Transcriptomic analysis of diplomonad parasites reveals a trans-spliced intron in a helicase gene in Giardia.

Scott William Roy

doi:10.7717/peerj.2861

Abstract

BackgroundThe mechanisms by which DNA sequences are expressed is the central preoccupation of molecular genetics. Recently, ourselves and others reported that in the diplomonad protist Giardia lamblia, the coding regions of several mRNAs are produced by ligation of independent RNA species expressed from distinct genomic loci. Such trans-splicing of introns was found to affect nearly as many genes in this organism as does classical cis-splicing of introns. These findings raised questions about the incidence of intron trans-splicing both across the G. lambliatranscriptome and across diplomonad diversity in general, however a dearth of transcriptomic data at the time prohibited systematic study of these questions.MethodsI leverage newly available transcriptomic data from G. lamblia and the related diplomonad Spironucleus salmonicidato search for trans-spliced introns. My computational pipeline recovers all four previously reported trans-spliced introns in G. lamblia, suggesting good sensitivity.ResultsScrutiny of thousands of potential cases revealed only a single additional trans-spliced intron in G. lamblia, in the p68 helicase gene, and no cases in S. salmonicida. The p68 intron differs from the previously reported trans-spliced introns in its high degree of streamlining: the core features of G. lamblia trans-spliced introns are closely packed together, revealing striking economy in the implementation of a seemingly inherently uneconomical molecular mechanism.DiscussionThese results serve to circumscribe the role of trans-splicing in diplomonads both in terms of the number of genes effected and taxonomically. Future work should focus on the molecular mechanisms, evolutionary origins and phenotypic implications of this intriguing phenomenon.

Highlights

Splicing of nuclear RNA transcripts by the spliceosomal machinery is a ubiquitous feature of the expression of nuclear genes in eukaryotes (Roy & Irimia, 2014; Nixon et al, 2002; Vanácová et al, 2005; see Lane et al, 2007; Akiyoshi et al, 2009)
I used bowtie and blat to identify Illumina reads that contained sequence from multiple genomic loci and which are suggestive of trans-splicing. This procedure identified some 495,066 potential boundaries in G. lamblia and 231,769 in S. salmonicida For both species, the vast majority of these cases were either supported by only a single read (400,460 and 212,801 respectively), had extended similarities at the 5 and 3 boundaries suggesting reverse transcriptase artifacts produced during library formation (‘RTfacts’; Roy & Irimia, 2008) (388,835 and 159,836 cases), and/or did not represent a clear splice junction (with >5 nucleotides in the middle of the read that did not map to either locus (35,740 and 8,307 cases)
Each of the five cases had an extended 5 splicing signal, an extended 3 splicing signal (CT[AG]ACACACAG), complementarity between the pairs of apparently trans-spliced loci, and presence of the G. lamblia 3 cleavage motif; no other cases showed any of these features

Summary

Introduction

Splicing of nuclear RNA transcripts by the spliceosomal machinery is a ubiquitous feature of the expression of nuclear genes in eukaryotes (Roy & Irimia, 2014; Nixon et al, 2002; Vanácová et al, 2005; see Lane et al, 2007; Akiyoshi et al, 2009). In the genome of the diplomonad intestinal parasite G. lamblia, systematic studies have revealed only six cis-spliced introns to date (Nixon et al, 2002; Russell et al, 2005; Morrison et al, 2007; Roy et al, 2012; Franzén et al, 2013); intriguingly small-scale studies revealed four cases of genic trans-splicing, including two in a single gene (Nageshan et al, 2011; Kamikawa et al, 2011; Roy et al, 2012; Hudson et al, 2015) These cases showed distinctive sequence features— most notably extended basepairing potential between the pairs of trans-spliced transcripts. Ourselves and others reported that in the diplomonad protist Giardia lamblia, the coding regions of several mRNAs are produced by ligation of independent RNA species expressed from distinct genomic loci Such trans-splicing of introns was found to affect nearly as many genes in this organism as does classical cis-splicing of introns. Future work should focus on the molecular mechanisms, evolutionary origins and phenotypic implications of this intriguing phenomenon

Methods

Results

Conclusion