Compositionally and functionally distinct editosomes in Trypanosoma brucei

Abstract

Uridylate insertion/deletion RNA editing in Trypanosoma brucei mitochondria is catalyzed by a multiprotein complex, the approximately 20S editosome. Editosomes purified via three related tagged RNase III proteins, KREN1 (KREPB1/TbMP90), KREPB2 (TbMP67), and KREN2 (KREPB3/TbMP61), had very similar but nonidentical protein compositions, and only the tagged member of these three RNase III proteins was identified in each respective complex. Three new editosome proteins were also identified in these complexes. Each tagged complex catalyzed both precleaved insertion and deletion editing in vitro. However, KREN1 complexes cleaved deletion but not insertion editing sites in vitro, and, conversely, KREN2 complexes cleaved insertion but not deletion editing sites. These specific nuclease activities were abolished by mutations in the putative RNase III catalytic domain of the respective proteins. Thus editosomes appear to be heterogeneous in composition with KREN1 complexes catalyzing cleavage of deletion sites and KREN2 complexes cleaving insertion sites while both can catalyze the U addition, U removal, and ligation steps of editing.

FIGURE 1.

T. brucei editosomes with TAP-tagged RNase III proteins. (A) Diagram showing the positions of the motifs and the tags. (B) SYPRO Ruby stained SDS-PAGE gel of TAP-tag purified complexes. The tagged proteins are indicated with an asterisk in the lanes with KREN1 (N1), KREPB2 (B2), and KREN2 (N2) samples, and the molecular weights of the protein size standards (M) are indicated. (C) Western analysis of samples from B using monoclonal antibodies specific for KREPA1, KREPA2, KREL1, and KREPA3 editosome proteins. (D) SDS-PAGE gel of adenylated proteins in samples from B showing the presence of KREL1 and KREL2.

FIGURE 2.

Sequence comparison between editosome proteins. (A) KREPA5 and KREPA6, (B) KREPB7 and KREPB8. The amino acid alignments indicate conserved (|), semiconserved (:), partially conserved (.).

FIGURE 3.

In vitro editing assays of tagged editosomes. Tagged KREN1 (N1), KREPB2 (B2), and KREN2 (N2) complexes and untagged 20S editosomes that were purified by biochemical methods were assayed for precleaved deletion (A) and insertion (B) editing, which requires coordinated U removal and ligation, or U addition and ligation, respectively. They were also assayed for full-round deletion (C) and insertion (D) editing using a modified assay designed for the accumulation of endonucleolytic cleavage fragments. (See Materials and Methods for details.) Untagged editosomes with gRNA served as a positive control (+) and, without gRNA, as a negative control (−). Input labeled substrate RNAs (I) and unprocessed ligated input RNA (L) are identified, as are the products from which up to 4U's were removed (−4U), up to 2 U's were added (+2U), result from endonucleolytic cleavage, or are edited by U deletion or insertion (E).

FIGURE 4.

Tagged complexes containing mutated RNase III proteins. (A) Aligned RNase III domains KREN1, KREPB2, and KREN2 proteins showing the double mutations of conserved amino acids Glutamic acid (E) and Glycine (G) to Valine (V). (B) Western analyses of the purified mutant tagged complexes with a monoclonal antibody mixture specific for KREPA1, KREPA2, KREPA3, and KREL1. (C) Adenylation assays of the purified mutant complexes revealing KREL1 and KREL2. (D) Modified full-round editing assays (see Materials and Methods for details) that enhance detection of the 3′ cleavage products of editing endonuclease activity (arrow).

FIGURE 5.

Functional association of KREN1. Western analysis showing high-resolution glycerol gradient sedimentation profile of tagged KREN1 (N1) (A) and mutated KREN1 (mut-N1) (B) complexes purified from cell lysate using IgG column. Lane T represents the TEV elute from IgG column. Protein bands corresponding to KREPA1, KREPA2, KREL1, and KREPA3 are indicated. Fractions from 5 to 10S region and ∼20S region as indicated by line were pooled and purified by Calmodulin affinity column. Western analysis of N1 (C) and mut-N1 5–10S and ∼20S (E) complexes, protein bands corresponding to KREPA1, KREPA2, KREL1, and KREPA3 are indicated. Adenylation of N1 (D) and mut-N1 5–10S and ∼20S (F) complexes. Adenylated KREL1 and KREL2 are indicated. In vitro precleaved deletion editing (G), precleaved insertion editing (H), deletion endonuclease (I), and insertion endonuclease (J) assays using N1, and mut-N1 5–10S and ∼20S complexes as indicated. Input RNA (I), edited RNA (E), and ligated 5′ and 3′ RNA in precleaved assays (L), and resulting 3′ cleavage product in endonuclease assay are indicated. In precleaved deletion assay RNA band resulting from removal of 4 U's (−4 U) from 5′ RNA and in precleaved insertion assay by addition of 2 U's (+2 U) to 5′ RNA are indicated.

FIGURE 6.

Mutations in KREN2 affect structure and function of editosome. Western analysis using MAbs against KREPA1, KREPA2, KREPA3, and KREL1, showing high-resolution glycerol gradient sedimentation profile of tagged KREN2 (N2) (A) and mutated KREN2 (mut-N2) (B) complexes purified from cell lysate using IgG column. Lane T represents the TEV elute from IgG column. Corresponding protein bands are indicated. Fractions from 5 to 10S region and ∼20S region as indicated by line were pooled and purified by Calmodulin affinity column. Western analysis using the MAbs as above of B3 (C) and mut-B3 5–10S and ∼20S (E) complexes, and corresponding protein bands are indicated. Adenylation of N2 (D) and mut-N2 5–10S and ∼20S (F) complexes, showing the presence of adenylated proteins KREL1 and KREL2 as indicated. In vitro assays using N2 and mut-N2 complexes: (G) precleaved deletion editing, (H) precleaved insertion editing, (I) deletion editing endonuclease, and (J) insertion editing endonuclease assays. Input RNA (I), edited RNA (E), and ligated 5′ and 3′ RNA in precleaved assays (L), and resulting 3′ cleavage product in endonuclease assay are indicated. In precleaved deletion assay RNA band resulting from removal of 4 U's (−4 U) from 5′ RNA and in precleaved insertion assay by addition of 2 U's (+2 U) to 5′ RNA are indicated. N2, mut-N2 5–10S, and ∼20S complexes as indicated are used in these assays.