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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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Graphical representation of newly identified splicing events in TF genes. General scheme of the new alternative splicing events identified for SATB1 (A); ELF2 (B); SP140L (C); ARID5B (D); NCOA2 (E) and ZNF266 (F). For all the genes, the genomic region encompassing the gene is shown in the upper part. Nucleotide sequences (and electropherograms by Sanger sequencing) of the new splice junctions are shown below the exon/intron structure for each gene. White numbers indicate exons’ numbers. Donor and acceptor splice sites are shown in bold. Red arrows indicate the primers annealing sites.
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ijms-16-01755-f003: Graphical representation of newly identified splicing events in TF genes. General scheme of the new alternative splicing events identified for SATB1 (A); ELF2 (B); SP140L (C); ARID5B (D); NCOA2 (E) and ZNF266 (F). For all the genes, the genomic region encompassing the gene is shown in the upper part. Nucleotide sequences (and electropherograms by Sanger sequencing) of the new splice junctions are shown below the exon/intron structure for each gene. White numbers indicate exons’ numbers. Donor and acceptor splice sites are shown in bold. Red arrows indicate the primers annealing sites.

Mentions: SATB1 gene validation assay revealed a shorter amplicon of 245 bp with respect to the 480 bp annotated one (ENST00000440737.1). Sanger sequencing confirmed the skipping of exon 2 in this new transcript (Figure 3A). For the ELF2 gene we found a more complex scenario. Indeed, we validated two new transcripts for this gene. Particularly, targeted PCR analysis revealed three amplicons (46, 149 and 205 bp). By cloning and sequencing we observed that only the shortest one (46 bp) corresponded to the annotated ELF2 transcript (NM_201999.2). Indeed, the analysis of nucleotide sequences confirmed RNA-Seq data, i.e., the presence of two novel exons (schematized in Figure 2 and Figure 3B). In detail, the longest amplicon (205 bp) corresponds to a new ELF2 transcript containing an additional exon (159 bp long) located within intron 3 of the ELF2 gene. Surprisingly, sequence analysis on the intermediate PCR amplicon (149 bp) revealed the presence of a novel transcript that originates by AS from the new above-described transcript, which has a cryptic acceptor splice site in the new exon. Therefore, this AS leads to the formation of another exon, 103 bp long. Notably, both these exons are reported in the AceView database, although these predictions refer to non-coding transcripts with 5' and 3' alternative exons with respect to the RefSeq transcript.


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)

Graphical representation of newly identified splicing events in TF genes. General scheme of the new alternative splicing events identified for SATB1 (A); ELF2 (B); SP140L (C); ARID5B (D); NCOA2 (E) and ZNF266 (F). For all the genes, the genomic region encompassing the gene is shown in the upper part. Nucleotide sequences (and electropherograms by Sanger sequencing) of the new splice junctions are shown below the exon/intron structure for each gene. White numbers indicate exons’ numbers. Donor and acceptor splice sites are shown in bold. Red arrows indicate the primers annealing sites.
© Copyright Policy
Related In: Results  -  Collection

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

ijms-16-01755-f003: Graphical representation of newly identified splicing events in TF genes. General scheme of the new alternative splicing events identified for SATB1 (A); ELF2 (B); SP140L (C); ARID5B (D); NCOA2 (E) and ZNF266 (F). For all the genes, the genomic region encompassing the gene is shown in the upper part. Nucleotide sequences (and electropherograms by Sanger sequencing) of the new splice junctions are shown below the exon/intron structure for each gene. White numbers indicate exons’ numbers. Donor and acceptor splice sites are shown in bold. Red arrows indicate the primers annealing sites.
Mentions: SATB1 gene validation assay revealed a shorter amplicon of 245 bp with respect to the 480 bp annotated one (ENST00000440737.1). Sanger sequencing confirmed the skipping of exon 2 in this new transcript (Figure 3A). For the ELF2 gene we found a more complex scenario. Indeed, we validated two new transcripts for this gene. Particularly, targeted PCR analysis revealed three amplicons (46, 149 and 205 bp). By cloning and sequencing we observed that only the shortest one (46 bp) corresponded to the annotated ELF2 transcript (NM_201999.2). Indeed, the analysis of nucleotide sequences confirmed RNA-Seq data, i.e., the presence of two novel exons (schematized in Figure 2 and Figure 3B). In detail, the longest amplicon (205 bp) corresponds to a new ELF2 transcript containing an additional exon (159 bp long) located within intron 3 of the ELF2 gene. Surprisingly, sequence analysis on the intermediate PCR amplicon (149 bp) revealed the presence of a novel transcript that originates by AS from the new above-described transcript, which has a cryptic acceptor splice site in the new exon. Therefore, this AS leads to the formation of another exon, 103 bp long. Notably, both these exons are reported in the AceView database, although these predictions refer to non-coding transcripts with 5' and 3' alternative exons with respect to the RefSeq transcript.

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