Widespread establishment and regulatory impact of Alu exons in human genes

Abstract

The Alu element has been a major source of new exons during primate evolution. Thousands of human genes contain spliced exons derived from Alu elements. However, identifying Alu exons that have acquired genuine biological functions remains a major challenge. We investigated the creation and establishment of Alu exons in human genes, using transcriptome profiles of human tissues generated by high-throughput RNA sequencing (RNA-Seq) combined with extensive RT-PCR analysis. More than 25% of Alu exons analyzed by RNASeq have estimated transcript inclusion levels of at least 50% in the human cerebellum, indicating widespread establishment of Alu exons in human genes. Genes encoding zinc finger transcription factors have significantly higher levels of Alu exonization. Importantly, Alu exons with high splicing activities are strongly enriched in the 5′-UTR, and two-thirds (10/15) of 5′-UTR Alu exons tested by luciferase reporter assays significantly alter mRNA translational efficiency. Mutational analysis reveals the specific molecular mechanisms by which newly created 5′-UTR Alu exons modulate translational efficiency, such as the creation or elongation of upstream ORFs that repress the translation of the primary ORFs. This study presents genomic evidence that a major functional consequence of Alu exonization is the lineagespecific evolution of translational regulation. Moreover, the preferential creation and establishment of Alu exons in zinc finger genes suggest that Alu exonization may have globally affected the evolution of primate and human transcriptomes by regulating the protein production of master transcriptional regulators in specific lineages. transcriptome evolution | transposable element | alternative splicing | deep sequencing | uORF

The Persistent Contributions of RNA to Eukaryotic Gen(om)e Architecture and Cellular Function

“…Such co-opted or exapted modules can be derived from inter-or intragenic space and constitute novel protein-coding exons and regulatory regions such as promoters and enhancers (Brosius 2005b(Brosius , 2009Baertsch et al 2008;Rebollo et al 2012). 5 A prominent case is the exonization of parts of Alu elements, usually as alternatively spliced exons (Makalowski et al 1994;Nekrutenko and Li 2001;Sorek et al 2002;Lev-Maor et al 2003;Krull et al 2005;Shen et al 2011). Many such events are, over evolutionary time, not stable.…”

Validation of predicted mRNA splicing mutations using high-throughput transcriptome data

“…A coverage of at least 20 reads should be sufficient. This estimate is based upon alternative splicing analyses in which this threshold was found to imply concordance with microarray and RT-PCR measurements 30– 33 . There are many potential legitimate reasons why a mutation may not be validated: (a) nonsense-mediated decay may result in a loss of expression of the entire transcript, (b) the gene itself may have multiple paralogs and reads may not be unambiguously mapped, (c) other non-splicing mutations could account for a loss of expression, and (d) confounding natural alternative splicing isoforms may result in a loss of statistical significance during read mapping of the control samples.…”

Functional transcriptomics in the post-ENCODE era

“…Furthermore, there is a well-established link between TEs and AS in protein-coding genes; certain TE families such as Alu contain DNA motifs that resemble splicing signals, making them a ready substrate for exon creation events (''exonization'') (Sorek 2007;Shen et al 2011). Since detectable TE insertions are typically seen to have occurred during recent evolution, it appears that most TE transcripts or exons are also young (Sorek 2007).…”