RNA-Seq for the identification of novel Mediator transcripts in endothelial progenitor cells

Highlights

• Novel transcripts of MED genes in endothelial progenitors through in silico analysis

• MED9, MED14 and MED27 genes differ from the annotated transcripts for the 3′-UTR.

• The newly identified MED transcripts are predicted to encode novel protein isoforms.

Abstract

Mediator (MED) complex is a multiprotein playing a key role in the eukaryotic transcription. Alteration of MED function may have enormous pathophysiological consequences and several MED genes have been implicated in human diseases. Here, we have combined computational and experimental approaches to identify and characterize, new transcripts generated by alternative splicing (AS) for all MED genes, through the analysis of our recently published RNA-Sequencing datasets of endothelial progenitor cells (EPCs). This combined strategy allowed us to identify novel transcripts for MED4, MED9, MED11, MED14, MED27 and CDK8 most of them generated by AS. All the newly identified transcripts, except MED11, are predicted to encode novel protein isoforms. The identification of novel MED variants could lead to the finding of other MED complexes with different functions depending on their subunit composition. Finally, the expression profile of all MED genes, together with an extensive gene expression analysis, may be useful to better classify the diverse subsets of cell populations that contribute to neovascularization.

Introduction

Mediator (MED) complex functions as a key component of transcription machinery (Casamassimi and Napoli, 2007, Napoli et al., 2012). MED is a multiprotein known to play a fundamental role in the regulation of eukaryotic mRNA synthesis. Different MED complexes have been isolated by a variety of methods (Napoli et al., 2012) and they comprise about 25–30 subunits (MEDs). Biochemical and structural studies showed that MEDs are organized into four main modules referred to as the “head”, “middle”, “tail” and “kinase module”. The head domain accounts for the most extensive contacts with RNA Pol II, whereas the tail domain represents a platform for interactions with gene specific regulators (Napoli et al., 2012). Although MED complex was initially found to play a critical role in the regulation of transcription initiation, recent evidence has revealed multiple roles in nearly all stages of transcription, including initiation, promoter escape, elongation, pre-mRNA processing and termination (Conaway and Conaway, 2013, Napoli et al., 2012).

MED complex undergoes a conformational change upon interaction with RNA Pol II (Conaway and Conaway, 2013, Napoli et al., 2012). A minimal module serves as a core Mediator enabling the cell to custom-design MED complexes (Conaway and Conaway, 2013, Napoli et al., 2012). Each complex is then formed in response to the signals from transcription factors, which are recruited in response to the continuous changes of the cellular environment both in mature and developing cells. Because of the importance of MED role in the transcription of the eukaryotic genes, alteration of MED function may have enormous pathophysiological consequences. In our previous studies on endothelial progenitor cell (EPC) differentiation, we identified novel transcripts for three MED genes – MED30, MED12 and MED19 – expressed in circulating immature CD133⁺ progenitor cells. Interestingly, the expression of these isoforms generated by alternative splicing (AS) was decreased or absent in cultured CD133⁺ cells (Rienzo et al., 2010, Rienzo et al., 2012). It is currently demonstrated that about 95% of multi-exonic human genes undergo AS, and in line with this, in silico analysis of human MED genes has predicted the presence of alternative transcripts, suggesting that MED complex composition is not yet fully described (Rienzo et al., 2010, Rienzo et al., 2012). Therefore, AS is virtually involved in all cellular processes and, noteworthy, in self-renewal and pluripotency (Chepelev and Chen, 2013), suggesting that this mechanism could also be involved in EPC maintenance.

Thus, elucidating the different expression patterns of genes encoding for MED subunits is essential to fully exploit the composition of MED complex, and subsequently its functionality. Moreover, understanding whether – and in what tissues or cells – alternative isoforms of MED proteins are produced, as well as the timing and specificity of their expression, would surely have a broad impact in many research fields. In this scenario, RNA-Sequencing (RNA-Seq) on next-generation sequencing (NGS) platforms represents the most innovative and powerful tool to investigate gene expression and to simultaneously detect alternative splice isoforms for hundreds of genes (Costa et al., 2013, Costa et al., 2010).

In this study, we have combined computational and experimental approaches to identify, and further characterize, new transcripts generated by AS for all MED genes, through the analysis of our recently published RNA-Seq datasets of EPCs (Costa et al., 2011). Investigating the alternative splicing of genes is very important for understanding the complex regulatory system of gene expression in cell differentiation. NGS technology has provided new opportunities for these studies. The combination of these approaches revealed to be useful to identify, validate and then characterize, six novel transcripts for six different MED genes, most of them generated by alternative splicing.