Comparative genomics of microsporidian genomes reveals a minimal non-coding RNA set and new insights for transcription in minimal eukaryotic genomes

Abstract

Microsporidia are ubiquitous intracellular pathogens whose opportunistic nature led to their increased recognition with the rise of the AIDS pandemic. As the RNA world was largely unexplored in this parasitic lineage, we developed a dedicated in silico methodology to carry out exhaustive identification of ncRNAs across the Encephalitozoon and Nosema genera. Thus, the previously missing U1 small nuclear RNA (snRNA) and small nucleolar RNAs (snoRNAs) targeting only the LSU rRNA were highlighted and were further validated using 5' and 3'RACE-PCR experiments. Overall, the 15 ncRNAs that were found shared between Encephalitozoon and Nosema spp. may represent the minimal core set required for parasitic life. Interestingly, the systematic presence of a CCC- or GGG-like motif in 5' of all ncRNA and mRNA gene transcripts regardless of the RNA polymerase involved suggests that the RNA polymerase machineries in microsporidia species could use common factors. Our data provide additional insights in accordance with the simplification processes observed in these reduce genomes and underline the usefulness of sequencing closely related species to help identify highly divergent ncRNAs in these parasites.

Keywords: Microsporidia; genome evolution; ncRNA prediction; ncRNA structure; transcriptional regulation signals.

Figure 1

Encephalitozoon cuniculi ncRNAs validated by 5′ and 3′ RACE-PCR and their potential regulation signals. Black boxes correspond to the potential TATA box signals for transcription initiation by RNA polymerases II or III. The potential transcriptional motifs (CCC/GGG-like signals) are boxed. For brevity, the ncRNA sequences were only represented by the transcription start site, the end of transcription and the corresponding gene name.

Figure 2

Proposed secondary structure for the E. cuniculi SRP non-coding RNA. Specific domains of the molecule are given.

Figure 3

Proposed secondary structure for the E. cuniculi U1 snRNA (a) and multiple sequence alignment with U1 snRNA from other microsporidia, human and yeast (b). In the secondary structure (a), the 5′ SS and Sm conserved motifs are shaded orange and blue, respectively. (b) Multiple sequence alignment of U1 snRNA from Encephalitozoon (Ec, Eh, Ei and Er), Nosema (Na, Nc and Nb), Human (Hs) and S. cerevisiae (Sc) species. Nucleotides are coloured according to the stem they belong to. Ec, E. cuniculi; Ei, E. intestinalis; Eh, E. hellem; Er, E. romaleae; Na, N. apis; Nc, N. ceranae; Nb, N. bombycis. [n]: insertion of n nucleotides in Sc.

Figure 4

Putative rRNA targets for the C/D and H/ACA snoRNAs encoded by E. cuniculi. Base-pairing interactions between: (a) Putative 2′-O methylated target regions in the 23S LSU rRNA and guide sequences within C/D box snoRNAs and (b) putative Ψ target regions in LSU rRNA and guide sequences within H/ACA box snoRNAs.