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Novel transcription factor variants through RNA-sequencing: the importance of being "alternative".

Scarpato M, Federico A, Ciccodicola A, Costa V - Int J Mol Sci (2015)

Bottom Line: The highest rate of alternative splicing occurs in transcription factors encoding genes, mostly in Krüppel-associated box domains of zinc finger proteins.Through computational approaches we also predicted their novel structural and functional properties.Our findings indicate that alternative splicing is a major determinant of transcription factor diversity, confirming that accurate analysis of RNA-Sequencing data can reliably lead to the identification of novel transcripts, with potentially new functions.

View Article: PubMed Central - PubMed

Affiliation: Institute of Genetics and Biophysics "Adriano Buzzati-Traverso", National Research Council, 80131 Naples, Italy. margherita.scarpato@igb.cnr.it.

ABSTRACT
Alternative splicing is a pervasive mechanism of RNA maturation in higher eukaryotes, which increases proteomic diversity and biological complexity. It has a key regulatory role in several physiological and pathological states. The diffusion of Next Generation Sequencing, particularly of RNA-Sequencing, has exponentially empowered the identification of novel transcripts revealing that more than 95% of human genes undergo alternative splicing. The highest rate of alternative splicing occurs in transcription factors encoding genes, mostly in Krüppel-associated box domains of zinc finger proteins. Since these molecules are responsible for gene expression, alternative splicing is a crucial mechanism to "regulate the regulators". Indeed, different transcription factors isoforms may have different or even opposite functions. In this work, through a targeted re-analysis of our previously published RNA-Sequencing datasets, we identified nine novel transcripts in seven transcription factors genes. In silico analysis, combined with RT-PCR, cloning and Sanger sequencing, allowed us to experimentally validate these new variants. Through computational approaches we also predicted their novel structural and functional properties. Our findings indicate that alternative splicing is a major determinant of transcription factor diversity, confirming that accurate analysis of RNA-Sequencing data can reliably lead to the identification of novel transcripts, with potentially new functions.

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Computational and experimental workflow. Schematic overview of the in silico procedures used to infer the presence of new transcription factors (TFs) transcripts from the re-analysis of our RNA-Seq datasets. The experimental approach used to validate the presence of the new variants is also depicted.
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ijms-16-01755-f001: Computational and experimental workflow. Schematic overview of the in silico procedures used to infer the presence of new transcription factors (TFs) transcripts from the re-analysis of our RNA-Seq datasets. The experimental approach used to validate the presence of the new variants is also depicted.

Mentions: We used our published RNA-Seq data [16] to identify new transcripts that encode transcription factors (see workflow scheme in Figure 1). First, we found that 1043 (out of ~1500) genes annotated in the Transcription Factor Class (TFClass) database [18] are expressed according to our RNA-Seq data. Normalized gene expression data revealed that most of genes encoding TFs have medium to low expression (Figure S1). Targeted re-analysis (Section 4.1 and [17]) of AS in genes encoding TFs revealed the presence of new putative isoforms, mainly originated by cassette exon skipping. A complete—or even partial—overlap with transcripts annotated in AceView [19] and expressed sequence tags (ESTs; [20]) databases was used as the selection parameter. In contrast, splice junctions with less than two mapped reads/sample and those that overlap genomic repeats were not considered for further studies. The 50 bp single-end libraries of our previous experiment did not allow us to automatically reconstruct the entire exon/intron structure of the new transcripts, nor to measure their relative abundance [16]. Thus, we in silico assessed the presence of new transcripts through a targeted analysis of “uniquely mapped” reads spanning along different exons. Fourteen new potential AS events in twelve genes encoding TFs were identified (Figure S1). We experimentally confirmed nine transcripts, whereas five could not be in vitro validated and were not considered in the further analyses.


Novel transcription factor variants through RNA-sequencing: the importance of being "alternative".

Scarpato M, Federico A, Ciccodicola A, Costa V - Int J Mol Sci (2015)

Computational and experimental workflow. Schematic overview of the in silico procedures used to infer the presence of new transcription factors (TFs) transcripts from the re-analysis of our RNA-Seq datasets. The experimental approach used to validate the presence of the new variants is also depicted.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4307332&req=5

ijms-16-01755-f001: Computational and experimental workflow. Schematic overview of the in silico procedures used to infer the presence of new transcription factors (TFs) transcripts from the re-analysis of our RNA-Seq datasets. The experimental approach used to validate the presence of the new variants is also depicted.
Mentions: We used our published RNA-Seq data [16] to identify new transcripts that encode transcription factors (see workflow scheme in Figure 1). First, we found that 1043 (out of ~1500) genes annotated in the Transcription Factor Class (TFClass) database [18] are expressed according to our RNA-Seq data. Normalized gene expression data revealed that most of genes encoding TFs have medium to low expression (Figure S1). Targeted re-analysis (Section 4.1 and [17]) of AS in genes encoding TFs revealed the presence of new putative isoforms, mainly originated by cassette exon skipping. A complete—or even partial—overlap with transcripts annotated in AceView [19] and expressed sequence tags (ESTs; [20]) databases was used as the selection parameter. In contrast, splice junctions with less than two mapped reads/sample and those that overlap genomic repeats were not considered for further studies. The 50 bp single-end libraries of our previous experiment did not allow us to automatically reconstruct the entire exon/intron structure of the new transcripts, nor to measure their relative abundance [16]. Thus, we in silico assessed the presence of new transcripts through a targeted analysis of “uniquely mapped” reads spanning along different exons. Fourteen new potential AS events in twelve genes encoding TFs were identified (Figure S1). We experimentally confirmed nine transcripts, whereas five could not be in vitro validated and were not considered in the further analyses.

Bottom Line: The highest rate of alternative splicing occurs in transcription factors encoding genes, mostly in Krüppel-associated box domains of zinc finger proteins.Through computational approaches we also predicted their novel structural and functional properties.Our findings indicate that alternative splicing is a major determinant of transcription factor diversity, confirming that accurate analysis of RNA-Sequencing data can reliably lead to the identification of novel transcripts, with potentially new functions.

View Article: PubMed Central - PubMed

Affiliation: Institute of Genetics and Biophysics "Adriano Buzzati-Traverso", National Research Council, 80131 Naples, Italy. margherita.scarpato@igb.cnr.it.

ABSTRACT
Alternative splicing is a pervasive mechanism of RNA maturation in higher eukaryotes, which increases proteomic diversity and biological complexity. It has a key regulatory role in several physiological and pathological states. The diffusion of Next Generation Sequencing, particularly of RNA-Sequencing, has exponentially empowered the identification of novel transcripts revealing that more than 95% of human genes undergo alternative splicing. The highest rate of alternative splicing occurs in transcription factors encoding genes, mostly in Krüppel-associated box domains of zinc finger proteins. Since these molecules are responsible for gene expression, alternative splicing is a crucial mechanism to "regulate the regulators". Indeed, different transcription factors isoforms may have different or even opposite functions. In this work, through a targeted re-analysis of our previously published RNA-Sequencing datasets, we identified nine novel transcripts in seven transcription factors genes. In silico analysis, combined with RT-PCR, cloning and Sanger sequencing, allowed us to experimentally validate these new variants. Through computational approaches we also predicted their novel structural and functional properties. Our findings indicate that alternative splicing is a major determinant of transcription factor diversity, confirming that accurate analysis of RNA-Sequencing data can reliably lead to the identification of novel transcripts, with potentially new functions.

Show MeSH