The Architecture of Trypanosoma brucei editosomes

Abstract

Uridine insertion and deletion RNA editing generates functional mitochondrial mRNAs in Trypanosoma brucei Editing is catalyzed by three distinct ∼20S editosomes that have a common set of 12 proteins, but are typified by mutually exclusive RNase III endonucleases with distinct cleavage specificities and unique partner proteins. Previous studies identified a network of protein-protein interactions among a subset of common editosome proteins, but interactions among the endonucleases and their partner proteins, and their interactions with common subunits were not identified. Here, chemical cross-linking and mass spectrometry, comparative structural modeling, and genetic and biochemical analyses were used to define the molecular architecture and subunit organization of purified editosomes. We identified intra- and interprotein cross-links for all editosome subunits that are fully consistent with editosome protein structures and previously identified interactions, which we validated by genetic and biochemical studies. The results were used to create a highly detailed map of editosome protein domain proximities, leading to identification of molecular interactions between subunits, insights into the functions of noncatalytic editosome proteins, and a global understanding of editosome architecture.

Keywords: RNA editing; Trypanosoma brucei; cross-linking; editosome; proteomics.

Fig. 1.

(A) BS3 interprotein cross-linking map of editosome complexes. Domains are highlighted as indicated based on previous studies or bioinformatics predictions. Black dots distributed across proteins indicate the positions of lysine residues. Cross-links observed in the KREPB5–TAP complexes are shown as red lines, KREN1–TAP complexes as gray lines, and both KREPB5–TAP and KREN1–TAP complexes as purple lines. Black dotted boxes outline the trimeric deletion (KREX2, KREPA2, and KREL1) and insertion (KRET2, KREPA1, and KREL2) subcomplexes. Colored boxes outline the endonuclease proteins with their associated proteins that are unique to each of the three types of 20S editosome. Green represents KREN3 and KREPB6; red represents KREN2 and KREPB7; and blue represents KREN1, KREPB8, and KREX1. (B) Network diagrams of all interprotein cross-links within editosome complexes. Interlinks involving KREN1, KREN2, and KREN3 have been separated for clarity. Network edge width is proportional to the number of interprotein cross-links.

Fig. 2.

CXMS results for heterotrimeric insertion and deletion subcomplexes in editosomes. (A) Cross-linking maps for the identified interprotein cross-links within the deletion subcomplex (Upper), and the insertion subcomplex (Lower). Anchor, anchor domain in KREPA1 and KREPA2; CTD, C-terminal domain. (B) Network of interprotein cross-links within the insertion and deletion subcomplexes, and those that bridge the insertion and deletion subcomplexes. (C) A cross-linking map of interprotein cross-links between the KREPA proteins.

Fig. 3.

(A) Network diagrams and (B) interprotein cross-linking maps of KREN1, KREN2, and KREN3 first neighbors. (C) Comparative models of RNase III domains in KREPB6, KREPB7, and KREPB8. The Campylobacter jejuni RNase III nuclease domain (PDB ID code 3O2R) was used as a template for comparative modeling of the RNase III domains. Modeled structures are shown in magenta, and template RNase III domain dimer is shown in gray. Dashed lines denote large loop regions (Dataset S3). (D) Network diagram and interprotein cross-linking map of KREX1 first neighbors.

Fig. 4.

(A) Diagram showing U1-like zinc finger, RNase III, and PUF motifs of KREN1 and KREN2 and the C-terminal regions exchanged in the chimeric KREN1-N2 and KREN2-N1 proteins. (B) Expressed TAP-tagged chimeric endonucleases were detected using PAP antibody, which is specific to the Protein A-tagged component of the tag. (C) Western analysis of calmodulin binding eluates isolated via TAP-tagged chimeric proteins using the monoclonal antibody mixture to KREPA1, KREPA2, KREL1, and KREPA3. The positive 20S control is from purified fractionated mitochondria. (D) Cumulative growth of cells constitutively expressing the KREN2-N1 chimeric endonuclease in BF KREN1 or KREN2 CN cells in which the tet-regulatable WT KREN1 or KREN2 allele was also expressed (E) in the presence of tet, or repressed (R) in the absence of tet.

Fig. 5.

(A) Network diagrams and (B) interprotein cross-linking maps of KREPB4 and KREPB5 first neighbors.

Fig. 6.

(A) Diagram showing the U1-like zinc finger, RNase III, and PUF motifs, and the C-terminal domain of KREPB5. The C-terminal truncation (CTT) mutant of KREPB5 lacks the C-terminal domain (residues 255–384*), but retains all other motifs. (B) Cumulative growth of BF and PF CN cells that contain a constitutively expressed V5-tagged WT or CTT mutant KREPB5 allele in addition to the tet-regulatable WT allele that is expressed (E) or repressed (R). (C) Heat maps showing the log10-transformed abundances of RNAs from BF and PF KREPB5 CN cells in which either V5-tagged WT or CTT mutant alleles (B5 EE) were exclusively expressed for 3 (BF) or 4 (PF) d relative to those from cells in which the tet-regulatable WT allele (B5 reg) was also expressed. RNAs were analyzed using qRT-PCR, and their levels normalized to the level of TERT RNA. The relative abundances of KREPB5, never-edited (COI and ND4), pre-edited, and edited RNAs were determined in quadruplicate. (D) Anti-V5 IP of editosome complexes from BF and PF cells that exclusively expressed V5-tagged WT or CTT mutant KREPB5. Anti-V5 immunoprecipitates from BF and PF cells, and BF and PF input cell lysates were probed with monoclonal antibodies against KREPA1, KREPA2, KREL1, KREPA3, and the V5 epitope tag, and a polyclonal antibody against KREPA6. Lysates from KREPB5 CN cells that had no tags and mock IPs without antibody (−) were used as negative controls.