Kinetoplastid guide RNA biogenesis is dependent on subunits of the mitochondrial RNA binding complex 1 and mitochondrial RNA polymerase

Abstract

The mitochondrial RNA binding complex 1 (MRB1) is a recently discovered complex of proteins associated with the TbRGG1 and TbRGG2 proteins in Trypanosoma brucei. Based on the phenotype caused by down-regulation of these two proteins, it was proposed to play an unspecified role in RNA editing. RNAi silencing of three newly characterized protein subunits, guide RNA associated proteins (GAPs) 1 and 2 as well as a predicted DExD/H-box RNA helicase, show they are essential for cell growth in the procyclic stage. Furthermore, their down-regulation leads to inhibition of editing in only those mRNAs for which minicircleencoded guide (g) RNAs are required. However, editing remains unaffected when the maxicircle-encoded cis-acting gRNA is employed. Interestingly, all three proteins are necessary for the expression of the minicircle-encoded gRNAs. Moreover, downregulation of a fourth assayed putative MRB1 subunit, Nudix hydrolase, does not appear to destabilize gRNAs, and downregulation of this protein has a general impact on the stability of maxicircle-encoded RNAs. GAP1 and 2 are also essential for the survival of the bloodstream stage, in which the gRNAs become eliminated upon depletion of either protein.Immunolocalization revealed that GAP1 and 2 are concentrated into discrete spots along the mitochondrion, usually localized in the proximity of the kinetoplast. Finally, we demonstrate that the same mtRNA polymerase known to transcribe the maxicircle mRNAs may also have a role in expression of the minicircle-encoded gRNAs.

Trypanosome REH1 is an RNA helicase involved with the 3′–5′ polarity of multiple gRNA-guided uridine insertion/deletion RNA editing

“…Interestingly, the REL1 RNA ligase, a component of the RECC, and the S17 and L3 kinetoplast ribosome proteins also exhibited a concentration in the kDNA region of the mitochondrion, suggesting the intriguing possibility of a physical linkage of kDNA transcription, translation, and editing pathways, but this remains to be investigated. Our localization results differ somewhat from several previous studies that showed that editing proteins are distributed throughout the mitochondrion with no apparent concentration (36,37) and that the RNA-linked editing proteins, GAP1 and GAP2, are localized in discrete particles throughout the mitochondrion (14). These differences could be speciesdependent or could be due to technical details.…”

“…Repression of REH1 expression showed no effect on the stability or length of the gRNA 3′ oligo U tails (Fig. 1C), as was reported for the trypanosome REH2 mitochondrial RNA helicase (14,16) Real-time RT-PCR was also used to quantitate the effect of REH1 down-regulation on the relative abundance of several preedited and mature edited mRNAs ( Fig. 1D and Table S1).…”

“…These include the MRP1/2 complex (11, 12) and the guide RNA binding complex (GRBC)/mitochondrial RNA binding (MRB1) complex (13)(14)(15)(16). Heterogeneous-sized high molecular weight complexes (L* or RECC*) consisting of the RECC together with several RNA-linked complexes have been visualized by electrophoresis in blue native gels (4).…”

“…An RNA helicase has been suggested to be involved in this process by displacing the initial gRNA, thereby allowing the adjacent upstream gRNA to form an anchor duplex with the edited sequence (19). Two trypanosome mitochondrial DExD/H-box proteins have been identified (14,16,20,21) and shown to be involved in RNA editing. RNA editing helicase 2 or REH2 is a component of GRBC or MRB1 complexes and is involved in gRNA biogenesis (13,14,16).…”

“…Two trypanosome mitochondrial DExD/H-box proteins have been identified (14,16,20,21) and shown to be involved in RNA editing. RNA editing helicase 2 or REH2 is a component of GRBC or MRB1 complexes and is involved in gRNA biogenesis (13,14,16). Hel61, another DEAD-box protein, was shown to be involved in RNA editing by a gene disruption experiment in T. brucei, which produced a slow growth phenotype and affected editing efficiency, and ectopic reexpression of Hel61 rescued a partially restored editing phenotype (21).…”