Identification of transcripts regulated by CUG-BP, Elav-like family member 1 (CELF1) in primary embryonic cardiomyocytes by RNA-seq

CUG-BP, Elav-like family member 1 (CELF1) is a multi-functional RNA binding protein that regulates pre-mRNA alternative splicing in the nucleus, as well as polyadenylation status, mRNA stability, and translation in the cytoplasm [1]. Dysregulation of CELF1 has been implicated in cardiomyopathies in myotonic dystrophy type 1 and diabetes [2–5], but the targets of CELF1 regulation in the heart have not been systematically investigated. We previously demonstrated that in the developing heart CELF1 expression is restricted to the myocardium and peaks during embryogenesis [6–8]. To identify transcripts regulated by CELF1 in the embryonic myocardium, RNA-seq was used to compare the transcriptome of primary embryonic cardiomyocytes following siRNA-mediated knockdown of CELF1 to that of controls. Raw data files of the RNA-seq reads have been deposited in NCBI's Gene Expression Omnibus [9] under the GEO Series accession number GSE67360. These data can be used to identify transcripts whose levels or alternative processing (i.e., alternative splicing or polyadenylation site usage) are regulated by CELF1, and should provide insight into the pathways and processes modulated by this important RNA binding protein during normal heart development and during cardiac pathogenesis

Identification of Targets of CUG-BP, Elav-Like Family Member 1 (CELF1) Regulation in Embryonic Heart Muscle

<div><p>CUG-BP, Elav-like family member 1 (CELF1) is a highly conserved RNA binding protein that regulates pre-mRNA alternative splicing, polyadenylation, mRNA stability, and translation. In the heart, CELF1 is expressed in the myocardium, where its levels are tightly regulated during development. CELF1 levels peak in the heart during embryogenesis, and aberrant up-regulation of CELF1 in the adult heart has been implicated in cardiac pathogenesis in myotonic dystrophy type 1, as well as in diabetic cardiomyopathy. Either inhibition of CELF activity or over-expression of CELF1 in heart muscle causes cardiomyopathy in transgenic mice. Nonetheless, many of the cardiac targets of CELF1 regulation remain unknown. In this study, to identify cardiac targets of CELF1 we performed cross-linking immunoprecipitation (CLIP) for CELF1 from embryonic day 8 chicken hearts. We identified a previously unannotated exon in <i>MYH7B</i> as a novel target of CELF1-mediated regulation. We demonstrated that knockdown of CELF1 in primary chicken embryonic cardiomyocytes leads to increased inclusion of this exon and decreased <i>MYH7B</i> levels. We also investigated global changes in the transcriptome of primary embryonic cardiomyocytes following CELF1 knockdown in a published RNA-seq dataset. Pathway and network analyses identified strong associations between CELF1 and regulation of cell cycle and translation. Important regulatory proteins, including both RNA binding proteins and a cardiac transcription factor, were affected by loss of CELF1. Together, these data suggest that CELF1 is a key regulator of cardiomyocyte gene expression.</p></div

CELF1 CLIP tags are enriched with UG motifs.

<p>(A) Incidence of dinucleotides within CELF1 CLIpP tags that map to known genes. The dotted green line indicates the incidence expected if all dinucleotides were equally represented. (B) Incidence of hexanucleotides within CELF1 CLIP tags that map to known genes. The 20 most-frequent hexanucleotides are indicated in red. The dotted green line indicates the incidence expected if all hexanucleotides were equally represented. The sequences of the top hexanucleotides are shown, with UG dinucleotides within those motifs in red. (C) The distributions of tags containing different numbers of UG or CA dinucleotides are shown.</p

The EIF2 signaling pathway is strongly inhibited following knockdown of CELF1.

<p>The EIF2 signaling pathway was identified as one of the top canonical pathways affected by CELF1 knockdown using Ingenuity Pathway Analysis software. This pathway is shown with a molecule activity predictor (MAP) overlay from differential gene expression analysis of the RNA-seq dataset. Green indicates reduced expression in si2-transfected versus mock-transfected cardiomyocytes, whereas red/pink would indicate increased expression. Blue indicates predicted inhibition, whereas orange indicates predicted activation. The degree of saturation indicates the level of observed change or predicted effect. The 40S and 60S ribosome subunits (highlighted in magenta) have been expanded to show individual members of the complexes.</p

CELF1 regulates the inclusion of an unannotated exon in chicken <i>MYH7B</i> transcripts.

<p>(A) A CELF1 CLIP tag (green) maps to an intron within the coding region of <i>MYH7B</i>. RT-PCR using primers in upstream and downstream exons (indicated by half arrows) revealed the presence of a previously unrecognized exon (blue box) that is alternatively included in the embryonic heart. (B) The extent of inclusion of the novel <i>MYH7B</i> alternative exon was determined by semi-quantitative RT-PCR in primary embryonic cardiomyocytes transfected with or without control (siCont) or anti-CELF1 (si1 and si2) siRNAs. Data represent mean values from three independent transfections. A representative autoradiogram from one of the transfection sets is shown; an empty lane between the mock and si1 sample has been excised. (C) Translation of the transcript sequence including this exon indicates that its inclusion would lead to the insertion of an in-frame stop codon close to the N-terminal end of the protein. (D) Total <i>MYH7B</i> transcript levels in primary cardiomyocytes transfected with control (siCont) or anti-CELF1 (si2) siRNA were compared to mock-transfected controls by qRT-PCR. An asterisk indicates P ≤ 0.05 compared to mock.</p

Top functions identified by Ingenuity Pathway Analysis software as affected by CELF1 knockdown.

<p>Top functions identified by Ingenuity Pathway Analysis software as affected by CELF1 knockdown.</p

Cross-linking immunoprecipitation (CLIP) of CELF1 from embryonic chicken heart.

<p>(A) CLIP was performed on embryonic day 8 chicken hearts using an anti-CELF1 antibody. Vertical red line indicates immunoprecipitated CELF1:RNA complexes following addition of an RNase inhibitor to block high levels of endogenous RNase activity (“low RNase” lanes); fully digested complexes (“high RNase”) run just above the size of immunoprecipitated CELF1 alone (Western blot). (B) Distribution of CLIP tags within known genes.</p

RT-PCR primer sequences used in this study.

<p>RT-PCR primer sequences used in this study.</p

The transcriptome is substantially altered following CELF1 knockdown.

<p>(A) RNA-seq analysis of primary embryonic chicken cardiomyocytes ± siRNA directed against CELF1 (si2) identified a large number of transcripts that were down-regulated (green) or up-regulated (red) following CELF1 knockdown, including 308 transcripts that were expressed only in mock-transfected cells and 1535 transcripts that were expressed only in cells depleted of CELF1. (B) The ten top canonical pathways affected in primary embryonic chicken cardiomyocytes following CELF1 knockdown were identified using IPA software. The intensity of the color of each bar reflects the absolute value of the z-score for that pathway; a negative z-score indicates that a pathway is predicted to be inactivated. The ratio indicates the fraction of molecules in a pathway that are significantly different following CELF1 knockdown.</p