Evolutionarily divergent spliceosomal snRNAs and a conserved non-coding RNA processing motif in Giardia lamblia

Abstract

Non-coding RNAs (ncRNAs) have diverse essential biological functions in all organisms, and in eukaryotes, two such classes of ncRNAs are the small nucleolar (sno) and small nuclear (sn) RNAs. In this study, we have identified and characterized a collection of sno and snRNAs in Giardia lamblia, by exploiting our discovery of a conserved 12 nt RNA processing sequence motif found in the 3′ end regions of a large number of G. lamblia ncRNA genes. RNA end mapping and other experiments indicate the motif serves to mediate ncRNA 3′ end formation from mono- and di-cistronic RNA precursor transcripts. Remarkably, we find the motif is also utilized in the processing pathway of all four previously identified trans-spliced G. lamblia introns, revealing a common RNA processing pathway for ncRNAs and trans-spliced introns in this organism. Motif sequence conservation then allowed for the bioinformatic and experimental identification of additional G. lamblia ncRNAs, including new U1 and U6 spliceosomal snRNA candidates. The U6 snRNA candidate was then used as a tool to identity novel U2 and U4 snRNAs, based on predicted phylogenetically conserved snRNA–snRNA base-pairing interactions, from a set of previously identified G. lamblia ncRNAs without assigned function. The Giardia snRNAs retain the core features of spliceosomal snRNAs but are sufficiently evolutionarily divergent to explain the difficulties in their identification. Most intriguingly, all of these snRNAs show structural features diagnostic of U2-dependent/major and U12-dependent/minor spliceosomal snRNAs.

The cellular landscape of mid‐size noncoding RNA

“…The evolution of introns and the requirement of splicing required the development of an intron removal machinery capable of detecting exon-intron boundaries (Rogozin, Carmel, Csuros, & Koonin, 2012). The U1, U2, U4, U5, and U6 snRNA of the major spliceosome clearly appeared early in eukaryotic evolution ( Figure 2) as they are found in higher eukaryotes as well as deep-branching organisms like Giardia lamblia (Brow & Guthrie, 1988;Hudson et al, 2012;Marz, Kirsten, & Stadler, 2008;Vanacova, Yan, Carlton, & Johnson, 2005). The U11, U12, U4atac, and U6atac of the minor spliceosome have only been identified in higher eukaryotes Metazoa, Fungi, Viridiplantaea, and Heterokonta (Lorkovic, Lehner, Forstner, & Barta, 2005; A. G. Russell, Charette, Spencer, & Gray, 2006;Turunen, Niemela, Verma, & Frilander, 2013).…”

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

“…1D); and (iii) the recently-discovered G. lamblia cleavage motif (with consensus TCCTTTACTCAA; Fig. 1D; Hudson et al, 2012). A BLASTX search of the mature trans-spliced transcript against Genbank revealed homology to p68 helicase (Fig.…”

“…(C) Comparison of splice boundaries for newly-discovered p68 trans-spliced intron with known trans- and cis-spliced introns, for G. lamblia isolate GS. (D) Trans-spliced intron sequences for newly-discovered p68 intron exhibits basepairing potential between intronic regions of 5′ and 3′ pre-mRNA transcripts and conserved cleavage motif reported by Hudson et al (2012). (E) Protein sequence alignment between the protein encoded by trans-spliced G. lamblia p68 gene and highest-scoring BLAST hits in Genbank ( Chryseobacterium caeni, Accession WP_027384510.1, Hymenobacter sp.…”

Robust and stable transcriptional repression inGiardiausing CRISPRi

“…A gRNA expression cassette with inverted BbsI sites for cloning specific gRNA 2 targeting sequences was added, followed by the gRNA scaffold sequence from pX330 (Cong et 3 al., 2013). To drive gRNA expression in Giardia and ensure proper termination, we included 4 approximately 200 bp of DNA located upstream and downstream of the Giardia U6 5 spliceosomal snRNA (Hudson et al, 2012). The upstream sequence includes 12 bp of U6 coding 6 sequence, and the downstream sequence includes the 12 bp motif thought to mediate 3' end 7…”

“…processing (Hudson et al, 2012). The gRNA cassette was synthesized (Biomatik) and inserted 8 into a unique Acc65I site in the vector.…”