Identification of novel components of Trypanosoma brucei editosomes

Abstract

The editosome is a multiprotein complex that catalyzes the insertion and deletion of uridylates that occurs during RNA editing in trypanosomatids. We report the identification of nine novel editosome proteins in Trypanosoma brucei. They were identified by mass spectrometric analysis of functional editosomes that were purified by serial ion exchange/gel permeation chromatography, immunoaffinity chromatography specific to the TbMP63 editosome protein, or tandem affinity purification based on a tagged RNA editing ligase. The newly identified proteins have ribonuclease and/or RNA binding motifs suggesting nuclease function for at least some of these. Five of the proteins are interrelated, as are two others, and one is related to four previously identified editosome proteins. The implications of these findings are discussed.

FIGURE 1.

Isolation of T. brucei editosomes. (A) Western analysis of the complexes purified by column chromatography (sequential ion-exchange and gel filtration), MAb affinity, and the TAP method showing the presence of editosome proteins TbMP81, TbMP63, TbREL1, and TbMP42. IgG heavy chain (hc) and tagged TbREL1 are indicated. (B) Adenylation of editosome preparations. The adenylated TbREL1, TbREL2, and tagged TbREL1 are indicated. (C) In vitro deletion RNA editing assays using the purified editosomes. Mitochondrial 20S fraction served as a positive control; no gRNA indicates the negative control. Input pre-mRNA, resulting edited RNA, and chimeras resulting from ligation of cleaved 3′ pre-mRNA to gRNA are indicated. (D) Silver-stained SDS-PAGE protein profile of MAb affinity-purified complexes. Proteins identified by LC-MS/MS are indicated, although some were not identified as discussed in the text. Size standards, bovine serum albumin (BSA) used as a blocking agent, and IgG heavy (hc) and light (lc) chains of the MAb are indicated. The relative migration of some proteins differs from that predicted from the gene sequence and some proteins comigrate.

FIGURE 2.

Identification of editosome protein TbMP90 by LC-MS/MS analysis. (A) CID spectrum of a tryptic peptide generated by the mass spectrometer. The spectrum matches that predicted for the peptide VLDLEEVYFR, both from N to C terminus (b ions) and C to N terminus (y ions), and corresponds to a peptide predicted from the CHR1.148 (TbMP90) gene sequence. (B) Fourteen tryptic peptides (shaded region, peptide 7 and 8 from left overlap) were identified across the protein that covered 16.8% of the sequence.

FIGURE 3.

Sequence similarities among TbMP90, TbMP67, TbMP61, TbMP46, and TbMP44. (A) Diagram showing a conserved mid-region (red), and weakly similar N (green) and C terminus (yellow) of these proteins. TbMP90 has a unique C terminus (blue). The putative catalytic center of the RNase III motif is indicated in black. The upstream and downstream sequence of this motif is similar between most of the proteins (orange). TbMP46 and TbMP44 have greater sequence conservation among them (dotted) than to the other three proteins. (B) Sequence conservation the in mid-region of TbMP61 (amino acids 162–355), TbMP67 (amino acids 123–325) and TbMP90 (amino acids 196–407). The amino acid alignments indicate conserved (*), semiconserved (:), and partially conserved (.) amino acids among these proteins. A line indicates the predicted catalytic RNase III domain, and it is related to the consensus pattern [DEQ]-[KRQT]-[LM]-E-[FYW]-[LV]-G-D-[SARH]. (C) Amino acids sequence similarities [conserved (|), semiconserved (:), and partially conserved (.)] of TbMP46 (amino acids 51–306) and TbMP44 (amino acids 14–258), and likely domains therein (indicated by a line). TbMP46 contains a probable Pumilio-family RNA binding domain (one repeat unit), and TbMP44 a RNase III domain.

FIGURE 3.

Sequence similarities among TbMP90, TbMP67, TbMP61, TbMP46, and TbMP44. (A) Diagram showing a conserved mid-region (red), and weakly similar N (green) and C terminus (yellow) of these proteins. TbMP90 has a unique C terminus (blue). The putative catalytic center of the RNase III motif is indicated in black. The upstream and downstream sequence of this motif is similar between most of the proteins (orange). TbMP46 and TbMP44 have greater sequence conservation among them (dotted) than to the other three proteins. (B) Sequence conservation the in mid-region of TbMP61 (amino acids 162–355), TbMP67 (amino acids 123–325) and TbMP90 (amino acids 196–407). The amino acid alignments indicate conserved (*), semiconserved (:), and partially conserved (.) amino acids among these proteins. A line indicates the predicted catalytic RNase III domain, and it is related to the consensus pattern [DEQ]-[KRQT]-[LM]-E-[FYW]-[LV]-G-D-[SARH]. (C) Amino acids sequence similarities [conserved (|), semiconserved (:), and partially conserved (.)] of TbMP46 (amino acids 51–306) and TbMP44 (amino acids 14–258), and likely domains therein (indicated by a line). TbMP46 contains a probable Pumilio-family RNA binding domain (one repeat unit), and TbMP44 a RNase III domain.

FIGURE 3.

Sequence similarities among TbMP90, TbMP67, TbMP61, TbMP46, and TbMP44. (A) Diagram showing a conserved mid-region (red), and weakly similar N (green) and C terminus (yellow) of these proteins. TbMP90 has a unique C terminus (blue). The putative catalytic center of the RNase III motif is indicated in black. The upstream and downstream sequence of this motif is similar between most of the proteins (orange). TbMP46 and TbMP44 have greater sequence conservation among them (dotted) than to the other three proteins. (B) Sequence conservation the in mid-region of TbMP61 (amino acids 162–355), TbMP67 (amino acids 123–325) and TbMP90 (amino acids 196–407). The amino acid alignments indicate conserved (*), semiconserved (:), and partially conserved (.) amino acids among these proteins. A line indicates the predicted catalytic RNase III domain, and it is related to the consensus pattern [DEQ]-[KRQT]-[LM]-E-[FYW]-[LV]-G-D-[SARH]. (C) Amino acids sequence similarities [conserved (|), semiconserved (:), and partially conserved (.)] of TbMP46 (amino acids 51–306) and TbMP44 (amino acids 14–258), and likely domains therein (indicated by a line). TbMP46 contains a probable Pumilio-family RNA binding domain (one repeat unit), and TbMP44 a RNase III domain.

FIGURE 4.

Sequence similarities [conserved (|), semiconserved (:), and partially conserved (.)] of the C termini of TbMP100 (amino acids 619–891) and TbMP99 (amino acids 618–906) that belong to the endonuclease/exonuclease/phosphatase family of proteins.