1. Spliced leader RNA trans-splicing in metazoa
Spliced leader (SL) RNA trans-splicing generates the mature 5’ ends of mRNAs by addition of a spliced leader sequence to the 5’ end of a pre-mRNA. Addition of the SL sequence also brings a new and an atypical cap to the RNA, a trimethylguanosine cap (m2,2,7GpppN) compared to the typical m7GpppN eukaryotic cap. Current studies involve analysis of
- Functional significance of trans-splicing
- Protein and mRNA metabolism adaptation to spliced leader trans-splicing
- Post-transcriptional role of trans-splicing in mRNA translation and stability
- Structure/function of mRNA cap-interacting proteins in trans-splicing and mRNA metabolism (eIF4E, eIF4G, nuclear cap binding complex, decapping, etc.)
2. Chromatin Diminution
Genome maintenance and stability are essential, and an organism’s genome rarely changes. But a few organisms undergo chromatin diminution, a programmed process that eliminates specific DNA sequences from the genome. In the parasitic nematode, Ascaris, 25% of the genome is eliminated in the somatic lineages during the 3rd through 5th cleavage (4 to 16 cell stage), while the germline genome remains intact. Both repetitive and unique sequences (genes) are lost during chromatin diminution. The elimination results in chromosome breakage and the loss of chromosome termini as well as the generation of new chromosomes. Studies are underway to compare the genome of the somatic cells which have undergone chromatin dimimution with the germline genome to determine
- What is the mechanism of chromosome breakage?
- Where are the chromosome breaks? What rearrangements occur?
- How are the breakpoints and sequences eliminated defined?
- What genes are lost?
- What are the somatic consequences of DNA elimination?
3. Small RNA function and silencing in nematodes
A variety of small RNAs including endogenous siRNA (22G-RNAs and 26G-RNAs) and miRNAs play key roles in silencing in the Ascaris germline, gametogenesis, and early development. Current studies are addressing the role and function of these small RNAs with a particular focus on the mechanisms of small RNA silencing and biogenesis in novel cell-free systems.
- Function and role of small RNAs in Ascaris development, gametogenesis, and the germ line
- Mechanisms of small RNA silencing and biogenesis in Ascaris
4. Regulation of Ascaris mRNA metabolism during gametogenesis and early embryo development
Zygote maturation and early development in Ascaris are very slow compared to C. elegans and enables the staging of large amounts of material for developmental studies. This enables us to carry out analysis of post-transcriptional gene regulation including
- Developmental mRNA analyses (RNA-seq analysis) combined with small RNA profiling
- Translation and stability during early development (polysome RNA-seq analysis)
- mRNA masking, P-bodies, and P-granules