Phosphorylation of the RSRSP stretch is critical for splicing regulation by RNA-Binding Motif Protein 20 (RBM20) through nuclear localization

RBM20 is a major regulator of heart-specific alternative pre-mRNA splicing of TTN encoding a giant sarcomeric protein titin. Mutation in RBM20 is linked to autosomal-dominant familial dilated cardiomyopathy (DCM), yet most of the RBM20 missense mutations in familial and sporadic cases were mapped to an RSRSP stretch in an arginine/serine-rich region of which function remains unknown. In the present study, we identified an R634W missense mutation within the stretch and a G1031X nonsense mutation in cohorts of DCM patients. We demonstrate that the two serine residues in the RSRSP stretch are constitutively phosphorylated and mutations in the stretch disturb nuclear localization of RBM20. Rbm20 S637A knock-in mouse mimicking an S635A mutation reported in a familial case showed a remarkable effect on titin isoform expression like in a patient carrying the mutation. These results revealed the function of the RSRSP stretch as a critical part of a nuclear localization signal and offer the Rbm20 S637A mouse as a good model for in vivo study

A missense mutation in the RSRSP stretch of Rbm20 causes dilated cardiomyopathy and atrial fibrillation in mice

Dilated cardiomyopathy (DCM) is a fatal heart disease characterized by left ventricular dilatation and cardiac dysfunction. Recent genetic studies on DCM have identified causative mutations in over 60 genes, including RBM20, which encodes a regulator of heart-specific splicing. DCM patients with RBM20 mutations have been reported to present with more severe cardiac phenotypes, including impaired cardiac function, atrial fibrillation (AF), and ventricular arrhythmias leading to sudden cardiac death, compared to those with mutations in the other genes. An RSRSP stretch of RBM20, a hotspot of missense mutations found in patients with idiopathic DCM, functions as a crucial part of its nuclear localization signals. However, the relationship between mutations in the RSRSP stretch and cardiac phenotypes has never been assessed in an animal model. Here, we show that Rbm20 mutant mice harboring a missense mutation S637A in the RSRSP stretch, mimicking that in a DCM patient, demonstrated severe cardiac dysfunction and spontaneous AF and ventricular arrhythmias mimicking the clinical state in patients. In contrast, Rbm20 mutant mice with frame-shifting deletion demonstrated less severe phenotypes, although loss of RBM20-dependent alternative splicing was indistinguishable. RBM20^ protein cannot be localized to the nuclear speckles, but accumulated in cytoplasmic, perinuclear granule-like structures in cardiomyocytes, which might contribute to the more severe cardiac phenotypes

Muscle-Specific Splicing Factors ASD-2 and SUP-12 Cooperatively Switch Alternative Pre-mRNA Processing Patterns of the ADF/Cofilin Gene in <em>Caenorhabditis elegans</em>

<div><p>Pre–mRNAs are often processed in complex patterns in tissue-specific manners to produce a variety of protein isoforms from single genes. However, mechanisms orchestrating the processing of the entire transcript are not well understood. Muscle-specific alternative pre–mRNA processing of the <em>unc-60</em> gene in <em>Caenorhabditis elegans</em>, encoding two tissue-specific isoforms of ADF/cofilin with distinct biochemical properties in regulating actin organization, provides an excellent <em>in vivo</em> model of complex and tissue-specific pre–mRNA processing; it consists of a single first exon and two separate series of downstream exons. Here we visualize the complex muscle-specific processing pattern of the <em>unc-60</em> pre–mRNA with asymmetric fluorescence reporter minigenes. By disrupting juxtaposed CUAAC repeats and UGUGUG stretch in intron 1A, we demonstrate that these elements are required for retaining intron 1A, as well as for switching the processing patterns of the entire pre–mRNA from non-muscle-type to muscle-type. Mutations in genes encoding muscle-specific RNA–binding proteins ASD-2 and SUP-12 turned the colour of the <em>unc-60</em> reporter worms. ASD-2 and SUP-12 proteins specifically and cooperatively bind to CUAAC repeats and UGUGUG stretch in intron 1A, respectively, to form a ternary complex <em>in vitro</em>. Immunohistochemical staining and RT–PCR analyses demonstrate that ASD-2 and SUP-12 are also required for switching the processing patterns of the endogenous <em>unc-60</em> pre-mRNA from UNC-60A to UNC-60B in muscles. Furthermore, systematic analyses of partially spliced RNAs reveal the actual orders of intron removal for distinct mRNA isoforms. Taken together, our results demonstrate that muscle-specific splicing factors ASD-2 and SUP-12 cooperatively promote muscle-specific processing of the <em>unc-60</em> gene, and provide insight into the mechanisms of complex pre-mRNA processing; combinatorial regulation of a single splice site by two tissue-specific splicing regulators determines the binary fate of the entire transcript.</p> </div

Schematic illustrations of the tissue-specific alternative processing of the <i>unc-60</i> pre-mRNA during the course of transcription.

<p>(<i>A</i>) A model of UNC-60A mRNA processing in non-muscle tissues. (<i>B</i>) A model of UNC-60B mRNA processing in muscles. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002991#s3" target="_blank">Discussion</a> for detail.</p

ASD-2 and SUP-12 regulate muscle-specific processing of the <i>unc-60</i> reporter in body wall muscles.

<p>(<i>A</i>) A micrograph of the <i>unc-60</i> reporter worms carrying the integrated allele <i>ybIs1831</i> in the wild-type (left) and <i>sup-12 (yb1253)</i> (right) backgrounds. (<i>B</i>) Schematic structure of the <i>asd-2</i> gene. The position of a nonsense mutation in <i>yb1540</i> is indicated. A region encoding STAR domain is coloured. (<i>C</i>) A micrograph of wild-type, <i>asd-2(yb1540)</i> mutant and <i>asd-2(RNAi)</i> worms carrying <i>ybIs1831</i>. (<i>D</i>) A micrograph of <i>asd-2(yb1540)</i> and <i>asd-2(yb1540); ybEx2266 [myo-2::mRFP myo-3::ASD-2b]</i> worms carrying <i>ybIs1831</i>. Arrowheads indicate RFP expression in pharynx as a marker for transgenesis. Scale bars in (<i>A</i>), (<i>C</i>) and (<i>D</i>), 50 µm. (<i>E</i>, <i>F</i>) Microphotographs of N2 (<i>E</i>) and <i>asd-2(yb1540)</i> (<i>F</i>) worms stained with anti-ASD-2b (ASD-2) and Hoechst 33258 (DNA). High-magnification and merged images are also indicated for N2 in bottom panels of (<i>E</i>). Arrowheads indicate nuclei of some of body wall muscle cells. Scale bars in (<i>E</i>) top panels and (<i>F</i>), 100 µm; in (<i>E</i>) bottom panels, 10 µm. (<i>G</i>) Western blotting with anti-ASD-2b. Lysates from synchronized L1 larvae of N2 (lane 1) and <i>asd-2(yb1540)</i> mutant (lane 2) were subjected to Western blotting with anti-ASD-2b (top) and anti-actin (bottom). (<i>H</i>) RT-PCR analysis of mRNAs derived from <i>ybIs1831</i> in the wild-type (lane 1), <i>asd-2 (yb1540)</i> (lane 2) and <i>sup-12 (yb1253)</i> (lane 3) backgrounds. RT-PCR products derived from <i>unc-60E1-E2A-RFP</i> (top) and <i>unc-60E1-E3B-GFP</i> (middle) and total RNAs (bottom) are shown. Splicing patterns of the mRNAs are schematically shown on the right. Triangles indicate positions and directions of the primers.</p

Visualization of tissue-specific alternative processing patterns of the <i>unc-60</i> transcript.

<p>(<i>A</i>) Schematic structure of the <i>unc-60</i> gene. Numbered boxes indicate exons. Predicted open reading frames (ORFs) are coloured in white and untranslated regions (UTRs) are in gray. The deleted region in <i>unc-60 (su158)</i> is indicated. (<i>B</i>) Schematic illustration of a pair of <i>unc-60</i> reporter minigenes, <i>unc-60E1-E2A-RFP</i> and <i>unc-60E1-E3B-GFP</i>, and UNC-60A- and UNC-60B-type mRNAs derived from them. cDNA cassettes and predicted ORFs for RFP and GFP are coloured in magenta and green, respectively. Triangles indicate positions and directions of primers used to check splicing patterns of mRNAs derived from the minigenes by RT-PCR. (<i>C</i> and <i>D</i>) Confocal images of transgenic <i>unc-60</i> reporter worms <i>ybEx1812 [unc-51::unc-60E1-E2A-RFP unc-51::unc-60E1-E3B-GFP]</i>. UNC-60A-RFP (left), UNC-60B-GFP (middle) and merged images (right) of an adult worm (<i>C</i>) and a head region at higher magnification (<i>D</i>). Anterior is to the left and dorsal is to the top. bwm, body wall muscles; int, intestine; N, neurons in head ganglia; pm, pharyngeal muscles; vnc, ventral nerve cord. Scale bars, 50 µm. (<i>E</i>) Confocal images of a transgenic <i>unc-60</i> reporter worm <i>ybIs1831 [myo-3::unc-60E1-E2A-RFP myo-3::unc-60E1-E3B-GFP]</i> shown as in (<i>C</i>) and (<i>D</i>).</p

SUP-12 represses excision of intron 1A from the endogenous <i>unc-60</i> transcript.

<p>(<i>A</i>–<i>D</i>) RT-PCR analyses of mature mRNAs (<i>A</i>) and partially spliced RNAs (<i>B</i>–<i>D</i>) from the endogenous <i>unc-60</i> gene. Total RNAs from synchronized L1 larvae of N2 (lanes 1 and 3) and <i>sup-12 (yb1253)</i> mutant (lanes 2 and 4) were subjected to RT-PCR without (lanes 1 and 2) or with (lanes 3 and 4) reverse transcriptase (RT). Positions of the primers are indicated on the left. Each band is numbered in the order of size. Schematic structures of the RNAs are indicated on the right. Black and blue triangles indicate positions and directions of exonic and intronic primers, respectively. Asterisks denote artificially amplified fragments.</p

ASD-2 regulates alternative pre-mRNA processing of the endogenous <i>unc-60</i> gene in body wall muscles.

<p>(<i>A</i>, <i>B</i>) Immunofluorescence images of UNC-60A (left) and MyoA (middle) and merged images (right) of wild-type (<i>A</i>) and <i>asd-2(RNAi)</i> (<i>B</i>) worms. MyoA, a heavy chain of muscle-specific myosin, is a marker for body wall muscles (encircled with dotted lines). Scale bar, 20 µm. (<i>C</i>–<i>F</i>) Micrographs of worms on bacterial lawns (left) and actin filaments in body wall muscles stained with tetramethylrhodamine-phalloidin (right) of N2 (<i>C</i>), <i>unc-60 (su158)</i> (<i>D</i>), <i>asd-2 (yb1540); unc-60 (su158)</i> (<i>E</i>) and <i>asd-2 (yb1540); unc-60 (su158); asd-2 (RNAi)</i> (<i>F</i>). Scale bars, 1 mm in left panels and 20 µm in right panels. (<i>G</i>, <i>H</i>) Micrographs of <i>unc-60 (su158); ybEx2149 [myo-3::UNC-60A]</i> (<i>G</i>) and <i>unc-60 (su158); ybEx2148 [myo-3::UNC-60B]</i> (<i>H</i>) worms on bacterial lawns. Scale bar, 1 mm.</p