Bill and Risa to Present at Student Seminar, 1.29.08
Bill Lanier and Risa Banasik will be presenting on Thursday, January 29th in 2289 CBRB @ noon.
Background for Bill’s talk:
MicroRNAs are a sub-classification of non-coding (nc) RNA sequences (~19-24 bp) that may have been present in the primordial RNA world. Unlike small interfering RNAs that regulate the transcription of transposable elements, miRNAs function post-transcriptionally to modulate host gene expression. The potential contribution of transposable elements (TEs) to the origin of miRNA loci and the targets they recognize suggest that miRNA regulatory networks may have evolved via transposon insertion. Here we examined the small RNA pool of Chlamydomonas reinhardtii (a single-celled green algae) raised under conditions of stress that are conducive to retrotransposition. Our goal was to identify miRNA loci with a TE signature. This work identified cre-MIR1146 as a member of a TE-derived family. In addition, we provide support for a TE contribution towards ~23% (11/48) of known miRNA loci in C. reinhardtii. Our results are consistent with the view that most miRNA loci (generally of unknown origin) may be attributed to a cryptic TE mediated system. By extension, this suggests that miRNAs and siRNAs likely share a common ancestry, still united in host defense, with miRNA regulatory networks retained as the byproduct of selection acting on randomly generated transposon insertions. The ancient origin of TE-derived miRNAs likely spans the major kingdoms of eukaryotes and is supported by the finding of independent origins of miRNA loci via the processing of Gypsy derived RNA hairpins.
Background for Risa’s talk:
My research currently focuses on using RNAi techniques to mediate target gene knockdown in polarized airway epithelial cell cultures. Polarized cultures are difficult to transfect with chemically synthesized siRNAs, and siRNA mediated knockdown is inherently transient. As an alternative, our lab is utilizing a lentiviral vector expression system that includes a transgene cassette that expresses a shRNA for gene knockdown and a fusion fluorescent/drug resistance gene to allow for easy generation and visualization of a pure population of transduced cells. This protocol could be used to address biological questions in a relevant cell model system, such as immortalized Calu3 cells, which are suitable for polarization, and human primary airway cells. We are currently examining several candidate genes for preliminary knockdown studies.
Lamin A/C is a nuclear localized, structural protein extensively targeted in siRNA studies. Knockdown is readily detectable, making lamin A/C an excellent proof-of-principle target. We designed two miRNAs and shRNAs based on the literature, and demonstrated through PsiCheck2 artificial knockdown studies and endogenous lamin A/C western blots the efficacy of one shRNA construct. This shRNA was produced as FIV lentiviral vector. Calu3 cells were transduced and selected on plastic to generate a pure population of cells prior to polarization. Preliminary western blot data confirms knockdown of lamin A/C in these cultures.
Additionally, we are examining knockdown of angiotensin-converting enzyme 2 (ACE2), the cellular receptor for the SARS coronavirus. A commercially available HIV lentiviral vector miRNA construct targeting ACE2, corresponding to a validated siRNA, was obtained and knockdown confirmed with the PsiCheck2 system. Polarized human primary epithelia cultures were transduced with this vector and selection is ongoing. Preliminary results demonstrate an increase in fluorescence, indicative of selected cell expansion. Parallel experiments in Calu3 cells demonstrate decreased levels of secreted ACE2 protein.