Faculty Bibliography

U-insertion/deletion RNA editing is a post-transcriptional mitochondrial RNA modification phenomenon required for viability of trypanosomatid parasites. Small guide RNAs encoded mainly by the thousands of catenated minicircles contain the information for this editing. We analyzed by NGS technology the mitochondrial genomes and transcriptomes of two strains, the old lab UC strain and the recently isolated LEM125 strain. PacBio sequencing provided complete minicircle sequences which avoided the assembly problem of short reads caused by the conserved regions. Minicircles were identified by a characteristic size, the presence of three short conserved sequences, a region of inherently bent DNA and the presence of single gRNA genes at a fairly defined location. The LEM125 strain contained over 114 minicircles encoding different gRNAs and the UC strain only ~24 minicircles. Some LEM125 minicircles contained no identifiable gRNAs. Approximate copy numbers of the different minicircle classes in the network were determined by the number of PacBio CCS reads that assembled to each class. Mitochondrial RNA libraries from both strains were mapped against the minicircle and maxicircle sequences. Small RNA reads mapped to the putative gRNA genes but also to multiple regions outside the genes on both strands and large RNA reads mapped in many cases over almost the entire minicircle on both strands. These data suggest that minicircle transcription is complete and bidirectional, with 3' processing yielding the mature gRNAs. Steady state RNAs in varying abundances are derived from all maxicircle genes, including portions of the repetitive divergent region. The relative extents of editing in both strains correlated with the presence of a cascade of cognate gRNAs. These data should provide the foundation for a deeper understanding of this dynamic genetic system as well as the evolutionary variation of editing in different strains.

Drosophila oogenesis provides many examples of essential processes in development. A myriad of genetic tools combined with recent advances in culturing egg chambers ex vivo has revealed several surprising mechanisms that govern how this tissue develops, and which could not have been determined in fixed tissues. Here we describe a straightforward protocol for dissecting ovaries, culturing egg chambers, and observing egg development in real time by fluorescent microscopy. This technique is suitable for observation of early- or late-stage egg development, and can be adapted to study a variety of cellular, molecular, or developmental processes. Ongoing analysis of oogenesis in living egg chambers has tremendous potential for discovery of new developmental mechanisms.

The inhibitory potency of an antisense oligonucleotide depends critically on its design and the accessibility of its target site. Here, we used an RNA interference-guided approach to select antisense oligonucleotide target sites in the coding region of the highly structured hepatitis C virus (HCV) RNA genome. We modified the conventional design of an antisense oligonucleotide containing locked nucleic acid (LNA) residues at its termini (LNA/DNA gapmer) by inserting 8-oxo-2'-deoxyguanosine (8-oxo-dG) residues into the central DNA region. Obtained compounds, designed with the aim to analyze the effects of 8-oxo-dG modifications on the antisense oligonucleotides, displayed a unique set of properties. Compared to conventional LNA/DNA gapmers, the melting temperatures of the duplexes formed by modified LNA/DNA gapmers and DNA or RNA targets were reduced by approximately 1.6-3.3 degrees C per modification. Comparative transfection studies showed that small interfering RNA was the most potent HCV RNA replication inhibitor (effective concentration 50 (EC50): 0.13 nM), whereas isosequential standard and modified LNA/DNA gapmers were approximately 50-fold less efficient (EC50: 5.5 and 7.1 nM, respectively). However, the presence of 8-oxo-dG residues led to a more complete suppression of HCV replication in transfected cells. These modifications did not affect the efficiency of RNase H cleavage of antisense oligonucleotide:RNA duplexes but did alter specificity, triggering the appearance of multiple cleavage products. Moreover, the incorporation of 8-oxo-dG residues increased the stability of antisense oligonucleotides of different configurations in human serum.