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. 2013 Jan 9;41(4):2659–2672. doi: 10.1093/nar/gks1362

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© The Author(s) 2013. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 3. — Rbm20 aggregated on newly synthesized titin mRNAs forms Rbm20 speckles and represses the splicing of titin mRNA. (A) Immunofluorescence shows two Rbm20 speckles in the nucleus of cardiomyocytes from LV. The size of Rbm20 speckles was similar to nucleoli; Rbm20 is stained red, and nucleoli are stained green with antibody against fibrillarin. 4′,6 diamidino-2-phenylindole (DAPI)-stained nuclei are blue. Black scale bar: 2 microns. (B) Immunostaining of Rbm20 protein combined with titin mRNA FISH shows the co-localization of titin mRNA with Rbm20 speckles. Rbm20 is stained red; titin mRNA is stained green; and the overlap of red and green yields yellow. (C) Rbm20 speckles are transcription dependent. When the transcription of HL-1 cell is inhibited, Rbm20 speckles (red) dissociate. The behavior of paraspeckles (PSPC1) and splicing speckles (SC35) verified that transcription in the specific cell is inhibited. After transcription inhibition, crescent-shaped paraspeckles were formed, and the splicing speckles (green) become concentrated. Rbm20 is stained red; PSPC1 and SC35, marker proteins for paraspeckles and splicing speckles, respectively, are stained green. (D) Rbm20 can be specifically immunoprecipitated by anti-Rbm20 but not by control antibody. RIP: with anti-Rbm20, CTL: with control antibody (anti-spectrin α). (E) When using RT-PCR to amplify the total titin mRNA (TTL) from LV and the titin mRNA co-immunoprecipitated with Rbm20 (RIP), for the total titin mRNA, both retained introns (upper band in each primer pair) and constitutive splicing pattern (lower band) can be detected. However, for the Rbm20-immunoprecipitated titin mRNA, only the retained intron pattern can be clearly detected, meaning the constitutively spliced titin mRNA can barely be precipitated by Rbm20. (F) The co-existence of retained intron and constitutive splicing patterns on the same titin message were found in the Wt. The splicing on introns before exon 70 remains inhibited when the splicing on the downstream introns was finished. This situation does not occur in the Hm.