KREH1 RNA helicase activity promotes utilization of initiator gRNAs across multiple mRNAs in trypanosome RNA editing

Figures

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  Figure 1.. Effects of KREH1 gene disruption on growth and U-indel RNA editing. (A) Schematic representation of the knock out (KO) and add back (AB) strategies. Linearized plasmid was transfected; shown is only the region encompassing and flanking the KREH1 gene and drug resistance markers. (B) KO confirmation by PCR of genomic DNA isolated from six different clones using UTR-based and ORF-based primers. (C) Growth of 29-13 (WT; black), KREH-KO (red), and KREH-AB (blue, -doxy; green, +doxy) parasites was monitored for 12 days. Growth rates were examined using one biological replicate each of clones 2 and 3 of KREH1-KO, two replicates of WT, and three replicates of KREH1-AB. (D) RNA was isolated from KREH1-KO and WT T. brucei cells and quantified by qRT-PCR using primer sets that specifically detect total, pre-edited, or edited versions of mRNAs. Relative RNA abundance represents RNA levels in KREH1-KO cells compared to levels in WT cells. RNA levels were normalized to 18S rRNA. Levels and numbers represent the mean and standard error of six determinations mRNAs (one biological replicate each of clones 2 and 3, each in technical triplicate). Grey shading indicates 1.5-fold change. (E) Edited…

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  Figure 2.. KREH1-KO impacts both editing initiation and progression through the gRNA-1 and gRNA-2 directed blocks of A6 mRNA. (A) Read counts for each sample were normalized to 100 000, and the average levels of normalized pre-edited, partially edited, and fully edited A6 reads were calculated for 29-13 (WT), KREH1-KO, and KREH1-AB samples. Partially edited sequences are defined as reads that are not fully edited but have some U insertion or deletion; fully edited sequences are defined as reads with canonical fully edited sequence up to the 5′ primer used for amplification of the A6 mRNA 3′ end. (B) Exacerbated Pause Sites (EPSs) were calculated for KREH1-KO samples with WT samples used as the control. Any Editing Stop Site for which the number of reads is significantly higher (Padj < 0.05) in the KREH1-KO condition than in the WT for both KO replicates is considered an EPS. Positions of identified EPSs are indicated with red diamonds above fully edited A6 sequence. Numbers indicate ES number. gRNA coverage (59) is shown with black bars; thick black portions represent gRNA anchor sequences. Gray bars represent areas of variable gRNA length. Quantification of the number of reads for each EPS…

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  Figure 3.. Junction analysis reveals a gRNA skipping phenotype in KREH1-KO. (A) Average number of normalized read counts in different junction length classes for WT and KREH1-KO samples. The length of a junction is the number of ES between the Editing Stop Site and the 5′ most ES with any U insertion or deletion. Each Student's t-test was performed on n = 5 WT and n = 2 KREH1-KO samples. ns, not significant; * Padj < 0.05; ** Padj < 0.01; *** Padj < 0.001. (B) The 10 most abundant A6 mRNA sequences with Editing Stop Site 24 in KREH1-KO samples are aligned with pre-edited and fully edited A6 sequences. A 52 nt portion of pre-edited A6 sequence as well as fully edited sequence beyond ES 39 were removed for ease of visualization. The position of the initiator gRNA (gRNA-1) is shown below the fully edited sequence, with the thick portion representing the gRNA anchor region. The junction lengths (JL), averaged normalized counts for KO and WT samples (KO Avg; WT Avg), and fold change (FC) are shown to the right of each sequence. Black text: pre-edited sequence; red text: fully edited sequence; blue text: junction sequence as determined by TREAT. The junction can contain a mixture of pre-edited,…

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  Figure 4.. Effect of KREH1-WT, KREH1-KA and KREH1-EQ overexpression on growth and RNA editing. (A) Schematic diagram of DEAD box RNA helicase structure. The DEAD box helicase core consists of two RecA-related domains containing a minimum of 12 conserved motifs that characterize the family. Two motifs, GSGKT and DEAD, are highlighted and demonstrate conservation from trypanosomes to humans. The K and E residues that were mutated are highlighted in yellow and blue, respectively. The figure is not drawn to scale and is adapted from (51). (B, D, F) Growth of triplicate samples of uninduced (-doxy) and induced (+doxy) cells overexpressing KREH1-WT (B), KREH1-KA (D) or KREH1-EQ (F) was monitored for 12 days. Insets: anti-myc western blots showing expression levels of KREH1 variants relative to p22 loading controls. All blots are from the same representative gel, which was cropped for ease of visualization. (C, E, G) Levels of total, pre-edited and edited mRNAs in cells overexpressing KREH1 variants. Levels of mRNAs encoding KREH1 variants are also shown. RNA was isolated from uninduced and induced cells after 3 days of induction and quantified by qRT-PCR. Relative RNA abundance represents RNA…

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  Figure 5.. DN KREH1 overexpression inhibits RNA editing initiation. (A) Read counts for each sample were normalized to 100 000, and the average levels of normalized pre-edited, fully edited and partially edited A6 reads were calculated for the six combined uninduced samples and for two doxycycline-induced KREH1 overexpressor samples. Partially edited and fully edited sequences defined as in Figure 2. (B) A6 EPSs were calculated for KREH1 overexpressor samples with all six uninduced samples used as the control. Any Editing Stop Site for which the number of reads is significantly higher (Padj < 0.05) in a KREH1 overexpressor cell line for both replicates is considered an EPS. Positions of identified EPSs are indicated with colored diamonds above fully edited A6 sequence. gRNA coverage is shown with black bars; thick black portions represent gRNA anchor sequences. Gray bars represent areas of variable gRNA length. (C) Levels of pre-edited, fully edited, and partially edited RPS12 reads were calculated as in (A). (D) RPS12 EPSs were calculated for KREH1 OE samples as in (B). (E) Levels of pre-edited, fully edited, and partially edited CYb reads were calculated as in (A). (F) CYb EPSs were…

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  Figure 6.. Overexpression of KREH1 dominant negative mutants results in gRNA skipping and initiator gRNA accumulation. Shown in A–C are the proportion of reads for a given mRNA that are pre-edited in the region directed by the first gRNA and contain a modification 5′ of this region. (A) The proportion of A6 mRNA reads with Editing Stop Site 24 and which are pre-edited between ES 25–39 and edited 5′ of ES39 was calculated for uninduced and induced KREH1 overexpressor samples. (B) Same as in (A), but measuring RPS12 mRNA reads with Editing Stop Site 9 and which are pre-edited between ES 9–22 and edited 5′ of ES22. (C) Same as in A, but measuring CYb reads with Editing Stop Site 558 and which are pre-edited between ES 558–569 and edited 5′ of ES569. (D) Abundance of A6 gRNA-1 was measured by qRT-PCR in the induced KREH1 overexpressor lines (plotted relative to the level in the corresponding uninduced cells), in KREH1-KO and KREH1-AB cells (plotted relative to that in WT 29-13 cells), and in induced RESC8 RNAi cells (plotted relative to the corresponding uninduced cells). Dotted line indicates no change compared to control. Student's t-tests in (A), (B) and (C) were performed on n = 6 combined…

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  Figure 7.. Effect of overexpression of KREH1-WT and KREH1-KA on intra-RESC protein–protein interactions. (A) Precipitation of RESC6-PTP was performed from lysates of KREH1-WT overexpressing cells either uninduced (–doxy) or induced (+doxy) after a 3-day induction. Bound proteins were released by TEV cleavage, and RESC6 was normalized in the elutions by western blot. Normalized elutions were analyzed by western blot with antibodies against RESC components. (B) As in A, but with lysates from KREH1-KA overexpressing cells. (C) 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. (D) Quantification of B. 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 RESC13-10XTY was performed from lysates of KREH1-WT uninduced (-doxy) and induced (+doxy) cells as in A. Bound proteins were released by 100 mM glycine (pH 2.5), and RESC13 was normalized in the elutions by western blot. Normalized elutions were analyzed by western blot with antibodies against…

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  Figure 8.. Effect of overexpression of KREH1-WT and KREH1-KA on RECC-RESC protein–protein interactions. (A) Precipitation of KREPB5-MHT was performed from lysates of KREH1-WT uninduced (−doxy) and induced (+doxy) cells, after a 3 day induction. Bound proteins were released by TEV cleavage, and KREPB5-Myc was normalized in the elutions by western blot. Normalized elutions were analyzed by western blot with antibodies against RESC components. (B) As in A, but using lysates of KREH1-KA cells. (C) 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. (D) Quantification of B. 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.

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  Figure 9.. Model of KREH1 function during utilization of initiator gRNAs. See text for details. Blue, never edited sequence; orange, edited sequence; black, pre-edited sequence; red, gRNA anchor region; yellow, gRNA guiding region.