potential.
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Project description:Circular RNAs (circRNAs), which play vital roles in many regulatory pathways, are widespread in many species. Although many circRNAs have been discovered in plants and animals, the functions of these RNAs have not been fully investigated. In addition to the function of circRNAs as microRNA (miRNA) decoys, the translation potential of circRNAs is important for the study of their functions; yet, few tools are available to identify their translation potential. With the development of high-throughput sequencing technology and the emergence of ribosome profiling technology, it is possible to identify the coding ability of circRNAs with high sensitivity. To evaluate the coding ability of circRNAs, we first developed the CircCode tool and then used CircCode to investigate the translation potential of circRNAs from humans and Arabidopsis thaliana. Based on the ribosome profile databases downloaded from NCBI, we found 3,610 and 1,569 translated circRNAs in humans and A. thaliana, respectively. Finally, we tested the performance of CircCode and found a low false discovery rate and high sensitivity for identifying circRNA coding ability. CircCode, a Python 3-based framework for identifying the coding ability of circRNAs, is also a simple and powerful command line-based tool. To investigate the translation potential of circRNAs, the user can simply fill in the given configuration file and run the Python 3 scripts. The tool is freely available at https://github.com/PSSUN/CircCode.
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Project description:We describe a transcriptional analysis platform consisting of a universal micro-array system (UMAS) combined with an enzymatic manipulation step that is capable of generating expression profiles from any organism without requiring a priori species-specific sequence information. The transcriptome is converted to cDNA and processed with restriction endonucleases to generate low-complexity pools (80–120) of equal length DNA fragments. The resulting material is amplified and detected with the UMAS system, comprising all possible 4,096 (4^6) DNA hexamers. Ligation to the arrays yields thousands of 14-mer sequence tags. The compendium of signals from all pools in the array-of-universal arrays comprises a full-transcriptome expression profile. The technology was validated by analysis of the galactose response of Saccharomyces cerevisiae, and the resulting profiles showed excellent agreement with the literature and real-time PCR assays. Keywords: other
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Project description:The extracellular microenvironment is an important regulator of cell functions. Numerous structural cues present in the cellular microenvironment, such as ligand distribution and substrate topography, have been shown to influence cell behavior. However, the roles of these cues are often studied individually using simplified, single-cue platforms that lack the complexity of the three-dimensional, multi-cue environment cells encounter in vivo. Developing ways to bridge this gap, while still allowing mechanistic investigation into the cellular response, represents a critical step to advance the field. Here, we present a new approach to address this need by combining optics-based protein patterning and lithography-based substrate microfabrication, which enables high-throughput investigation of complex cellular environments. Using a contactless and maskless UV-projection system, we created patterns of extracellular proteins (resembling contact-guidance cues) on a two-and-a-half-dimensional (2.5D) cell culture chip containing a library of well-defined microstructures (resembling topographical cues). As a first step, we optimized experimental parameters of the patterning protocol for the patterning of protein matrixes on planar and non-planar (2.5D cell culture chip) substrates and tested the technique with adherent cells (human bone marrow stromal cells). Next, we fine-tuned protein incubation conditions for two different vascular-derived human cell types (myofibroblasts and umbilical vein endothelial cells) and quantified the orientation response of these cells on the 2.5D, physiologically relevant multi-cue environments. On concave, patterned structures (curvatures between κ = 1/2500 and κ = 1/125 μm-1), both cell types predominantly oriented in the direction of the contact-guidance pattern. In contrast, for human myofibroblasts on micropatterned convex substrates with higher curvatures (κ ≥ 1/1000 μm-1), the majority of cells aligned along the longitudinal direction of the 2.5D features, indicating that these cells followed the structural cues from the substrate curvature instead. These findings exemplify the potential of this approach for systematic investigation of cellular responses to multiple microenvironmental cues.
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