Domain function and predicted structure of three heterodimeric endonuclease subunits of RNA editing catalytic complexes in Trypanosoma brucei

Figures

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  Figure 1.. Schematic of random mutagenesis and complementation screen. (A) B6, B7 and B8 mutant allele libraries were generated by error-prone PCR and transfected into bloodstream form (BF) conditional null (CN) cell lines for constitutive expression. CN cells require tetracycline (tet) for expression of a regulatable WT allele. Removal of tet results in exclusive expression of mutant alleles enabling identification of loss of function (LOF) mutations as cell growth defects. (B) Alleles containing LOF mutations identified in (A) were recreated by site-directed mutagenesis and transfected into BF and procyclic form (PF) CN cell lines for further analyses of effects on growth, editing, and RECCs in BF versus PF.

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  Figure 2.. Effect on growth of exclusive expression of alleles with a mutated single amino acid. The log2 ratio of the effect on cumulative growth in –tet versus +tet is indicated by the scale showing reduction in blue, increase in orange, and no effect on growth in white. Growth of parental CN cells are at the top and above that of cells exclusively expressing wild-type (WT) or mutant alleles as indicated, including B6 (G202R), B7 (G166R) and B8 (G232R) which disrupt function (15) as controls. Data are representative of a minimum of three experiments.

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  Figure 3.. Alignments of B6, B7 and B8 motifs, amino acid sequences and positions of loss-of-function mutations. (A) Schematic alignment of T. brucei B6, B7 and B8 amino acid sequences showing gaps larger than two residues with the zinc-finger (ZnF), RNase III and RNase III-Associated Motif (RAM) regions indicated. The RNase III motif boundaries are from (22) and those for ZnF and RAM are established herein. (B) Schematic of B6, B7 and B8 with regions of conservation indicated according to the heat map scale of % identity. The three multispecies alignments show: (top) all RECC RNase III endonuclease paralogs and orthologs of N1, N2, N3, B4, B5, B6, B7 and B8; (middle) the B6, B7 and B8 paralogs and orthologs; (bottom) the orthologs of B6, B7 or B8. The positions of loss of function single amino acid mutations identified here are indicated in black, published mutations are in gray (15), and double ZnF mutations are highlighted in yellow.

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  Figure 4.. Comparison of BF and PF growth upon exclusive expression of mutated alleles. (A) Reduction (blue) or increase (orange) in growth upon knockdown, or exclusive expression of WT or mutated alleles. The time scales are adjusted account for the approximate 2× growth rate of BF versus PF. Color scale as in Figure 2. Data are representative of a minimum of three experiments. (B) Relative effect on growth of PF versus BF. Heat map of the log2 ratio of cumulative growth in –tet versus +tet for PF on day 10 and BF on day 5.

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  Figure 5.. Consequences to mRNA editing upon exclusive expression of mutant alleles in BF or PF as shown by RT-PCR. Brackets indicate gel regions that span the sizes from pre-edited (P) to fully edited (E) RT-PCR products from A6, MURF2, RPS12, ND7-5′, ND8 or CYb mRNAs. Never-edited (NE) ND4 mRNA is included as a mitochondrial mRNA control.

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  Figure 6.. SDS-PAGE Western analyses of mutant cell lysates. Blots were probed with antibodies for V5 tagged B6, B7 or B8; HA tagged cognate endonuclease N3, N2, or N1, respectively; RECC proteins A1, A2, L1 and A3, and anti-HSP70 for mitochondrial heat-shock protein 70 as a loading control. See Figure 7 for BN-PAGE western analysis of the same lysates.

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  Figure 7.. Consequences to RECC protein associations upon exclusive expression of mutant alleles. BN-PAGE and Western blots of cell lysates were probed using antibodies against V5 tagged B6, B7 or B8 (top panels); HA tagged cognate endonuclease N3, N2 or N1, respectively (middle panels); and the A2 core component of RECC (bottom panels). Small boxes above blots represent the loss of tagged B or N proteins in Figure 6 with darker blue colors indicating greater loss; see Figure 8 for additional information. RECC generally migrates at ∼1 MDa and some samples have a ∼800 kDa subcomplex.

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  Figure 8.. Summary of phenotypes resulting from exclusive expression of mutant B6, B7, or B8 alleles. The magnitude of impacts on growth, B6/7/8 and cognate N1/2/3 protein abundances and their associations with RECC, as well as editing of A6, MURF2, RPS12, ND7-5′, ND8, and CYb mRNAs are indicated using a heat map which indicates large (+++), medium (++), small (+), marginal (+/−), or no impact (−); na = not applicable. Locations of mutations are indicated in B6/7/8 schematic alignment from Figure 3. See Figures 4–7 for primary data. Zinc Finger (ZnF) and RNase III motifs and the RNase III Associated Motif (RAM) are indicated.

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  Figure 9.. Predicted Alphafold structures of T. cruzi B6, B7 and B8 compared to the crystal structure of S. cerevisiae Rnt1p RNase III monomer. The lower structures are rotated by 90° toward the viewer with the ZnF motif in yellow, RNase III motif in blue, and RAM in orange. The functionally important amino acids that were characterized in both BF and PF T. brucei are highlighted in magenta, and the universally conserved glycine in the RNase III signature motif is highlighted in red in all structures.

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  Figure 10.. Models of B6/N3, B7/N2, and B8/N1 dimers with RNA based on the predicted T. cruzi B6-B8 and N1-N3 Alphafold structures, and the crystal structure of the S. cerevisiae Rnt1p RNase III dimer with RNA substrate (PDB structure 5T16). The lower structures are rotated 90° toward the viewer and the RECC ZnF motif is yellow, the RNase III motif is blue, and RAM is orange. Locations of functionally important amino acids in both BF and PF are colored magenta. Dimeric Rnt1p RNase III domains cleave both RNA strands but only the N partner of the B6/N3, B7/N2 and B8/N1 heterodimers has a catalytic amino acid that is positioned for cleavage of the red RNA strand. The catalytically non-functional B partner cannot cleave the cyan RNA strand. Note that the placements of ZnF motifs in RECC structures mirror the positions of the dsRBDs of Rnt1p that bind substrate RNA.