The contribution of Alu exons to the human proteome

Abstract

Background: Alu elements are major contributors to lineage-specific new exons in primate and human genomes. Recent studies indicate that some Alu exons have high transcript inclusion levels or tissue-specific splicing profiles, and may play important regulatory roles in modulating mRNA degradation or translational efficiency. However, the contribution of Alu exons to the human proteome remains unclear and controversial. The prevailing view is that exons derived from young repetitive elements, such as Alu elements, are restricted to regulatory functions and have not had adequate evolutionary time to be incorporated into stable, functional proteins.

Results: We adopt a proteotranscriptomics approach to systematically assess the contribution of Alu exons to the human proteome. Using RNA sequencing, ribosome profiling, and proteomics data from human tissues and cell lines, we provide evidence for the translational activities of Alu exons and the presence of Alu exon derived peptides in human proteins. These Alu exon peptides represent species-specific protein differences between primates and other mammals, and in certain instances between humans and closely related primates. In the case of the RNA editing enzyme ADARB1, which contains an Alu exon peptide in its catalytic domain, RNA sequencing analyses of A-to-I editing demonstrate that both the Alu exon skipping and inclusion isoforms encode active enzymes. The Alu exon derived peptide may fine tune the overall editing activity and, in limited cases, the site selectivity of ADARB1 protein products.

Conclusions: Our data indicate that Alu elements have contributed to the acquisition of novel protein sequences during primate and human evolution.

Fig. 1

Identification and analysis of putative coding Alu exons in human genes. a The bioinformatics work flow to identify and characterize putative coding Alu exons using RNA-seq and proteomics data. b The Venn diagram illustrating the number of putative coding Alu exons with different types of RNA-seq or proteomic evidence. c The splicing patterns and deduced peptide sequences of putative coding Alu exons with supporting peptide sequences from the PRIDE database. The stop codon of each isoform is indicated by a STOP sign

Fig. 2

Ribo-seq data (HeLa cells) provide evidence for the translational activities of putative coding Alu exons. a Comparison of Alu exon inclusion levels in Ribo-seq and RNA-seq data of HeLa cells. Each dot represents an Alu exon. Seventy-six putative coding Alu exons with at least 10 reads mapped to one of the three splice junctions in both Ribo-seq and RNA-seq data are shown in the plot. Exons with PRIDE peptide sequences are indicated in red. b-e The UCSC genome browser view of the Ribo-seq and RNA-seq data of four representative Alu exons. The upstream junction read count (UJC), downstream junction read count (DJC), and skipping junction read count (SJC) are also indicated. The stop codon of each mRNA isoform is indicated by a STOP sign

Fig. 3

A protein-coding Alu exon in SUGT1 supported by multiple lines of proteomics and Ribo-seq evidence. a The splicing pattern and deduced peptide sequence of a putative coding Alu exon in SUGT1 and its corresponding peptide evidence from PRIDE and PeptideAtlas. b Tandem mass spectrometry (MS/MS) spectrum of the peptide TSSDPPALDSQSAGITGADAN from PRIDE (experiment ID: 26855, spectrum ID: 7275). c The UCSC genome browser view of the Ribo-seq and RNA-seq data of the SUGT1 Alu exon. d RT-PCR analysis of the SUGT1 Alu exon in four different tissues in human (Hs), chimpanzee (Pt), and rhesus macaque (Rm). Error bars show standard error of the mean from at least three replicate experiments

Fig. 4

Minigene splicing reporter analysis of the human-specific splicing change of SUGT1 protein-coding Alu exon (Exon 6). a Schematic diagrams of the pI-11-H3 minigene splicing reporter and the wild-type/mutant minigene constructs of SUGT1 Alu exon. The nucleotide differences between human and chimpanzee are indicated by asterisks on the chimpanzee sequence. b Representative gel image for fluorescently labeled RT-PCR analyses in Hela cells using the SUGT1 Alu exon wild-type and mutant minigene constructs

Fig. 5

ADARB1 Alu exon inclusion isoform encodes an active RNA editing enzyme with altered editing activity. a The schematic diagram of the protein domain structure of ADARB1 isoforms and supporting peptide sequences from the PRIDE database. b The change in overall RNA editing levels of 7,618 RNA editing sites in HEK293 cells upon ectopic expression of the exon skipping (Short) or the exon inclusion (Long) ADARB1 isoform as compared to the empty vector (EV) control. Error bars show standard errors calculated based on the 7,618 known RNA editing sites used in this analysis. c Common and isoform-specific differentially edited sites upon ectopic expression of the Alu exon inclusion (Long) or skipping (Short) ADARB1 isoform. Isoform-specific differentially edited sites are further classified (low-confidence, high-confidence) based on the strength of the RNA-seq evidence