Minicircle-encoded guide RNAs from Crithidia fasciculata.

Although the mitochondrial uridine insertion/deletion, guide RNA (gRNA)-mediated type of RNA editing has been described in Crithidia fasciculata, no evidence for the encoding of gRNAs in the kinetoplast minicircle DNA has been presented. There has also been a question as to the capacity of the minicircle DNA in this species to encode the required variety of gRNAs, because the kinetoplast DNA from the C1 strain has been reported as essentially containing a single minicircle sequence class. To address this problem, the genomic and mature edited sequences of the MURF4 and RPS12 cryptogenes were determined and a gRNA library was constructed from mitochondrial RNA. Five specific gRNAs were identified, two of which edit blocks within the MURF4 mRNA, and three of which edit blocks within the RPS12 mRNA. The genes for these gRNAs are all localized with identical polarity within one of the two variable regions of specific minicircle molecules, approximately 60 bp from the "bend" region. These minicircles were found to represent minor sequence classes representing approximately 2% of the minicircle DNA population in the network. The major minicircle sequence class also encodes a gRNA at the same relative genomic location, but the editing role of this gRNA was not determined. These results confirm that kinetoplast minicircle DNA molecules in this species encode gRNAs, as is the case in other trypanosomatids, and suggest that the copy number of specific minicircle sequence classes can vary dramatically without an overall effect on the RNA editing system.     

A model for RNA editing in kinetoplastid mitochondria: "guide" RNA molecules transcribed from maxicircle DNA provide the edited information.

A class of small RNA molecules possibly involved in RNA editing is present in the mitochondrion of Leishmania tarentolae. These "guide" RNA (gRNA) molecules are encoded in intergenic regions of the mitochondrial maxicircle DNA and contain sequences that represent precise complementary versions of the mature mRNAs within the edited regions. In addition, the 5' portions of several gRNAs can form hybrids with mRNAs just 3' of the preedited region. A model is presented in which a partial hybrid formed between the gRNA and preedited mRNA is substrate for multiple cycles of cleavage, addition or deletion of uridylates, and religation, eventually resulting in a complete hybrid between the gRNA and the mature edited mRNA.     

Maxicircle (mitochondrial) genome sequence (partial) of Leishmania major: gene content, arrangement and composition compared with Leishmania tarentolae

We report 8420 bp of DNA sequence data from the maxicircle (mitochondrial) genome of Leishmania major (MHOM/SU/73/5ASKH), a much larger portion of this genome than has been reported previously from any Leishmania species infecting humans. This region contains 10 partial and complete genes: 5 protein-encoding genes (COII, COIII, ND1, ND7 and Cyt b); two ribosomal RNA subunits (12S and 9S) and three unidentified open reading frames (MURF1, MURF4 (ATPase6) and MURF5), as in the lizard-infecting species L. tarentolae. The genes from L. major exhibit 85-87% identity with those of L. tarentolae at the nucleotide level and 71-94% identity at the amino acid level. Most differences between sequences from the two species are transversions. The gene order and arrangement within the maxicircle of L. major are similar to those in L. tarentolae, but base composition and codon usage differ between the species. Codons assigned for initiation for protein-coding genes available for comparison are similar in five genes in the two species. Pre-editing was identified in some of the protein-coding genes. Short intergenic non-coding regions are also present in L. major as they are in L. tarentolae. Intergenic regions between 9S rRNA and MURF5, MURF1 and ND1 genes are G+C rich and considered to be extensive RNA editing regions. The RNA editing process is likely to be conserved in similar pattern in L. major as in L. tarentolae.     

Implications of novel guide RNA features for the mechanism of RNA editing in Crithidia fasciculata.

We have determined the relative steady state concentration of the two Crithidia fasciculata guide (g)RNAs involved in editing the two domains of mRNAs for NADH dehydrogenase (ND) subunit 7. We found that, although there was an 8-fold difference between the molar ratio of these two gRNAs relative to the (pre)-mRNA, the two domains are edited with a very similar frequency (around 50%). Also, for the editing of a given domain, many gRNA species exist with the same 5' end but with a different 3' uridylation site. Approximately 20% of these short gRNAs do not contain the information required for editing a complete domain, which may explain the high incidence of partially edited RNAs. Remarkably, genomically encoded Us are missing from two sites of a few of the gRNAs involved in editing apocytochrome b RNA. We speculate that these species are created by editing-like events. Both the short and complete forms of the ND7 gRNAs are found in chimeric molecules, in which the gRNA is covalently linked via its 3'-terminus to an editing site of pre-edited ND7 RNA. Some features of the chimeric molecules are at odds with current models of RNA editing: (i) U residues are completely absent from the connecting sequence of a number of these molecules, (ii) the ND7 gRNAs are frequently hooked up to the wrong editing domain of ND7 RNA, although other gRNAs are not found at these positions and (iii) in some chimeric molecules the gRNA appears to be linked to the 5' end of pre-edited RNA.     

A novel member of the RNase D exoribonuclease family functions in mitochondrial guide RNA metabolism in Trypanosoma brucei

RNA turnover and RNA editing are essential for regulation of mitochondrial gene expression in Trypanosoma brucei. RNA turnover is controlled in part by RNA 3' adenylation and uridylation status, with trans-acting factors also impacting RNA homeostasis. However, little is known about the mitochondrial degradation machinery or its regulation in T. brucei. We have identified a mitochondrial exoribonuclease, TbRND, whose expression is highly up-regulated in the insect proliferative stage of the parasite. TbRND shares sequence similarity with RNase D family enzymes but differs from all reported members of this family in possessing a CCHC zinc finger domain. In vitro, TbRND exhibits 3' to 5' exoribonuclease activity, with specificity toward uridine homopolymers, including the 3' oligo(U) tails of guide RNAs (gRNAs) that provide the sequence information for RNA editing. Several lines of evidence generated from RNAi-mediated knockdown and overexpression cell lines indicate that TbRND functions in gRNA metabolism in vivo. First, TbRND depletion results in gRNA tails extended by 2-3 nucleotides on average. Second, overexpression of wild type but not catalytically inactive TbRND results in a substantial decrease in the total gRNA population and a consequent inhibition of RNA editing. The observed effects on the gRNA population are specific as rRNAs, which are also 3'-uridylated, are unaffected by TbRND depletion or overexpression. Finally, we show that gRNA binding proteins co-purify with TbRND. In summary, TbRND is a novel 3' to 5' exoribonuclease that appears to have evolved a function highly specific to the mitochondrion of trypanosomes.     

RBP16 is a multifunctional gene regulatory protein involved in editing and stabilization of specific mitochondrial mRNAs in Trypanosoma brucei

RBP16 is a Trypanosoma brucei mitochondrial RNA-binding protein that associates with guide RNAs (gRNAs), mRNAs, and ribosomal RNAs. Based on its inclusion in the multifunctional Y-box protein family and its ability to bind multiple RNA classes, we hypothesized that RBP16 plays a role in diverse aspects of mitochondrial gene regulation. To gain insight into RBP16 function, we generated cells expressing less than 10% of wild-type RBP16 levels by tetracycline-regulated RNA interference (RNAi). Poisoned primer extension analyses revealed that edited, but not unedited, CYb mRNA is reduced by approximately 98% in tetracycline-induced RBP16 RNAi cells, suggesting that RBP16 is critical for CYb RNA editing. The down-regulation of CYb editing in RBP16 RNAi transfectants apparently entails a defect in gRNA utilization, as gCYb[560] abundance is similar in uninduced and induced cells. We observed a surprising degree of specificity regarding the ability of RBP16 to modulate editing, as editing of mRNAs other than CYb is not significantly affected upon RBP16 disruption. However, the abundance of the never edited mitochondrial RNAs COI and ND4 is reduced by 70%-80% in RBP16 RNAi transfectants, indicating an additional role for RBP16 in the stabilization of these mRNAs. Analysis of RNAs bound to RBP16 immunoprecipitated from wild-type cells reveals that RBP16 is associated with multiple gRNA sequence classes in vivo, including those whose abundance and usage appear unaffected by RBP16 disruption. Overall, our results indicate that RBP16 is an accessory factor that regulates the editing and stability of specific populations of mitochondrial mRNAs.