A unique mRNA decapping complex in trypanosomes

Abstract

Removal of the mRNA 5' cap primes transcripts for degradation and is central for regulating gene expression in eukaryotes. The canonical decapping enzyme Dcp2 is stringently controlled by assembly into a dynamic multi-protein complex together with the 5'-3'exoribonuclease Xrn1. Kinetoplastida lack Dcp2 orthologues but instead rely on the ApaH-like phosphatase ALPH1 for decapping. ALPH1 is composed of a catalytic domain flanked by C- and N-terminal extensions. We show that T. brucei ALPH1 is dimeric in vitro and functions within a complex composed of the trypanosome Xrn1 ortholog XRNA and four proteins unique to Kinetoplastida, including two RNA-binding proteins and a CMGC-family protein kinase. All ALPH1-associated proteins share a unique and dynamic localization to a structure at the posterior pole of the cell, anterior to the microtubule plus ends. XRNA affinity capture in T. cruzi recapitulates this interaction network. The ALPH1 N-terminus is not required for viability in culture, but essential for posterior pole localization. The C-terminus, in contrast, is required for localization to all RNA granule types, as well as for dimerization and interactions with XRNA and the CMGC kinase, suggesting possible regulatory mechanisms. Most significantly, the trypanosome decapping complex has a unique composition, differentiating the process from opisthokonts.

Graphical Abstract

Figure 1.

Predicted structure of ALPH1. (A) Schematics of ALPH1: the protein consists of a catalytic domain with N- and C- terminal extensions. (B) Secondary structure predictions for ALPH1. The N-terminal region of ALPH1 is predicted to be mostly unstructured, exposed and disordered, with the longest almost continuous disordered region at residues 57–166. The central region responsible for the catalytic activity contains a predicted α/β-domain which overlaps with the annotated Metallo-dependent phosphatase-like region (Interpro superfamily IPR029052, residues 253–519). The catalytic domain is followed by a disordered linker and a small C-terminal domain comprised of α-helices. (C-E) Structural model of ALPH1 predicted by AlphaFold. Shown in cartoon depiction are the catalytic domain (residues 253–543, in pink) and the C-terminal domain (residues 598–734, in rainbow). Regions with low model confidence (the N-terminal region and two disordered linkers at residues 421–437 and 544–597) were omitted. The central part of the catalytic domain overlaps well with ALPH2 crystal structure (PDB entry 2QJC, (47)) with 44% sequence identity in this region (residues 263–511, in hot pink). Highlighted are key residues forming the active site coming from the 4 phosphatase motifs (in khaki) and 2 ALPH motifs (in teal) (6). The inset (D) shows superposition of these residues with those from ALPH2 (PDB entry 2QJC, in light blue), including two Mn²⁺ ions (purple), 3 water molecules (their oxygens in red) and a phosphate ion (orange). (E) The predicted aligned error for the AlphaFold model (available at the AlphaFold Protein Structure Database entry for UniProt ID Q583T9) indicates two domains with little confidence regarding their mutual orientation (values above 15Å in the region corresponding to the inter-domain accuracy for the catalytic and C-terminal domains, indicated by the blue rectangle).

Figure 2.

ALPH1 is a dimer in solution. Size exclusion chromatography coupled with multiangle light scattering was used for protein molecular mass determination of wild type ALPH1 (WT), ALPH1ΔN (amino acids 222–734), ALPH1ΔC* (amino acids 120–552) and ALPH1cat (amino acids 222–552). All proteins were expressed in E. coli fused to a His-SUMO tag, purified using Nickel-affinity chromatography followed by Ion-exchange chromatography (Q or Heparin column) and the His-SUMO tag was cleaved off with SUMO protease. All proteins were loaded at a concentration of 5 mg/ml. Full length ALPH1 and ALPH1ΔN elute as a dimer, while ALPH1ΔC* and ALPH1cat elute as monomers.

Figure 3.

ALPHΔN/– cells are viable. (A) Cumulative growth of procyclic cells of WT (one clone), ALPHΔN/+ (2 clones) and ALPHΔN/– (4 clones). (B) Cumulative growth of bloodstream form cells of WT (one clone), ALPHΔN/+ (one clone), ALPH1 WT/– (2 clones) and ALPHΔN/– (3 clones). (C) ALPH1 was detected in WT, ALPHΔN/+ and ALPHΔN/– cells by immunofluorescence with ALPH1 antiserum. One representative images is shown for each cell line.

Figure 4.

Localization of ALPH1 truncations. (A, D) Overview about ALPH1 truncations used in this study. Numbers refer to amino acid positions. Wild type ALPH1 and all truncations were expressed with an inducible expression system in cells expressing mChFP-DHH1 from the endogenous locus. (B, E) Left: Representative microscopy images of untreated cells. Right: Posterior pole localization of ALPH1 (truncations) was quantified by dividing the percentage of ALPH1 at the PP granule through the percentage of DHH1 in the same region. Any value above 1 (red line) indicates localization to the posterior pole. Each dot represents one cell. The number of cells is indicated in the figure (N). A two-tailed unpaired homoscedastic TTEST (p) was used to test the significance of any decrease or increase in PP localization in comparison to WT ALPH1. (C, F) Left: representative microscopy images of starved cells. DHH1 served as a stress granule marker. Middle: Posterior pole localization of ALPH1 (truncations) was quantified as above. A significant increase in PP localization in comparison to untreated cells is marked with ***, ** or * correlating to the result of a two-tailed unpaired homoscedastic TTEST of <0.0005, <0.005 or <0.05, respectively. Right: The percentage of ALPH1 (truncations) in stress granules was quantified. Granules were identified by the granule Marker DHH1. Each dot represents one cell. The number of analysed cells is indicated in the figure (N). A two-tailed unpaired homoscedastic TTEST (p) was used to test the significance of any decrease or increase in stress granule localization in comparison to WT ALPH1.

Figure 5.

BioID proximity labelling with ALPH1. (A) Western blot loaded with cell extracts from cells expressing TurboID-HA fusions of ALPH1 (endogenous expression and inducible overexpression) and truncated ALPH1 (inducible overexpression). Wild type cells and cells expressing eYFP-TurboID-HA served as controls. The blot was probed with IRDye 800CW streptavidin to detect biotinylated proteins (shown in red) and with rat-anti-HA (3F10, Sigma) followed by IRDye 680RD goat anti-rat IgG to detect the bait proteins (shown in green). (B) Hawaii-plot (multiple comparative volcano plots) for statistical analysis of BioID experiments with full-length ALPH1 and the three truncated fragments ΔN2 (lacking the N-terminal domain), ΔC (lacking the C-terminal domain) and the catalytic fragment (cat; lacking both, the N- and C-terminal domain). All samples were prepared in triplicates. To generate the volcano plots, the − log₁₀P-value was plotted versus the t-test difference (difference between means), comparing each respective bait experiment to the wt control. Potential interactors were classified according to their position in the plot, applying cut-off curves for ‘significant class A’ (SigA; FDR = 0.01, s0 = 0.1) and ‘significant class B’ (SigB; FDR = 0.05, s0 = 0.1). Detected protein groups are coloured for their respective significance class in the full-length experiment (red = SigA; blue = SigB). Selected proteins are labelled (green for apparent C-terminal interactors; orange for apparent N-terminal interactors; blue for interactors with posterior pole localization, that do not rely on the presence of the terminal regions). Three further proteins that may be part of the decapping complex based on experiments with other baits (see below) are shown in black. For all data see Supplementary Table S1; a comparison to a BioID experiment with endogenously expressed ALPH1, and a respective correlation analysis, is shown in Supplementary Figure S4.

Figure 6.

Analysis of the ALPH1 interactome. (A) The final high-confidence table of potential ALPH1 interacting proteins identified by BioID (Supplementary Table S1D). The color-scheme indicates whether a protein was identified with the respective ALPH1 BioID bait and in which significance group (green/red), whether the protein is involved in mRNA metabolism (light brown), and whether the protein is known to localize to either the posterior pole (PP, light blue) or to starvation stress granules (SGs, dark blue) as judged by Tryptag (44) or own data (published here or elsewhere). None of the proteins was detected or enriched with the eYFP control, with the exception of the Tc40-antigen like protein. (B) Tb927.10.10870 was expressed as a HaloTag® fusion and stained with TMR in a cell line also expressing ALPH1-eYFP from the endogenous locus. Representative images of one untreated and one heat shocked cell (2 h 41°) are shown (Z-stack projection sum slices of 5 slices a 140 nm); note that colors are switched for clarity. Tb927.10.10870 fused to eYFP was also co-expressed with PABP2-mCHFP (a marker for starvation stress granules (31)) and starvation was induced by incubation in PBS for 2 h. Images are presented as deconvolved Z-stack projections (sum slices). (C) Tb927.9.12070 and Tb927.11.3490 were expressed fused to a C-terminal eYFP tag from the endogenous locus in a cell line also expressing ALPH1-mChFP or the stress granule marker protein PABP2-mChFP from the endogenous locus. Fluorescent images of representative cells are shown under untreated conditions, after 2 h heat shock at 41°C and after 2 h of PBS starvation as projections (sum slices) of deconvolved Z-stacks. Note that the autofluorescence of the lysosome is visible in the red channel, because the ALPH1-mCHFP fluorescence is very weak.

Figure 7.

The trypanosome decapping complex. (A) Volcano plot of the CMGC-type kinase interactome. The CMGC-type protein kinase Tb927.10.10870 was expressed as TurboID fusion and biotinylated proteins were purified by streptavidin affinity and identified by LC-MSMS. Shown is a volcano plot generated by plotting the − log₁₀P-value versus the t-test difference, comparing the bait experiment (TurboID- tagged Tb927.10.10870) to a wild type (wt) control. All proteins of the final ALPH1 BioID list are labelled. The statistical cut-off (SigC; FDR = 0.05 S0 = 2.0) is indicated by a gray curved line. The full dataset is shown in Supplementary Table S2. (B) Volcano plot of the XRNA interactome in T. brucei and T. cruzi.XRNA was extracted from trypanosome cells via GFP (T. cruzi) or mNeonGreen (T. brucei) nanobody beads and co-purified proteins were identified by LC-MSMS. Shown are volcano plots for XRNA affinity capture in T. brucei (left) and T. cruzi (right). Plots were generated by plotting the −log₁₀P-value versus the t-test difference, comparing the bait experiment (XRNA) to a wild type (wt) control. All proteins that are also in the final stringent list of the ALPH1 BioID are labelled. The statistical cut-off (FDR = 0.05 S0 = 2) is indicated by a grey line. The full datasets are shown in Supplementary Table S3. (C) A model of the ALPH1 core complex. Overlapping proteins from interactors of ALPH1, CMGC-type protein kinase and XRNA are shown as the most likeliest components of the ALPH1 core complex. Proteins with slightly less robust evidence for being complex subunits are shown in dashed circles. Note that the dimerization of ALPH1 via the C-terminus observed in vitro is not shown here for clarity. Moreover, we cannot exclude that binding sites of either ALPH1 domain are used in competition rather than simultaneously. (D) Summary of the ALPH1 interactome. ALPH1 is depicted as a schematic homodimer and all interacting proteins identified in this work are shown connected to the respective ALPH1 domain(s). Localization of proteins to the posterior pole and/or to stress granules is indicated at the left. (E) Evolution of distinct components in decapping complexes in Kinetoplastida. Coulson plot representation of subunit presence layered onto a simplified eukaryotic phylogeny, to emphasize subunit losses and replacements of subunits between Metamonads and Discoba. In teal are canonical subunits including Dcp1/2 and Edc, while in magenta are the subunits reported here as associated with APLH1. Significantly, XRNA/XRN1 (orange) is retained by all lineages. The selective pressure and order of events, such as by a gradual or more catastrophic change its unknown, but parallels several additional systems such as the lamina and kinetochore (78,79). Significantly, in Metamonada, Heterolobosea and Euglenida subunit retention is sparse and may reflect the presence of an additional set of divergent components remaining to be identified. For a list of gene IDs for decapping complex component homologs see Supplementary Table S5.

Figure 8.

The posterior pole granule. (A) Changes in ALPH1 localization during the cell cycle. Cells were induced to overexpress ALPH1-eYFP for 24 h and representative images of the different cell cycle phases are shown. The different ALPH1 localizations were quantified from 376 cells and are shown as percentages underneath the images. (B) ALPH1 localizes posterior to XMAP215. Expression of ALPH-eYFP was induced by tetracyclin for 24 h in cells also expressing the microtubules plus end marker protein mChFP-XMAP215 from the endogenous locus. One representative cell is shown with the region of the posterior pole granule enlarged. (C, D) Changes in ALPH1 localization to the PP with mRNA-metabolism drugs. Cells co-expressing ALPH1-eYFP-4Ty1 and mChFP-DHH1 were left untreated or exposed for 1 h to 41°C (HS), starved for 2 h in PBS (starvation), incubated for 1 h with actinomycin D (ActD), sinefungin (SF) or cycloheximide (CHX). ALPH1 was either expressed from the endogenous locus (C) or overexpressed (24 h TET) (D). The percentage of ALPH1 at the posterior pole was quantified. The P-values are the result of an unpaired, two-tailed t-TEST and indicate the likelihood that the fraction of ALPH1 localization to the posterior pole is different between treated and untreated cells. (E) The PP-granule contains no mRNAs. Cells expressing ALPH1-4Ty1-eYFP were probed for total mRNA by oligos antisense to either the miniexon sequence (left) or the poly (A) tail (right). ALPH1 was detected by immunofluorescence using anti-Ty1 (BB2). Both untreated and heat-shocked cells were used; the later to increase the amount of ALPH1 at the posterior pole. Note that the miniexon probe also recognizes the nuclear-localized SL RNA next to total mRNAs; heat shock reduces total mRNA levels, but not SL RNA levels and thus causes an increase in the nuclear signal (31). (F) XRNA-eYFP was expressed from the endogenous locus in either wild type cells or ALPH1ΔN/– cells. XRNA localization was monitored after 70 minutes of heat shock. An arrow points to XRNA-eYFP localized at the posterior pole.