Native mitochondrial RNA-binding complexes in kinetoplastid RNA editing differ in guide RNA composition

Abstract

Mitochondrial mRNAs in kinetoplastids require extensive U-insertion/deletion editing that progresses 3'-to-5' in small blocks, each directed by a guide RNA (gRNA), and exhibits substrate and developmental stage-specificity by unsolved mechanisms. Here, we address compositionally related factors, collectively known as the mitochondrial RNA-binding complex 1 (MRB1) or gRNA-binding complex (GRBC), that contain gRNA, have a dynamic protein composition, and transiently associate with several mitochondrial factors including RNA editing core complexes (RECC) and ribosomes. MRB1 controls editing by still unknown mechanisms. We performed the first next-generation sequencing study of native subcomplexes of MRB1, immunoselected via either RNA helicase 2 (REH2), that binds RNA and associates with unwinding activity, or MRB3010, that affects an early editing step. The particles contain either REH2 or MRB3010 but share the core GAP1 and other proteins detected by RNA photo-crosslinking. Analyses of the first editing blocks indicate an enrichment of several initiating gRNAs in the MRB3010-purified complex. Our data also indicate fast evolution of mRNA 3' ends and strain-specific alternative 3' editing within 3' UTR or C-terminal protein-coding sequence that could impact mitochondrial physiology. Moreover, we found robust specific copurification of edited and pre-edited mRNAs, suggesting that these particles may bind both mRNA and gRNA editing substrates. We propose that multiple subcomplexes of MRB1 with different RNA/protein composition serve as a scaffold for specific assembly of editing substrates and RECC, thereby forming the editing holoenzyme. The MRB3010-subcomplex may promote early editing through its preferential recruitment of initiating gRNAs.

Keywords: RNA editing; Trypanosoma brucei; deep RNA sequencing; guide RNA; mitochondrial RNA-binding complex MRB1.

FIGURE 1.

Native immunopurified subcomplexes of MRB1 with either H2 or 3010 subunits. (A) Western analysis of H2 and 3010 in IPs by the indicated antibodies, and in mitochondrial extract (ME). H2 (∼240 kDa) is often fragmented, and 3010 (57.5 kDa) migrates slightly below IgG in the IPs. A fainter band below 3010 is a breakage product of this protein in ME (*). The blot was split into halves treated with either anti-H2 or anti-3010 antibodies. (B) Western analysis of the RECC subunit REL1 ligase in IPs and ME. (C) [³²P]G-capping of gRNA 5′ triphosphate with guanylyltransferase on 15% UREA-PAGE to concentrate gRNA in a discrete band. (D) Western analysis of the MRB1 subunit GAP1 in test and mock IPs. Mock IPs (Mk) used an irrelevant affinity-purified antibody. (E) Site-specific crosslinking of H2 and 3010 IPs with a pre-edited mRNA construct whose first editing site contains ³²P in its phosphodiester bond and 4-thioU in the flanking 5′ nucleotide. After RNase trimming, the protein-RNA adducts were resolved on 10% SDS-PAGE. (F) Crosslinks as in E, but on a high-resolution 6% SDS-PAGE. Native (wt) and ectopic (tagged) H2 differ in mobility due to an ∼20 kDa tag. Tagged H2 was affinity-purified (AP). Intact and fragmented H2 are marked. Molecular markers are in kDa. All IPs (200 mM KCl, 5% BSA) used precleared extract.

FIGURE 2.

Analysis of edited mRNA coverage by gRNAs in Illumina libraries. (A–G) Steady-state gRNA from PF parasites and gRNA in immunopurified MRBs are annotated in blocks of edited sequence, each directed by a gRNA. Cumulative standard and G·U pairs between edited mRNA and gRNAs are scored in nucleotide frequency plots (NFPs Log₂), including initiating gRNAs (i.e., block 1 or B1) and major upstream gRNAs (B2 or B3) in our libraries. Some gRNAs may guide alternative editing (e.g., B1.alt and B2.alt), and the ND7 5′ domain uses at least two similar initiating gRNAs (B1a and B1b). The entire editing domain in cytochrome B (CYb) and the 3′ terminus of other domains are plotted. The sequence of CYb (including all 34 U-insertions as lowercase t's) and the ND7 5′ domain are shown. Equal protein loads were applied to the IPs, and the gRNA was gel-isolated and extracted. gRNA from IPs and total RNA were adjusted to apply comparable amounts in the libraries (e.g., Supplemental Fig. S3; see Materials and Methods). gRNA from the IPs was directly ligated to the adapters, whereas total gRNA from PF parasites was first treated with 5′ monophosphate specific Terminator exonuclease, which degrades rRNA but not 5′ triphosphate gRNA ends. (H,I) Northern blots of select initiating gRNAs from IPs and PF cells. The blot was hybridized with the A6 gRNA probe, stripped, and re-used with the ND7 gRNA probe. 15% UREA-PAGE was run as in Figure 1C.

FIGURE 3.

gRNA alignments at editing blocks B1 and B2 with currently annotated edited mRNAs, and predicted alternative editing patterns. Base-paired gRNA sequences are in 3′→5′ orientation. Regions of interest are identified as follows: editing domain (bold), mature mRNA sequence (underlined), gRNA guide domain (gray box), length of the 3′ U tail (subscript), and stop codon (double line). Proposed alternative U-insertions and U-deletions (“t” and “⋆”, respectively, in a box) result in higher quality duplexes, i.e., longer guide domains and changes in encoded C-terminal amino acids (in ND7 3′ domain [B] and RPS12 [F]) or 3′ UTR (in CO3 [D], ND8 [E], and A6 [G]). No alternative editing is predicted for CyB (A) and ND7 5′ domain (C). Homology alignments of gRNAs are given in 5′-3′ orientation with identities (dotted boxes) and guide domains (gray boxes). Aligned gRNAs from the study in EATRO 164 cells by Koslowsky et al. (2013) are indicated. The gRNA numbering uses standard nomenclature indicating paired nucleotide positions in edited mRNAs. The arrow in ND8 indicates an extra guiding “A” in B1.alt. The arrow in RPS12 indicates a position in the guide domain of gRPS12(267–322) that forms an A·C mismatch with mRNA.

FIGURE 4.

H2-MRB and 3010-MRB copurify with pre-edited and edited mRNAs. (A) Fold-enrichment in IPs of the samples normalized to the mock IP in qPCR assays. (E) Edited, (P) pre-edited (P), (COI) never-edited COI mRNA, two reference transcripts: (TUB) tubulin mRNA and (18S) nuclear 18S rRNA (Fold = 2^−ddCq, where ddCq = Cq test IP−Cq mock IP). (B) dCq of steady-state mRNAs in mitochondrial extract relative to background tubulin mRNA or nuclear 18S rRNA used as reference (dCq = 2^{(Target Cq−Ref Cq)}). For example, edited ND7 is more abundant than other edited mRNAs (i.e., has a lower Cq). All end-point amplicons in this study were single products during linear amplification, gel-purified and sequenced. Cq duplicates (STDEV ≤ 0.1) were averaged, and dilutions were adjusted to 100%.

FIGURE 5.

Working model. Dynamic higher-order MRB1 “organizers” composed of several specialized subcomplexes (MRBs) with different RNA/protein composition may coordinate recruitment of editing substrates (pre-edited mRNA and gRNA) and catalytic RECCs forming an editing holoenzyme. MRBs may bind edited mRNAs and route them into ribosomes by undefined “hand-over” mechanisms. Native helicase REH2 binds RNA, presumably gRNA, mRNA, or both, and forms a stable MRB with editing substrates, unwinding activity, and several subunits including other RNA-binding proteins (stars). REH2-associated unwinding seems to promote stable assembly of editing substrates with REH2-MRB but could also affect other MRBs and editing progression. The MRB3010 subunit affects an early editing step and may not bind RNA directly but forms a separate stable MRB. The current study showed that the MRB3010-subcomplex associates with editing substrates and edited mRNA and suggests that it promotes early editing through its preferential recruitment of initiating gRNAs. Both purified REH2-MRB and MRB3010-MRB contain GAP1/2 subunits that bind and stabilize gRNA. A common RNA-binding core in MRBs may include GAP proteins and several RNA crosslinking subunits that we detected but need to be identified. Dynamic interactions between these MRBs and additional factors (MRBs and non-MRBs not shown in the cartoon) may control substrate recruitment and interactions during initiation and progression between blocks (e.g., B1-to-B4). A proposed TbRGG2 MRB-like subcomplex does not contain REH2, MRB3010, or GAP proteins (Madina et al. 2011; Ammerman et al. 2012). Distinct RNA/protein composition of MRBs may also impact transient associations with RECCs, mitoribosomes, and other mitochondrial factors. Recombinant proteins known to crosslink with synthetic RNA (stars) include the following: GAPs with gRNA, and the 4160/8160 paralogs and TbRGG2 preferentially with mRNA-like fragments. The initiation gRNA anchor anneals just 3′ of the editing domain. Later anchors often need edited sequence. Whether gRNAs remain annealed to edited mRNA is unknown.