Functional complementation of Trypanosoma brucei RNA in vitro editing with recombinant RNA ligase

Abstract

The Ϸ20S RNA ligase-containing complex (L-complex) in trypanosomatid mitochondria interacts by means of RNA linkers with at least two other multiprotein complexes to mediate the editing of mitochondrial cryptogene transcripts. The L-complex contains Ϸ16 proteins, including the two RNA-editing ligases (RELs), REL1 and REL2. Leishmania tarentolae REL1 and REL2 and Trypanosoma brucei REL1 were expressed as enzymatically active tandem affinity purification-tagged proteins in a Baculovirus system. When these proteins were added to mitochondrial lysates from T. brucei procyclic cells that were depleted of the cognate endogenous ligase by RNA interference down-regulation of expression, the added proteins were integrated into the L-complex, and, in the case of REL1, there was a complementation of in vitro-precleaved U-insertion and U-deletion editing activities of the 20S L-complex. Integration of the recombinant proteins did not occur or occurred at a very low level with noncognate ligase-depleted L-complex or with wild-type L-complex. A C-terminal region of the T. brucei recombinant REL1 downstream of the catalytic domain was identified as being involved in integration into the L-complex. The ability to perform functional complementation in vitro provides a powerful tool for molecular dissection of the editing reaction.editing ͉ RNA-editing ligase 1 ͉ RNA-editing ligase 2 ͉ trypanosome S everal macromolecular complexes have been identified that are involved in U-insertion͞deletion RNA editing in trypanosomatid mitochondria (1-3). The mitochondrial RNA-binding protein complex contains two small RNA-binding proteins and three associated proteins, and may possibly be involved in the initial annealing of the guide RNA and the mRNA (4-9). The RNAediting terminal uridylyl transferase 1 (TUTase 1) (RET1) complex contains the RET1 tetramer and one or more unidentified components (10). RET1 has been shown to be involved in the addition of nonencoded Us to the 3Ј ends of guide RNAs (11). The RNA ligase-containing L-complex was initially detected as an ␣-32 P-ATP-labeled particle sedimenting Ϸ20S in glycerol gradients and migrating as a single band with an approximate size of 1,200-2,000 kDa in native gels (12,13). This complex has been labeled the editosome but it seems appropriate to reserve this nomenclature for the entire RNA͞protein supercomplex once it is more defined. The L-complex has been isolated from Trypanosoma brucei and Leishmania tarentolae mitochondria by column chromatography (13-16) and by tandem affinity purification (TAP) chromatography (17). At least 16 protein components have been identified, including the two RNA-editing ligases (RELs) (REL1 and REL2), a second 3Ј TUTase (RET2), three to four RNase III-motif proteins, three related zinc finger-motif proteins, two AP-endo-exonucleasephosphatase-motif proteins, two RNA binding-motif proteins, and several proteins lacking identifiable motifs.The two ligases are localized in subcomplexes that have been partially defined by coimmunoprecipitation experiments and...

The Architecture of Trypanosoma brucei editosomes

“…2 A and B). These cross-links reinforce the importance of the KREL1 C-terminal and ligase domains, which were previously shown to be important for KREL1 interaction with KREPA2, and integration into editosomes (37,39,58). We observed extensive cross-linking between KREPA2 and KREX2, primarily between the same KREPA2 region and the N-terminal exonuclease domain of KREX2 ( Fig.…”

A Protein-Protein Interaction Map of Trypanosome ∼20S Editosomes

“…This was not unexpected, because the N-terminal two-thirds of KREL1 have been identified as the catalytic domain of the protein (Fig. 1A) (51,52), and recombinant KREL1 with a C-terminal truncation failed to bind to KREL1-depleted editosomes (53). The corresponding interacting region in KREPA2 is located just C-terminal to the first zinc finger.…”

Uridylate-specific 3′–5′-Exoribonucleases Involved in Uridylate-deletion RNA Editing in Trypanosomatid Mitochondria

“…Restriction sites are indicated in boldface type. Genes were released with BamHI and XbaI and inserted into the corresponding sites of a modified pFastBac1 vector (Invitrogen) containing C-terminal tandem affinity purification (TAP) (calmodulin-binding peptide (cbp), TEV protease site, protein A) epitope tags to generate pFastBac1-LmREX2*-TAP, pFastBac1-TbREX1-TAP, and pFastBac1-TbREX2-TAP vectors (9). Double amino acid mutations were made in the TbREX1 and TbREX2 EEP domains using the QuickChange site-directed mutagenesis kit (Stratagene) and the primers 5Ј-GCATATT-TGTTCCCATCCGCTAACTACGGCGTAAGTATA-3Ј and 5Ј-GAGTATTTGTACCCGTCTGCCAATTTCGGTCTCC-TTGTTG-3Ј (mutations indicated in boldface) to generate the plasmids pFastBac1-TbREX1(D881A,H882N)-TAP and pFastBac1-TbREX2(D896A,H897N)-TAP respectively.…”