Trypanosome RNAEditing Substrate Binding Complex integrity and function depends on the upstream action of RESC10

Figures

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  Figure 1.. Effects of RESC10 knockdown on T. brucei growth and editing factor levels, and relative abundance compared to other RESC proteins. (A) Growth curve of PF T. brucei upon RESC10 RNAi induction. Cell growth was measured in triplicate for 10 days for uninduced (-tetracycline; Tet) and induced cells (+tetracycline). (B) Top, Determination of the number of molecules of RESC10 present per cell. Known amounts of recombinant RESC10 were probed with anti-RESC10 antibodies alongside known cell and mitochondrial equivalents. The experiment was performed twice, and shown is a representative experiment. Bottom, Estimated RESC10 molecules per cell compared to that of previously published RESC proteins (12). (C) Effect of doxycycline (doxy)-mediated RESC10 knockdown on the abundance of selected RESC proteins, including both GRBC and REMC components, and the KREPA6 RECC protein. p22 is a loading control.

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  Figure 2.. RESC10 is an RNA binding protein that exhibits RNA dependent and independent interactions with other RESC proteins. (A) Recombinant MBP-RESC10, GST-RESC13, or his-p22 were incubated with radiolabeled gA6[14] gRNA or a 79-nt fragment of pre-edited A6 mRNA, UV-cross-linked, treated with RNase A, and separated on a 12% SDS-acrylamide gel. The left panel shows a Coomassie-stained gel image, and the right panel shows a phosphorimage. Asterisks indicate the positions of full length proteins. Molecular mass standards are on the left (kDa). (B) Top, Immunoprecipitation (IP) of RESC10 with α-RESC10 antibodies from cell lysates of T. brucei either treated with RNase inhibitor (−) or an RNase cocktail (+). Elutions were normalized for the levels of RESC10, and RESC proteins were probed with the respective antibodies shown on the left. Arrows indicate proteins whose RESC10 interaction is decreased following RNase treatment. Bottom, RESC13 was probed again, with both original loading (top two panels) and higher loading of the elution (3×) and lower loading of input (1/4X) (bottom panel). (C) Quantification of the experiment in B (top panels). The levels in the -RNase samples were set to 1.0.…

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  Figure 3.. Effect of RESC10 depletion on RESC protein-protein interactions. (A) IP of RESC13-HTM was performed from lysates of RESC10 replete (–doxy) and depleted (+doxy) cells (three day induction). Bound proteins were released by TEV cleavage, and RESC13 was normalized in the elutions by western blot. Normalized elutions were analyzed by western blot with antibodies against RESC proteins, including GRBC and REMC components. (B) Quantification of A. Levels in -doxy samples were set to 1.0. Bar graphs represent the average and standard deviation of two biological replicates each with two technical replicates. (C) IP of RESC6-PTP was performed in lysates of RESC10 replete (–doxy) and depleted (+doxy) cells as in A. (D) Quantification of C. Levels in -doxy samples were set to 1.0. Bar graphs represent the average and standard deviation of two biological replicates each with two technical replicates. (E) IP of RESC5-MHT IP of RESC13-HTM was performed in lysates of RESC10 replete (-doxy) and depleted (+doxy) cells as in (A). The RESC12A panel contains three times the amount of eluate as do the other panels. (F) Quantification of (E). Levels in -doxy samples were set to 1.0. Bar graphs represent…

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  Figure 4.. Effect of RESC10 depletion on RESC protein-RNA interactions. (A) Comparison of mRNAs and gRNAs immunoprecipitated with RESC6-PTP from cells grown with or without doxycycline to induce RESC10 knockdown for 3 days. Fold change with RESC10 knockdown (KD) represents the RNA levels detected in the RIP from doxycycline induced cells compared to the RIP from uninduced cells. RNA levels were standardized against 18S rRNA, and numbers represent the mean and standard deviation of two biological replicates, each with three technical replicates. (B) Comparison of mRNAs and gRNAs immunoprecipitated with RESC13-HTM from cells grown with or without doxycycline for three days to induce RESC10 knockdown. Quantification as in (A). Shaded area indicates 1.5-fold change.

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  Figure 5.. Effect of RESC10 depletion on mitochondrial RNA levels. (A) RNA was quantified by qRT-PCR using primer sets that specifically detect the pre-edited (Pre) and edited (Edit) versions of the pan-edited and moderately edited RNAs indicated. Relative RNA abundance represents RNA levels in tetracycline-induced cells compared to levels in uninduced cells (three day induction). RNA levels were normalized to 18S rRNA levels, and numbers represent the mean and standard deviation of two biological replicates, each with three technical replicates. The level of RESC10 knockdown was quantified using RESC10 specific primers. (B) RNA was isolated from RESC10 RNAi cells either grown in the absence or presence of tetracycline for three days, and then labeled with [α32P]-GTP using guanylyltransferase to identify gRNAs and resolved on a denaturing gel. A cytoplasmic RNA that was used for normalization and the labeled gRNA are indicated.

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  Figure 6.. High-throughput sequence analysis of RPS12 mRNA in RESC10 depleted cells. (A) Average number of normalized pre-edited, partially edited, and fully edited RPS12 transcripts for ten uninduced (Avg UI) and two RESC10 RNAi induced samples. P-values (Student t-test): 0.002 for RPS12 partially edited (∗). (B) RPS12 mRNA 3′ edited sequence showing EPS arising upon RNAi of RESC10 (black diamonds and outlines), RESC13 (dark blue diamonds), RESC11A (orange diamonds), RESC2 (turquoise diamonds), RESC8 (red diamonds), and RESC14 (green diamonds). Exacerbated Pause Sites (EPSs) are defined as having padj <0.05 in both biological replicates (12). Those EPS found in five of the six knockdown cell lines are indicated by their ES number above the outline. Bars below the sequence indicate gRNAs, numbered 3′ to 5′ (42). gRNA anchor regions are depicted with bold lines. Gray regions at the 5′ and 3′ ends represent variation in gRNA lengths within a gRNA class. RPS12 stop codon is underlined; additional underscores are shown for clarity in stretches of unedited sequence to align numbers with the correct ES. (C) Significance of the overlaps in EPSs between six different RESC protein knockdowns…

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  Figure 7.. Comparison of RPS12 mRNA junction sequences at ES30, ES53 and ES78 in numerous RESC protein knockdown cell lines. Shown are those ES that were quantified as EPS in five of six cell lines tested. Abundant sequences that were present in >25 average normalized counts in knockdown cell lines are shown. The region matching fully edited is shown in red, pre-edited in black, and the junction region (containing mis-edited sequence) in blue. The editing stop site (ESS) number and the junction length (JL) are shown to the right of each sequence. To the right is indicated the average number of each sequence detected from 10 uninduced samples (UI) and two induced samples (KD). The fold change (FC) of each sequence compared to the average of uninduced is also indicated for all RESC proteins (12,25,29).

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  Figure 8.. High-throughput sequence analysis of ND7–5′, CYb and MURF2 mRNAs in RESC10 depleted cells. (A) Average number of normalized pre-edited, partially edited, and fully edited ND7–5′ transcripts for ten uninduced and two RESC10 RNAi induced samples. P-values (Student t-test): 0.00087 for ND7–5′ pre-edited (∗∗) and 0.00088 for ND7–5′ partially-edited (∗∗). (B) Average number of normalized pre-edited, partially edited, and fully edited CYb transcripts for ten uninduced and two RESC10 RNAi induced samples. P-values (Student t-test): 0.0062 for CYb pre-edited (∗) and 0.00035 for CYb fully-edited (∗∗). (C) Average number of normalized pre-edited, partially edited, and fully edited MURF2 transcripts for ten uninduced and two RESC10 RNAi induced samples. P-values (Student's t-test): 0.000002 for MURF2 pre-edited (∗∗∗) and 0.000041 for MURF2 fully-edited (∗∗∗). (D) CYb edited mRNA sequence with exacerbated pause sites (EPS) arising upon RESC10 RNAi in two biological replicates indicated with black diamonds. EPSs are defined as having padj <0.05 in both biological replicates (12). (E) MURF2 edited mRNA sequence with exacerbated pause sites (EPS) arising upon RESC10 RNAi in two biological…

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  Figure 9.. Working model of RESC10 action. RESC10 associates with GRBC to promote an editing-competent conformation of the GRBC module. Once GRBC is in the proper conformation, REMCs and organizer proteins can assemble into RESC. Dashed lines indicate that the precise timing and extent of RESC10 dissociation and association of distinct REMC and organizer proteins is not fully established. However, RESC10 appears to minimally interact with REMC factor, RESC13. The absence of a robust RESC10-RESC13 interaction, and the low abundance of RESC10 are consistent with RESC10 dissociation from the assembling RESC complex. Once RESC is assembled, RECC can associate with RESC/mRNA/gRNA and catalyze editing. This cycle may occur at many or most editing sites, and it appears to be especially critical when a deletion RECC needs to be substituted for an insertion RECC. Proteins are indicated by their RESC numbers, and ‘REMCs’ indicates heterogeneous REMC complexes. Blue line ending in AAA indicates mRNA shown 5′ to 3′; red line starting with UUU indicates gRNA shown 3′ to 5′. See text for additional details.