The zinc-fingers of KREPA3 are essential for the complete editing of mitochondrial mRNAs in Trypanosoma brucei

Abstract

Most mitochondrial mRNAs in trypanosomes undergo uridine insertion/deletion editing that is catalyzed by approximately 20S editosomes. The editosome component KREPA3 is essential for editosome structural integrity and its two zinc finger (ZF) motifs are essential for editing in vivo but not in vitro. KREPA3 function was further explored by examining the consequence of mutation of its N- and C-terminal ZFs (ZF1 and ZF2, respectively). Exclusively expressed myc-tagged KREPA3 with ZF2 mutation resulted in lower KREPA3 abundance and a relative increase in KREPA2 and KREL1 proteins. Detailed analysis of edited RNA products revealed the accumulation of partially edited mRNAs with less insertion editing compared to the partially edited mRNAs found in the cells with wild type KREPA3 expression. Mutation of ZF1 in TAP-tagged KREPA3 also resulted in accumulation of partially edited mRNAs that were shorter and only edited in the 3'-terminal editing region. Mutation of both ZFs essentially eliminated partially edited mRNA. The mutations did not affect gRNA abundance. These data indicate that both ZFs are essential for the progression of editing and perhaps its accuracy, which suggests that KREPA3 plays roles in the editing process via its ZFs interaction with editosome proteins and/or RNA substrates.

Figure 1. Effects of ZF mutation of KREPA3 with a myc-tag on growth, editing, and editosome integrity.

C-terminal myc-tagged WT KREPA3 protein or KREPA3 with mutated ZF1, ZF2, or ZF1&2 were constitutively expressed from the β-tubulin locus and were exclusively expressed upon repression of KREPA3 Reg allele expression by tet withdrawal. (A) Growth of RKO-KREPA3 WT-myc, ZFm1-myc, ZFm2-myc, and ZFm1&2-myc cell lines in which KREPA3 Reg was expressed (E) (grey line and solid symbol) or repressed (R) (black line and open symbol). Only WT-myc cells survive after KREPA3 Reg is repressed. (B) Western analysis using KREPA3-specific MAb to probe samples from the cells in panel A with KREPA3 Reg expressed (E) or repressed (R) for three days; parental cell line KREPA3-RKO is used as a control. (C) Real time PCR analysis of in vivo RNA editing in RKO-A3 WT-myc or ZFm2-myc cells in which KREPA3 Reg was repressed for three days. The relative amounts of pre-edited and edited mRNAs A6, COIII, COII, and MURF2 and never-edited ND4 mRNAs was normalized to 18S rRNA and compared to the corresponding cells in which KREPA3 Reg was expressed. The same analysis was done with KREPA3-RKO cells as a control. Note the log scale and that 1 represents no difference, >1 an increase, and <1 a decrease in relative RNA amount. (D) Western analysis of the glycerol gradient fractions of crude mitochondrial lysates from RKO-KREPA3 cells in which KREPA3 Reg was expressed (E) or repressed for three days (R) or from RKO-A3 WT-myc or ZFm2-myc cell in which myc tagged WT or ZF2 mutated KREPA3 was exclusively expressed, respectively, by three days repression (R). The analyses used a mixture of MAbs specific for the KREPA1, KREPA2, KREL1, and KREPA3 ∼20S editosome proteins. Purified ∼20S editosomes used as a control show the size of the untagged KREPA3 protein.

Figure 2. Partially edited A6 and MURF2 mRNAs accumulate in cells exclusively expressing KREPA3ZFm2.

(A) Schematic showing the unedited flanking sequences used as the upstream/downstream primers for RT-PCR. (B) Agarose gel analysis of the RT-PCR products of A6 and MURF2 mRNAs following 1 to 4 days repression of KREPA3 Reg expression in KREPA3-RKO, RKO-A3 WT-myc and ZFm2-myc cells. The locations of the edited, partially edited, and pre-edited cDNA bands are indicated. Never-edited ND4 mRNA was used as a control. Expression of untagged and myc-tagged KREPA3 protein was monitored by Western analyses using a MAb specific for KREPA3 (lowest panel). (C) Comparison of the protein level of exclusively expressed KREPA3ZFm2-myc in RKO-A3 ZFm2-myc cells to the untagged KREPA3 in RKO-KREPA3 cells induced with 4 ng/ml tet by Western analysis using KREPA3 specific MAb (top panel) and agarose gel analysis of the corresponding RT-PCR products of A6 and MURF2 mRNAs (lower panel).

Figure 3. Sequences of gel isolated RT-PCR products of the partially edited A6 mRNAs.

A6 mRNA RT-PCR products from KREPA3-RKO and RKO-A3ZFm2-myc cell with KREPA3 Reg expressed (E) or repressed for three days (R), respectively, were isolated from agarose gels. BF 427 RNA was used as a control. The region of the gel from which the DNA was cut, cloned and sequenced is indicated with the bracket. The relative location of the partially edited sequence in the junction between the unedited and edited sequences is shown schematically. Inserted u's are shown in lower case, each deleted U is shown as an asterisk (*), and those not matching fully edited RNA are underlined. The number of the clones found with each shown sequence is indicated in parentheses on the right. In some cases (indicated by text instead of sequence), pre-edited A6 sequences were obtained due to mis-priming at a site downstream of the A6 coding sequence, thereby creating PCR products large enough to be found in the excised band.

Figure 4. Sequences of gel isolated RT-PCR products of the edited MURF2 mRNAs.

The edited MURF2 RT-PCR products (indicated by brackets) from RKO-KREPA3ZFm2-myc cells with KREPA3 Reg expressed (E) and repressed (R) were cut from agarose gel, cloned and sequenced. The inserted, deleted, and non-matching positions and numbers of sequenced clones are indicated as in Figure 3.

Figure 5. Effect of ZF1 mutation in KREPA3 on RNA editing progression.

(A) Agarose gel analysis of A6 and MURF2 mRNAs RT-PCR products from cells that exclusively expressed WT TAP-tagged KREPA3 or mutants with one or both ZFs mutated (see [49]). RT-PCR products from KREPA3-RKO cells with KREPA3 expressed (E) or repressed (R) were used as controls. (B). Sequence analysis of gel isolated partially edited A6 mRNAs that accumulated (indicated by bracket) upon mutation of KREPA3 ZF1. The location of the partially edited junction is shown schematically. Sequence designations as in Figures 3 and 4.

Figure 6. Loss of KREPA3 or mutation of KREPA3 ZF2 has no effect on gRNA levels.

Total cellular RNA from KREPA3-RKO, RKO-KREPA3 WT-myc and ZFm2-myc cells with KREP3 Reg expressed (E) or repressed (R) were examined by a capping assay using guanylyltransferse and [α-³²P]GTP. The similarly labeled ssRNA ladder was used to size the gRNAs which show their characteristics size heterogeneity due to their variable oligo-U tails.