RNA editing in the free-living bodonid Bodo saltans

Abstract

In parasitic kinetoplastid protozoa, mitochondrial (mt) mRNAs are post-transcriptionally edited by insertion and deletion of uridylate residues, the information being provided by guide (g) RNAs. In order to further explore the role and evolutionary history of this process, we searched for editing in mt RNAs of the free-living bodonid Bodo saltans. We found extensive editing in the transcript for NADH dehydrogenase (ND) subunit 5, which is unedited in trypanosomatids. In contrast, B.saltans cytochrome c oxidase (cox) subunit 2 and maxicircle unidentified reading frame (MURF) 2 RNAs display limited editing in the same regions as their trypanosomatid counterparts. A putative intramolecular cox2 gRNA and the gene for gMURF2-I directing the insertion of only one U in the 5′ editing domain of MURF2 RNA, are conserved in B.saltans. This lends (further) evolutionary support to the proposed role of these sequences as gRNAs. Phylogenetic analysis showed that B.saltans is more closely related to trypanosomatids than the cryptobiids Trypanoplasma borreli and Cryptobia helicis, in line with the trypanosomatid-like cox2 and MURF2 RNA editing patterns. Nevertheless, other features like the apparent absence of a catenated mtDNA network, are shared with bodonid and cryptobiid species. ND5 RNA editing may represent yet another example of editing 'on the way out' during kinetoplastid evolution, but in view of the fact that cox2 RNA is unedited in T.borreli and C.helicis, we infer that the editing of this RNA may have arisen relatively recently. Our results provide the first examples of RNA editing in a free-living kinetoplastid, indicating that there is no direct link between U-insertion/ deletion editing and a parasitic lifestyle.

Phylogeny of the bodonid flagellates (Kinetoplastida) based on small-subunit rRNA gene sequences.

“…It is clear that a resolution of this problem will require finding an outgroup more closely related to kinetoplastids or analysing another dataset. It should be noted that the analysis of several mitochondrial genes ( Lukes) et al, 1994 ;Blom et al, 1998) also supported the bodonid root. Unfortunately, the limited number of available protein sequences is insufficient for a more detailed analysis.…”

“…uncinatus) were cultivated in the ATCC medium 802 with the feeder bacteria Alcaligenes xylosoxidans denitrificans and Aerobacter aerogenes at 16 and 20 mC, respectively. The origin and cultivation of the Bodo saltans strain Konstanz ( K) have been described previously (Blom et al, 1998 (Zhukov, 1975) is morphologically and ultrastructurally close to B. caudatus and P. nitrophilus (A. Mylnikov, personal communication). C. marina, B. designis and B. sorokini were cultivated with the feeder bacteria A. x. denitrificans in the ATCC medium 1525 at 16 or 22 mC (B. sorokini).…”

“…uncinatus) were cultivated in the ATCC medium 802 with the feeder bacteria Alcaligenes xylosoxidans denitrificans and Aerobacter aerogenes at 16 and 20 mC, respectively. The origin and cultivation of the Bodo saltans strain Konstanz ( K) have been described previously (Blom et al, 1998 DNA extraction, amplification and sequencing. Isolation of total cell DNA, PCR amplification of the small-subunit (SSU) rRNA genes and partial large-subunit (LSU) rRNA genes and sequencing with a set of conserved oligomer primers were performed as described previously ( Maslov et al, 1996( Maslov et al, , 1999Lukes) et al, 1997).…”

“…( Wright et al, 1999), and the freeliving species Bodo caudatus (Fernandes et al, 1993), Dimastigella trypaniformis (Berchtold et al, 1994) and , 1997). By putting into phylogenetic context the data obtained by the analysis of RNA editing Lukes) et al, 1994 ;Blom et al, 1998Blom et al, , 2000, kDNA structure ( Yasuhira & Simpson, 1996 ;Lukes) et al, 1998), and glycosomes ( Wiemer et al, 1995) a better understanding of these processes and structures can be achieved. The aim of this work is to determine phylogenetic relationships within the suborder Bodonina, and to provide a framework for molecular and biochemical studies on bodonids.…”

Mitochondrial minicircles in the free-living bodonid Bodo saltans contain two gRNA gene cassettes and are not found in large networks

“…We are studying the mt genetic system of the freeliving bodonid B. saltans+ In a previous report, we described the analysis of a section of the B. saltans maxicircle and the features of edited RNAs (Blom et al+, 1998a)+ During this work, we noticed the presence in B. saltans DNA of large amounts of noncatenated, 1+4-kb minicircle-like molecules, indicating that also in this species a large mt DNA network may be absent+ In addition, B. saltans did not appear to possess a kDNA disc, but rather a more oval structure in a dilated region of the mitochondrion (Brooker, 1971;Blom et al+, 1998b)+ In this article, we identify the 1+4-kb DNAs as the B. saltans equivalent of minicircles by showing that they contain gRNA genes and we demonstrate that the large majority of these circles are present as monomers+ Therefore, we draw the conclusion that a kDNA network structure is not required for the ordered segregation of minicircles in B. saltans+…”