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.
Highlights
Human genome sequencing and large-scale international projects have highlighted that eukaryotic complexity does not correlate with genome size and gene number [1,2,3]
We found that 1043 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 transcription factors (TFs) have medium to low expression (Figure S1)
Summary
Human genome sequencing and large-scale international projects have highlighted that eukaryotic complexity does not correlate with genome size and gene number [1,2,3]. Genes encoding transcription factors (TFs)—in human and mouse genomes—have the highest rate of AS [10] This mechanism has been proposed to facilitate tissue- or cell-specific gene expression regulation, during development [11]. The KRAB (Krüppel-associated box) is one of the domains most affected by AS in humans [10] It is located at the amino-terminal region of the majority of Cys2His zinc finger (ZNF) proteins and is responsible for transcriptional repression through binding to corepressor proteins [14]. Despite that large-scale studies from research groups and/or international consortia [1,2] have expanded the landscape of AS in humans, most predicted isoforms still remain to be experimentally confirmed and characterized This challenge is relevant for genes encoding TFs, given the higher rates of AS in this group compared to other human genes. Starting from the re-analysis of our previously published RNA-Seq datasets [16], here we describe the identification and experimental validation of novel transcripts of seven TFs encoding genes generated by differential exon usage
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