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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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Multiple alignment of the N-terminal residues of the novel predicted ZNF266 variants. The evolutionary conservation of the new 67 amino acids is shown in (A); Krüppel-associated box (KRAB)-A and -B boxes (dashed lines) alignment with other human genes is shown in (B). Identical residues are indicated by “*”, conservation between groups of strongly and weakly similar properties by “:” and “.” respectively. Black arrow indicates the start of the canonical ZNF266 protein isoform.
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ijms-16-01755-f004: Multiple alignment of the N-terminal residues of the novel predicted ZNF266 variants. The evolutionary conservation of the new 67 amino acids is shown in (A); Krüppel-associated box (KRAB)-A and -B boxes (dashed lines) alignment with other human genes is shown in (B). Identical residues are indicated by “*”, conservation between groups of strongly and weakly similar properties by “:” and “.” respectively. Black arrow indicates the start of the canonical ZNF266 protein isoform.

Mentions: In detail, we amplified five PCR products (613, 458, 454, 324 and 158 bp; Figure S4A). Cloning and further sequencing revealed that four out of five amplicons corresponded to known ZNF266 transcripts (uc010dwq.4, NM_006631.3, NM_001271314.1 and ENST00000592292.1). The shortest one confirmed the presence of the new transcript indicated by RNA-Seq, also supported by an AceView entry (ZNF266.bAug10; Figure 2 and Figure 3F). Sequence analysis of the new transcript revealed that the new AS event is predicted to cause an alternative AUG usage, in a strong Kozak context for the initiation of translation [24]. Indeed, 201 bp upstream the canonical AUG, we found a new putative translation-initiating site. Its usage would lead to the addition of 201 nucleotides to the ORF of the annotated ZNF266. The protein is predicted to have 67 new amino acids at N-terminus (Supplementary File S1). Local alignment of these 67 aa revealed a very high evolutionary conservation (Figure 4A). Diversity per residue calculation in multiple alignment showed that the amino acid diversity decreases in proximity of the canonical methionine (Figure S4B). The ZNF266 protein (UniProt ID Q14584) is 549 aa long, whereas the new isoform is 616 amino acids long. The annotated protein has a KRAB domain in position 1–42, corresponding to amino acids from 68 to 109 of the new protein. Interestingly, the analysis of conserved domains and comparative sequence analysis on the annotated ZNF266 protein revealed that the KRAB domain is incorrectly annotated in the UniProt database. Indeed, only the new predicted ZNF266 protein—with 67 additional amino acids at the N-terminus—has complete KRAB-A and KRAB-B box domains (amino acids from 39 to 77 and from 79 to 94, respectively; Figure 4B and Figure 5A). Moreover, the already annotated protein has 14 C2H2-type zinc finger motifs. Notably, solvent accessibility calculation per residue (Supplementary File S2) revealed a recurrence of maximum and minimum values in amino acids corresponding to these motifs. A similar occurrence was also present in three regions located in a hinge region (residues 88–100, 106–123 and 129–151 of the annotated protein, corresponding to amino acids 145–167, 173–190 and 196–218 of the new isoform). This observation suggested to us the presence of three additional zinc finger motifs located upstream of the already annotated ones (Figure 5A). In particular, amino acid alignments and solvent accessibility data revealed that predicted motifs are partially degenerated. In silico 3D structure of the new ZNF266 isoform predicted a structural rearrangement—due to the presence of 67 additional residues—in the region that mediates the transcriptional repression (Figure 5B). These evidences support the hypothesis that these additional residues may confer a functional role to the new ZNF266 variant herein identified.


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)

Multiple alignment of the N-terminal residues of the novel predicted ZNF266 variants. The evolutionary conservation of the new 67 amino acids is shown in (A); Krüppel-associated box (KRAB)-A and -B boxes (dashed lines) alignment with other human genes is shown in (B). Identical residues are indicated by “*”, conservation between groups of strongly and weakly similar properties by “:” and “.” respectively. Black arrow indicates the start of the canonical ZNF266 protein isoform.
© Copyright Policy
Related In: Results  -  Collection

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

ijms-16-01755-f004: Multiple alignment of the N-terminal residues of the novel predicted ZNF266 variants. The evolutionary conservation of the new 67 amino acids is shown in (A); Krüppel-associated box (KRAB)-A and -B boxes (dashed lines) alignment with other human genes is shown in (B). Identical residues are indicated by “*”, conservation between groups of strongly and weakly similar properties by “:” and “.” respectively. Black arrow indicates the start of the canonical ZNF266 protein isoform.
Mentions: In detail, we amplified five PCR products (613, 458, 454, 324 and 158 bp; Figure S4A). Cloning and further sequencing revealed that four out of five amplicons corresponded to known ZNF266 transcripts (uc010dwq.4, NM_006631.3, NM_001271314.1 and ENST00000592292.1). The shortest one confirmed the presence of the new transcript indicated by RNA-Seq, also supported by an AceView entry (ZNF266.bAug10; Figure 2 and Figure 3F). Sequence analysis of the new transcript revealed that the new AS event is predicted to cause an alternative AUG usage, in a strong Kozak context for the initiation of translation [24]. Indeed, 201 bp upstream the canonical AUG, we found a new putative translation-initiating site. Its usage would lead to the addition of 201 nucleotides to the ORF of the annotated ZNF266. The protein is predicted to have 67 new amino acids at N-terminus (Supplementary File S1). Local alignment of these 67 aa revealed a very high evolutionary conservation (Figure 4A). Diversity per residue calculation in multiple alignment showed that the amino acid diversity decreases in proximity of the canonical methionine (Figure S4B). The ZNF266 protein (UniProt ID Q14584) is 549 aa long, whereas the new isoform is 616 amino acids long. The annotated protein has a KRAB domain in position 1–42, corresponding to amino acids from 68 to 109 of the new protein. Interestingly, the analysis of conserved domains and comparative sequence analysis on the annotated ZNF266 protein revealed that the KRAB domain is incorrectly annotated in the UniProt database. Indeed, only the new predicted ZNF266 protein—with 67 additional amino acids at the N-terminus—has complete KRAB-A and KRAB-B box domains (amino acids from 39 to 77 and from 79 to 94, respectively; Figure 4B and Figure 5A). Moreover, the already annotated protein has 14 C2H2-type zinc finger motifs. Notably, solvent accessibility calculation per residue (Supplementary File S2) revealed a recurrence of maximum and minimum values in amino acids corresponding to these motifs. A similar occurrence was also present in three regions located in a hinge region (residues 88–100, 106–123 and 129–151 of the annotated protein, corresponding to amino acids 145–167, 173–190 and 196–218 of the new isoform). This observation suggested to us the presence of three additional zinc finger motifs located upstream of the already annotated ones (Figure 5A). In particular, amino acid alignments and solvent accessibility data revealed that predicted motifs are partially degenerated. In silico 3D structure of the new ZNF266 isoform predicted a structural rearrangement—due to the presence of 67 additional residues—in the region that mediates the transcriptional repression (Figure 5B). These evidences support the hypothesis that these additional residues may confer a functional role to the new ZNF266 variant herein identified.

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