slORFfinder: a tool to detect open reading frames resulting from trans-splicing of spliced leader sequences

Abstract

Trans-splicing of a spliced leader (SL) to the 5' ends of mRNAs is used to produce mature mRNAs in several phyla of great importance to human health and the marine ecosystem. One of the consequences of the addition of SL sequences is the change or disruption of the open reading frames (ORFs) in the recipient transcripts. Given that most SL sequences have one or more of the trinucleotide NUG, including AUG in flatworms, trans-splicing of SL sequences can potentially supply a start codon to create new ORFs, which we refer to as slORFs, in the recipient mRNAs. Due to the lack of a tool to precisely detect them, slORFs were usually neglected in previous studies. In this work, we present the tool slORFfinder, which automatically links the SL sequences to the recipient mRNAs at the trans-splicing sites identified from SL-containing reads of RNA-Seq and predicts slORFs according to the distribution of ribosome-protected footprints (RPFs) on the trans-spliced transcripts. By applying this tool to the analyses of nematodes, ascidians and euglena, whose RPFs are publicly available, we find wide existence of slORFs in these taxa. Furthermore, we find that slORFs are generally translated at higher levels than the annotated ORFs in the genomes, suggesting they might have important functions. Overall, this study provides a tool, slORFfinder (https://github.com/songbo446/slORFfinder), to identify slORFs, which can enhance our understanding of ORFs in taxa with SL machinery.

Keywords: open reading frames; ribosome footprints; spliced leader; trans-splicing.

Figure 1

Overview of slORFfinder. (A) SL sequences in different taxa. The bolder letters represent the cognate start codons. (B) The workflow of slORFfinder. Four input files and SL sequences, indicated in red in the workflow, are used for slORFfinder: the reference genome sequence (in fasta format), the genome annotation (in gtf format), the alignment of RNA-Seq reads and Ribo-Seq reads (in bam format), and the sequence of SL. The genome sequences and annotations are used to extract the sequences of nascent mRNAs, the alignments of RNA-Seq reads are used to identify the SL trans-splicing sites according to the SL sequences provided, and the alignments of Ribo-Seq reads are used to identify the P-sites on mature mRNAs to predict ORFs.

Figure 2

Evidence of the use of non-canonical start codons. (A) The offsets from the 5′ terminuses of RPFs to the translating codons calculated from metagene plots of C. elegans, C. brenneri and C. remanei. (B). Examples of footprints at translation initiation sites showing the use of non-canonical start codons. (C) The usage of codons at translation initiation sites.

Figure 3

The recall rate of slORFfinder tested in simulated datasets.

Figure 4

The distribution of RPFs in some examples of the slORFs identified in (A) C. elegans, (B) C. brenneri, (C) O. dioica and (D) T. brucei. The cyan, orange and purple lines represent frames 0, 1 and 2, respectively. The labels of ‘out-frame’ and ‘in-frame’ in the parentheses in each plot indicate whether these slORFs are translated in a frame different from its corresponding annotated ORF (out-frame) or not (in-frame).

Figure 5

Comparison of the translation levels between the mRNAs with or without slORFs in (A) C. elegans, (B) C. brenneri (C) O. dioica and (D) T. brucei.

Figure 6

Comparison of the translation levels between slORFs and other types of ORFs in (A) C. elegans, (B) C. brenneri, (C) O. dioica and (D) T. brucei. uORF: upstream ORF, aORF: annotated ORF, dORF: downstream ORF.