Here is a list of main acheivements:
A role of new Piwi proteins in Paramecium - my lab discovered that development-specific small RNAs (scnRNAs and iesRNAs) are physically associated with different sets of Piwi proteins in a mutually exclusive manner; the former bind to Ptiwi01 and Ptiwi09, and the latter bind to Ptiwi10 and Ptiwi11.
Circular Concatemers of Ultra-Short DNA Segments Produce Regulatory RNAs - In the ciliated protozoan Paramecium tetraurelia, Piwi-associated small RNAs are generated upon the elimination of tens of thousands of short transposon-derived DNA segments as part of development. These RNAs then target complementary DNA for elimination in a positive feedback process, contributing to germline defense and genome stability. In this work, we investigate the formation of these RNAs, which we show to be transcribed directly from the short (length mode 27 bp) excised DNA segments. Our data support a mechanism whereby the concatenation and circularization of excised DNA segments provides a template for RNA production. This process allows the generation of a double-stranded RNA for Dicer-like protein cleavage to give rise to a population of small regulatory RNAs that precisely match the excised DNA sequences.
Dicer-like Enzymes with Sequence Cleavage Preferences - Dicer proteins are known to produce small RNAs (sRNAs) from long double-stranded RNA (dsRNA) templates. These sRNAs are bound by Argonaute proteins, which select the guide strand, often with a 5' end sequence bias. However, Dicer proteins have never been shown to have sequence cleavage preferences. In Paramecium development, two classes of sRNAs that are required for DNA elimination are produced by three Dicer-like enzymes: Dcl2, Dcl3, and Dcl5. Through in vitro cleavage assays, we demonstrate that Dcl2 has a strict size preference for 25 nt and a sequence preference for 5' U and 5' AGA, while Dcl3 has a sequence preference for 5' UNG. Dcl5, however, has cleavage preferences for 5' UAG and 3' CUAC/UN, which leads to the production of RNAs precisely matching short excised DNA elements with corresponding end base preferences. Thus, we characterize three Dicer-like enzymes that are involved in Paramecium development and propose a biological role for their sequence-biased cleavage products.
Epigenetic control of DNA elimination - Another of my studies shows that relative to IES length, there are pronounced differences in the IES sub-terminal base frequencies, suggesting that the IES excisase's recognition/excision preferences change with IES length. My work suggests that some IESs are more difficult to recognize and/or excise than others, thus requiring the greatest targeting support from scnRNAs and iesRNAs. With time, and owing to the effects of purifying selection acting upon IES excisase preferences, longer, epigenetically controlled IESs tend to become shorter and non-epigenetically controlled. There is always demand for scnRNA-dependent IES excision, due to ongoing genomic influxes of invasive DNAs and mutations converting non-epigenetically controlled IESs to epigenetically controlled IESs.
Non-standard genetic codes and genetic code evolution - In addition, I’ve been investigating codon reassignment and evolution of the genetic code using ciliates as models. This diverse group of eukaryotes include multiple examples of non-standard genetic codes which makes them excellent models for this type of investigation. In my recent paper, Genetic Codes with No Dedicated Stop Codon: Context-Dependent Translation Termination, 2016 Cell, we describe two codes with efficient translation of all 64 codons as standard amino acids and recognition of either one or all three stop codons. The work provided evidence that mRNA 3' ends may contribute to distinguishing stop from sense in a context-dependent manner and further propose that such context-dependent termination/readthrough suppression near transcript ends enables genetic code evolution.
