Molecular mechanism of long non-coding RNAs during cardiomyocyte differentiation
Bioinformatics and Theoretical Biology
Final Report Abstract
Cardiovascular disease remains the number one cause of death and disability in all Western countries, including Germany. In addition, the economic and societal burden of cardiovascular disease is enormous. In view of these facts, intensive research has been conducted to identify the causes of cardiovascular disease and to cure these ailments. Although no treatment so far is available that can completely eliminate cardiovascular disease, mounting evidence suggests that regenerating the failing heart via the activation of resident adult stem cells might be key to reverse the adverse effects of heart failure produced by various cardiac disorders. In this project, we identified and characterized long non-coding RNAs (lncRNAs) that interact with epigenetic and transcription factors to control the expressions of various genes. In this project, we uncovered that a novel lncRNA AK020709 binds to the basic building block of chromatin called "histone H3" to control the stemness of mouse embryonic stem (ES) cells as its deletion led to cell death. Although we were able to knockdown AK020709 with LNA GapmeR and siRNA, the amount of knockdown cannot be kept for several days, which impaired the further functional study during the differentiation of ES cells into cardiomyocytes. In addition, we were unable to derive homozygous deletion clone of AK020709 using CRISPR/Cas9 system, which suggests the critical function of this lncRNA. Currently, we are confirming this finding with further experiments. By studying another lncRNA "Myolinc", we demonstrate that: (A) It is expressed highly in the heart and skeletal muscle; (B) It is preferentially localized in the nucleus of mouse myoblast cell line C2C12; (C) Its silencing and deletion in C2C12 cells cause impair differentiation into myotubes; and (D) It binds to the DNA/RNA-binding protein TDP-43 to control transcription of muscle marker genes. The results of this lncRNA have been submitted to eLife and being revised. Currently, we are pursuing to understand the function of Myolinc in the heart. Our preliminary results of Myolinc demonstrate that: (A) It is preferentially localized in the nucleus of mouse cardiomyocytic cell line HL-1; (B) Its silencing in HL-1 cells causes various transcriptional changes, especially for those genes involved in cellular morphology and metabolism; (C) Its expression is down-regulated in infarcted hearts compared to sham-operated ones, especially in cardiomyocytes; and (D) It binds to the DNA/RNA-binding protein TDP-43, which has so far not implicated in cardiovascular biology. Given the above preliminary results, Myolinc might function as molecular scaffold to recruit TDP-43 for the progress of cardiovascular disease, which may be a way to intervene this devastating disease if the mechanism focusing on Myolinc-TDP-43 complex is understood. Lastly, "Airn" (antisense Igf2r RNA, also known as "Air") is an imprinted gene transcribed from the paternal chromosome. Our preliminary results of Airn demonstrate that: (A) It is highly expressed in the heart; (B) It is preferentially expressed in the nucleus of HL-1 cells; (C) Its silencing in HL-1 cells causes increased cell death, vulnerability to cellular stress, and reduced cell migration; (D) It binds to the RNA-binding protein Igf2bp2 and the DNA-binding protein Rpa1; and (E) Its silencing causes less binding of Igf2bp2 to mRNAs. Taken all together, in this project, we functionally characterized three lncRNAs. Using bioinformatics methods, we identified mouse lncRNAs that have potential homologs in humans. These human lncRNAs may have the same functions as those mouse lncRNAs that are characterized in this study. If so, these lncRNAs have potential usage as RNA molecules to activate/inhibit a particular signaling pathway, which might be important for the function of heart, especially in cardiomyocytes, as well as might be involved in the differentiation of cardiac adult stem cells into cardiomyocytes. If we were able to show such mechanism in humans, it will be possible to use these lncRNAs for "RNA therapeutics" for cardiovascular disease.
Publications
- C-It-Loci: a knowledge database for tissueenriched loci. Bioinformatics. 2015 Nov 1;31(21):3537-43
Weirick T, John D, Dimmeler S, Uchida S
(See online at https://doi.org/10.1093/bioinformatics/btv410) - Exercise controls non-coding RNAs. Cell Metab. 2015 Apr 7;21(4):511-2
Uchida S, Dimmeler S
(See online at https://dx.doi.org/10.1016/j.cmet.2015.03.014) - The identification and characterization of novel transcripts from RNA-seq data. Brief Bioinform. 2016 Jul;17(4):678-85
Weirick T, Militello G, Müller R, John D, Dimmeler S, Uchida S
(See online at https://doi.org/10.1093/bib/bbv067)
