Patterns of conservation of spliceosomal intron structures and spliceosome divergence in representatives of the diplomonad and parabasalid lineages

Abstract

Background Two spliceosomal intron types co-exist in eukaryotic precursor mRNAs and are excised by distinct U2-dependent and U12-dependent spliceosomes. In the diplomonad Giardia lamblia , small nuclear (sn) RNAs show hybrid characteristics of U2- and U12-dependent spliceosomal snRNAs and 5 of 11 identified remaining spliceosomal introns are trans -spliced. It is unknown whether unusual intron and spliceosome features are conserved in other diplomonads. Results We have identified spliceosomal introns, snRNAs and proteins from two additional diplomonads for which genome information is currently available, Spironucleus vortens and Spironucleus salmonicida , as well as relatives, including 6 verified cis- spliceosomal introns in S. vortens . Intron splicing signals are mostly conserved between the Spironucleus species and G. lamblia . Similar to ‘long’ G. lamblia introns, RNA secondary structural potential is evident for ‘long’ (> 50 nt) Spironucleus introns as well as introns identified in the parabasalid Trichomonas vaginalis . Base pairing within these introns is predicted to constrain spatial distances between splice junctions to similar distances seen in the shorter and uniformly-sized introns in these organisms. We find that several remaining Spironucleus spliceosomal introns are ancient. We identified a candidate U2 snRNA from S. vortens , and U2 and U5 snRNAs in S. salmonicida ; cumulatively, illustrating significant snRNA differences within some diplomonads. Finally, we studied spliceosomal protein complements and find protein sets in Giardia , Spironucleus and Trepomonas sp. PC1 highly- reduced but well conserved across the clade, with between 44 and 62 out of 174 studied spliceosomal proteins detectable. Comparison with more distant relatives revealed a highly nested pattern, with the more intron-rich fornicate Kipferlia bialata retaining 87 total proteins including nearly all those observed in the diplomonad representatives, and the oxymonad Monocercomonoides retaining 115 total proteins including nearly all those observed in K. bialata . Conclusions Comparisons in diplomonad representatives and species of other closely-related metamonad groups indicates similar patterns of intron structural conservation and spliceosomal protein composition but significant divergence of snRNA structure in genomically-reduced species. Relative to other eukaryotes, loss of evolutionarily-conserved snRNA domains and co...

The role of nuclear organization in trans-splicing based expression of heat shock protein 90 in Giardia lamblia

“…Giardia has minimal spliceosomal machinery as well as canonical nucleotide signatures in 6 cis-spliced genes and 3 trans-spliced genes identified so far [3][4][5][6]8,9]. Previous bioinformatic analysis has revealed the presence of minimal spliceosomal machinery; the conserved snRNAs and major snRNA-associated proteins in Giardia [3,26]. Further, Vanessa Gomez et al, showed the in vivo expression of core spliceosomal proteins SmB, SmD3, SmD1, SmD2, SmE, SmF in a complex with U1, U2 and U4 snRNAs [13].…”

Characterization of Pre‐mRNA Splicing and Spliceosomal Machinery in Porphyridiumpurpureum and Evolutionary Implications for Red Algae

“…1998; Hudson et al. 2019; Madhani and Guthrie 1992; Mitrovich and Guthrie 2007; Stark et al. 2015), and the U2 snRNA candidate from P .…”

“…2; File S4). The U2 snRNA, which recognizes and interacts directly with nucleotides adjacent to the branch point adenosine, is highly conserved in structure across diverse eukaryotes (Fast et al 1998;Hudson et al 2019;Madhani and Guthrie 1992;Mitrovich and Guthrie 2007;Stark et al 2015), and the U2 snRNA candidate from P. purpureum is no exception (Fig. 2B).…”

Coupling of spliceosome complexity to intron diversity

“…The results from the BLAST searches were further screened by analyzing domain content (HMMsearch, HMMer 3.1b2 – default parameters), size comparisons against human protein sequence length (within 25% variation), and reciprocal best-hit BLAST searches (RBH) to the query proteome (Bork et al, 1998; Johnson et al, 2010; Tatusov et al, 1997). To avoid bias in protein domain content, domains used for HMM searches were defined as described (Hudson et al, 2019). Briefly, a conserved set of domains for each spliceosomal protein was assembled by using only those domains present in all three of the human, yeast, and Arabidopsis orthologs.…”