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Fig. 3 | Genome Biology

Fig. 3

From: Intronic tRNAs of mitochondrial origin regulate constitutive and alternative splicing

Fig. 3

NimtRNAs increase host gene mRNA abundance by increasing splicing efficiency. a Black boxes represent the single letter amino acid code of the respective nimtRNA: one nimtRNA (Y), two (YC), three (YCN), four (YCNA), or five (YCNAW) nimtRNAs from the mouse Myo3a intron 30 were cloned into the intronic region of the Low0-eGFP splicing reporter, exhibiting an efficient 5′-splice site. NimtRNA genes were inserted as indicated by triangles. b Northern blot analysis of eGFP mRNA constructs as depicted in a performed with digoxigenin (DIG)-labeled probes. Normalization of RT-qPCR and northern blot analyses was performed to Dsred2Express transfection control mRNA. Experiments were performed in triplicates. c RT-qPCR analysis of constructs as depicted in a was performed with the forward primer binding to exon 1 and the reverse primer binding to exon 2, depicted by black arrows shown in a. d Five nuclear tRNA genes (designated as nucl. YCNAW) were inserted into the Low0-eGFP splicing reporter and their influence on splicing was compared to the reporter containing five nimtRNAs (YCNAW). The Low0-eGFP splicing reporter was taken as a reference. e Stable cell lines were generated containing a single copy of the Low0-eGFP splicing reporter and either five nimtRNAs or five nuclear tRNAs. Normalization was performed to β-Actin. The stably integrated Low0-eGFP splicing reporter was taken as reference. f mRNA, pre-mRNA, and total transcript (mRNA+pre-mRNA) levels from cells transfected with the respective constructs were assessed by RT-qPCR. Error bars represent the standard deviation from the mean of three independent experiments. g The Low2-eGFP splicing reporter differs from the Low0-eGFP splicing reporter by possessing a less efficient 5′-splice site as indicated. Averages and standard deviations were determined from three independent sets of experiments. Error bars represent the SD and *P < 0.05; ***P < 0.001; ****P < 0.0001 (ANOVA)

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