Transcriptomic and genomic identification of spliceosomal genes from Euglena gracilis

Abstract

Diverse splicing types in nuclear and chloroplast genes of protist Euglena gracilis have been recognized for decades. However, the splicing machinery responsible for processing nuclear precursor messenger RNA introns, including trans-splicing of the 5' terminal outron and spliced leader (SL) RNA, remains elusive. Here, we identify 166 spliceosomal protein genes and two snRNA genes from E. gracilis by performing bioinformatics analysis from a combination of next-generation and full-length transcriptomic RNA sequencing (RNAseq) data as well as draft genomic data. With the spliceosomal proteins we identified in hand, the insensitivity of E. gracilis to some splicing modulators is revealed at the sequence level. The prevalence of SL RNA-mediated trans-splicing is estimated to be more than 70% from our full-length RNAseq data. Finally, the splicing proteomes between E. gracilis and its three evolutionary cousins within the same Excavata group are compared. In conclusion, our study characterizes the spliceosomal components in E. gracilis and provides the molecular basis for further exploration of underlying splicing mechanisms.

Keywords: -splicing; SF3B; intron; spliceosome; transcriptome; txcavata.

Figure 1

Spliceosomal proteins collected for this work The splicing pathway including thirteen spliceosomal complexes is depicted in the upper panel. Asterisks show the 12 groups of spliceosomal paralogues (U1A/U2B″, SRPK1/SRPK2, HSPA8/HSPA1A, SF1/Quaking/Sam68/Sam-2, CCAR1/CCAR2, p68/p72, RBM5/RBM10, RBM23/RBM39, FAM50A/FAM50B, CIR1/RP9, hnRNP A0/A1/A3/A/B/A2/B1 and PTBP1/PTBP2).

Figure 2

Working procedure for transcriptomic and genomic identification of E. gracilis spliceosomal protein genes We identified a total of 166 spliceosomal protein genes in E. gracilis ( Supplementary Table S2). We found all snRNP proteins except three (Prp39 of U1, Prp24 of U6, and snRNP27 of U4/U6/U5 snRNP) and all ATP helicases are present in E. gracilis. Except two (PRCC of NTR and CCDC16 of IBC), the NTC/NTR/IBC/RES complexes are also conserved. Notably, large proteins, such as Prp8 and Brr2, could be assembled in full-length, suggesting that the sequencing depth of the RNAseq data we used is well qualified for this analysis. Compared to human spliceosome proteins, most missing proteins in E. gracilis are from SR and related, hnRNP, miscellaneous, B*/C specific, and EJC/TREX proteins. Additionally, we noticed that there are some gene duplications in E. gracilis, including two copies of 10 proteins (SmD3, PUF60, CD2BP2, NY-CO-10, PPIL2, PPIL3, DDX57, PRP43, E1B-AP5 and PPP1CA) and even more copies of seven other proteins (p68/p72, U2AF35, HSPA8/HSPA1A, PRP22, PABP1, DDX3 and PTBP1/PTBP2). A total of 213 sequences of these 166 E. gracilis spliceosomal protein genes were identified in next-generation RNAseq. The presence of multiple copies of the above 17 and the lack of 76 spliceosomal protein genes indicate that the E. gracilis spliceosome may have different features during the splicing process. We concluded that E. gracilis harbors a majority of spliceosomal protein genes, most of which are conserved compared to their human counterparts.

Figure 3

Genomic identification of E. gracilis spliceosomal protein genes SF3B5 and SF3B6 and snRNA genes U2 and U6 (A) Genomic alignments of SF3B5 and SF3B6. mRNA sequences of SF3B5 and SF3B6 were projected to the corresponding genomic contigs visualized by kablammo [24]. (B) Genomic organization of E. gracilis U1 and U2 in the genomic contig sga_contig_369779. (C) Secondary structure of E. gracilis U2 and U6. Putative RNA motifs are indicated as boxed (Sm binding site of U2 and LSm binding site of U6), underlined (branch point interaction region of U2 and 5′ splice site interaction region of U6), or with arrows (U2/U6 helix Ia, Ib and II).

Figure 4

PCR verification of 27 spliceosomal proteins and two snRNA genes Spliceosomal genes were amplified with the respective primers listed in Supplementary Table S1. Numbers after U2 and U6 are genomic contig numbers according to GCA_900893395.1.

Figure 5

Sequence comparison of SF3B1/PHF5A between humans and E. gracilis (A) Structure of human SF3B1/PFH5A with PB. PHF5A is colored red, and SF3B1 is colored olive. SF3B1 Val 1078 and PHF5A Cys 36 are labeled with dashed circles. PB, which is harbored between SF3B1 and PHF5A, is indicated with an arrow. (B) Structure of human CDK11B with OTS964. OTS964 is indicated with an arrow. CDK11B domains that contain OTS964 are colored green and blue. His 572 and Gly 579 are labeled with dashed circles. (C) Sequence alignments of human and E. gracilis SF3B1, PHF5A and CDK11. For each alignment, the upper sequence(s) were from human proteins, and the lower sequence was from E. gracilis. Residues that influence the interaction with PB or OTS964 are indicated with red stars. Other residues in SF3B1 that interact with PB are indicated with dark triangles. The alignments were generated by ESPript [14].