Unique features of conventional and nonconventional introns in Euglena gracilis

Abstract

Background: Nuclear introns in Euglenida have been understudied. This study aimed to investigate nuclear introns in Euglenida by identifying a large number of introns in Euglena gracilis (E. gracilis), including cis-spliced conventional and nonconventional introns, as well as trans-spliced outrons. We also examined the sequence characteristics of these introns.

Results: A total of 28,337 introns and 11,921 outrons were identified. Conventional and nonconventional introns have distinct splice site features; the former harbour canonical GT/C-AG splice sites, whereas the latter are capable of forming structured motifs with their terminal sequences. We observed that short introns had a preference for canonical GT-AG introns. Notably, conventional introns and outrons in E. gracilis exhibited a distinct cytidine-rich polypyrimidine tract, in contrast to the thymidine-rich tracts observed in other organisms. Furthermore, the SL-RNAs in E. gracilis, as well as in other trans-splicing species, can form a recently discovered motif called the extended U6/5' ss duplex with the respective U6s. We also describe a novel type of alternative splicing pattern in E. gracilis. The tandem repeat sequences of introns in this protist were determined, and their contents were comparable to those in humans.

Conclusions: Our findings highlight the unique features of E. gracilis introns and provide insights into the splicing mechanism of these introns, as well as the genomics and evolution of Euglenida.

Keywords: Extended U6/5' ss duplex; Outron; Polypyrimidine tract; SL-RNA; Splice site.

Fig. 1

Working procedure used to identify nuclear introns in E. gracilis

Fig. 2

Length distribution of nuclear introns in E. gracilis. Intron groups (GT-AG, GC-AG, ss-invariant, and ss-variant) and their respective numbers are labelled in each panel

Fig. 3

Sequence conservation of the 5’ ss region of conventional introns. (A) Sequence conservation of the 5’ ss region of the GT-AG and GC-AG introns in E. gracilis. (B) Sequence conservation of the 5’ ss region of introns in humans, A. thaliana, S. pombe, and S. cerevisiae. (C) Base pairing between the consensus sequence motif of the 5’ ss of GT-AG introns and U1 in E. gracilis. (D) Extensive base pairing between sequences upstream of the U6 ACAGA box and downstream of the 5’ ss of SL-RNA in E. gracilis. The ACAGA box of U6 and the 5’ ss of SL-RNA are underlined. All sequence logos are plotted on a vertical scale with 0–2 bits of information

Fig. 4

Sequence conservation and composition of the 3’ ss region of conventional introns. (A) Sequence conservation of the E. gracilis 3’ ss regions of the GT-AG and GC-AG introns. (B) Sequence conservation of the 3’ ss regions of introns in humans, A. thaliana, S. pombe, and S. cerevisiae. (C) Sequence conservation of the 3’ ss regions of outrons in E. gracilis, C. elegans, T. brucei, and L. donovani. (D) Ratios of C/T in the 3’ ss regions of introns and/or outrons in E. gracilis, humans, A. thaliana, S. pombe, S. cerevisiae, C. elegans, T. brucei, and L. donovani. All sequence logos are plotted on a vertical scale with 0–2 bits of information

Fig. 5

Sequence conservation and base pairing of the ss region of E. gracilis nonconventional introns. (A) Sequence conservation of the 5’ ss region of nonconventional ss-invariant introns. (B) Sequence conservation of the 3’ ss region of nonconventional ss-invariant introns. (C) Sequence conservation of the 5’ and 3’ ss regions of nonconventional ss-variant introns. (D) Illustration of terminal base pairing generated by RNAfold and the energies associated with different levels of base pairing. (E) Plots of the energy and frequency of terminal base pairing of ss-invariant, ss-variant and GT-AG introns in E. gracilis. All sequence logos are plotted on a vertical scale with 0–2 bits of information

Fig. 6

AS patterns of E. gracilis nuclear introns. Schematic examples of overlapping (A), A5SS (B), A3SS (C), SE (D), atypical SE (E), and RI (F) are shown. Exons are depicted in dark grey, and introns are depicted in light grey. The genomic accessions for each pattern are indicated above the transcripts, and introns are labelled on the transcripts

Fig. 7

TRs in the nuclear introns of E. gracilis. (A) The distribution of TR lengths and numbers in GT-AG, ss-invariant and ss-variant introns of E. gracilis. The distribution of TRs in human introns is included for comparison. (B) Ratios of TRs containing introns and TRs containing bases