Identification of pentatricopeptide repeat proteins in Trypanosoma brucei.

A new class of organellar proteins, characterized by pentatricopeptide repeat (PPR) motifs, has been identified in plants. These proteins contain multiple 35-amino acid repeats that are proposed to form a super helix capable of binding a strand of RNA. All PPR proteins characterized to date appear to be involved in RNA processing pathways in organelles. Twenty-three PPR proteins have been identified in Trypanosoma brucei and database research indicates that most of these proteins are predicted to contain the traditional mitochondrial target sequence. Orthologues of each of the 23 proteins have also been identified in Leishmania major and Trypanosoma cruzi, indicating that these proteins represent a highly conserved class of proteins within the kinetoplastid family. Preliminary experiments using RNAi to specifically silence one identified PPR gene (TbPPRl- Tb927.2.3180), indicate that cells depleted of TbPPRl transcripts show a slow growth phenotype and altered mitochondrial maxicircle RNA profiles. This initial characterization suggests that PPR proteins will play important roles in the complex RNA processing required for mitochondrial gene expression in trypanosomes.

Evidence for U-tail stabilization of gRNA/mRNA interactions in kinetoplastid RNA editing.

The most dramatic example of RNA editing is found in the mitochondria of trypanosomes. In these organisms, U-insertions/deletions can create mRNAs that are twice as large as the gene that encodes them. Guide RNAs (gRNAs) that are complementary to short stretches of the mature message direct the precise placements of the U residues. The binding of gRNA to mRNA is a fundamental step in RNA editing and understanding the relative importance of the elements that confer affinity and specificity on this interaction is critical to our understanding of the editing process. In this study, we have analyzed the relative binding affinities of two different gRNA/mRNA pairs. The affinity of gA6-14 for its message (ATPase 6) is high, with an apparent K(D) in the 5-10 nM range. In contrast, gCYb-558 has a low affinity for its cognate mRNA. Deletion of the gRNA U-tail caused a significant reduction in the binding affinity for only the gCYb-558 pair, and was observed only under physiological magnesium conditions. These results indicate that the U-tail contribution can differ substantially between the different gRNA/mRNA pairs. In addition, our results suggest that the efficiency of gRNA/mRNA interaction is highly dependent on thermodynamic parameters determined by the local sequences and their adopted structures surrounding the anchor-binding site.

The impact of mRNA structure on guide RNA targeting in kinetoplastid RNA editing.

Mitochondrial mRNA editing in Trypanosoma brucei requires the specific interaction of a guide RNA with its cognate mRNA. Hundreds of gRNAs are involved in the editing process, each needing to target their specific editing domain within the target message. We hypothesized that the structure surrounding the mRNA target may be a limiting factor and involved in the regulation process. In this study, we selected four mRNAs with distinct target structures and investigated how sequence and structure affected efficient gRNA targeting. Two of the mRNAs, including the ATPase subunit 6 and ND7-550 (5' end of NADH dehydrogenase subunit 7) that have open, accessible anchor binding sites show very efficient gRNA targeting. Electrophoretic mobility shift assays indicate that the cognate gRNA for ND7-550 had 10-fold higher affinity for its mRNA than the A6 pair. Surface plasmon resonance studies indicate that the difference in affinity was due to a four-fold faster association rate. As expected, mRNAs with considerable structure surrounding the anchor binding sites were less accessible and had very low affinity for their cognate gRNAs. In vitro editing assays indicate that efficient pairing is crucial for gRNA directed cleavage. However, only the A6 substrate showed gRNA-directed cleavage at the correct editing site. This suggests that different gRNA/mRNA pairs may require different "sets" of accessory factors for efficient editing. By characterizing a number of different gRNA/mRNA interactions, we may be able to define a "bank" of RNA editing substrates with different putative chaperone and other co-factor requirements. This will allow the more efficient identification and characterization of transcript specific RNA editing accessory proteins.

Interactions of mRNAs and gRNAs involved in trypanosome mitochondrial RNA editing: structure probing of a gRNA bound to its cognate mRNA.

Expression of mitochondrial genes in Trypanosoma brucei requires RNA editing of its mRNA transcripts. During editing, uridylates are precisely inserted and deleted as directed by the gRNA template to create the protein open reading frame. This process involves the bimolecular interaction of the gRNA with its cognate pre-edited mRNA and the assembly of a protein complex with the enzymatic machinery required. While a considerable amount of work has been done identifying the protein components of the editing complex, very little is known about how a functional editosome is assembled. In addition, the importance of RNA structure in establishing a functional editing complex is poorly understood. Work in our lab suggests that different mRNA/gRNA pairs can form similar secondary structures suggesting that a common core architecture may be important for editosome recognition and function. Using solution structure probing, we have investigated the structure of the initiating gRNA, gCYb-558, in the mRNA/gRNA complex with pre-edited apocytochrome b mRNA. Our data indicate that the stem-loop formed by the guiding region of the gRNA alone is maintained in its interaction with the pre-edited message. In addition, our data suggest that a gRNA stem-loop structure is maintained through the first few editing events by the use of alternative base-pairing with the U-tail.

Connexin 26 35delG does not represent a mutational hotspot.

Non-syndromic hearing impairment (NSHI) is the most common form of deafness and presents with no other symptoms or sensory defects. Mutations in the gap junction gene GJB2 account for a high proportion of recessive NSHI. The GJB2 gene encodes connexin 26, which forms plasma membrane channels between cochlear cells. In Caucasian populations a single mutation, 35delG, accounts for most cases of NSHI. This mutation appears to be most prevalent in individuals of Mediterranean European descent, with carrier frequencies estimated as being as high as one in thirty. The 35delG region may be a mutational hotspot. The mutation arises from the deletion of a guanine from a six-guanine stretch and nearby microsatellite markers show little evidence for linkage disequilibrium. We believe that 35delG is an old mutation in a chromosomal region of high recombination. The genetic context of the 35delG mutation was examined to distinguish between an old or a recurring mutation. We identified two single-nucleotide polymorphisms (SNPs) immediately upstream of the first exon of GJB2. Polymerase chain reaction/restriction fragment length polymorphism analysis determined the SNP genotype of 35delG containing chromosomes from various populations, including Italy, Brazil, and North America. We found the same, relatively rare, polymorphism associated with the 35delG mutation in all populations studied. We have also examined microsatellite markers D13S175, which is 80 kb telomeric to GJB2, and D13S1316, which is 80 kb centromeric to GJB2. D13S175 appears to be in weak linkage disequilibrium with 35delG, while D13S1316 is less so. SNPs located between the 35delG mutation and the microsatellite markers show strong evidence of linkage disequilibrium. Taken together, these results indicate there has been substantial recombination near the 35delG mutation; however, we present evidence that the 35delG mutation arose in European and Middle Eastern populations from a single mutational event on a founder chromosome.

Diverse microbial communities inhabiting ferromanganese deposits in Lechuguilla and Spider Caves.

Lechuguilla Cave is an ancient, deep, oligotrophic subterranean environment that contains an abundance of low-density ferromanganese deposits, the origin of which is uncertain. To assess the possibility that biotic factors may be involved in the production of these deposits and to investigate the nature of the microbial community in these materials, we carried out culture-independent, small subunit ribosomal RNA (SSU rRNA) sequence-based studies from two sites and from manganese and iron enrichment cultures inoculated with ferromanganese deposits from Lechuguilla and Spider Caves. Sequence analysis showed the presence of some organisms whose closest relatives are known iron- and manganese-oxidizing/reducing bacteria, including Hyphomicrobium, Pedomicrobium, Leptospirillum, Stenotrophomonas and Pantoea. The dominant clone types in one site grouped with mesophilic Archaea in both the Crenarchaeota and Euryarchaeota. The second site was dominated almost entirely by lactobacilli. Other clone sequences were most closely related to those of nitrite-oxidizing bacteria, nitrogen-fixing bacteria, actinomycetes and beta- and gamma-Proteobacteria. Geochemical analyses showed a fourfold enrichment of oxidized iron and manganese from bedrock to darkest ferromanganese deposits. These data support our hypothesis that microorganisms may contribute to the formation of manganese and iron oxide-rich deposits and a diverse microbial community is present in these unusual secondary mineral formations.