Understanding miRNA Turnover: A Study of miRNA Half-Life Dominic McDonald Massachusetts Institute of Technology Mentor: Jun Lu, Ph. D Todd Golub Laboratory Broad Institute of MIT and Harvard Better titles.

What are miRNAs? miRNAs are short non-coding RNAs Post-transcriptional regulators of gene expression miRNAs regulate diverse biological and disease processes

miRNA Biogenesis Talk about: 20-25 nucleotide single stranded RNA molecules; first discovered in 1993 in C.Elegans by Lee, Feinbaum, and Ambros; major role in biological systems is gene regulations; Has been known to be possible causes to cancer because the miRNA may inhibit a gene from translating proteins that may induce apoptosis, metastasis, or that prevents cell proliferation; Not much is known about miRNA turnover and how fast they degrade Zamore PD & Haley B. Science 2005

How do miRNAs turnover? Put more space between words, background information as to why it is important. The Big Picture Purpose. Possible applications

Approach to Measure miRNA t1/2 Culture Cells in Triplicates Treat Cells with Actinomycin D Harvest RNA at 6 time points throughout the day Actinomycin-D is an antibiotic which binds to DNA and prevents RNA synthesis, therefore no more RNA molecules are being produced. We then harvest the RNA at different timepoints with TriZOL, and then using RT-PCR are able to measure the gene expression levels at each of the timepoints which then can be used as a marker to see the degradation of miRNA. Actinomycin-D has it’s problems as it is very toxic to the cells and will kill them within a day. Therefore this method can only be used during the course of 1 day. To measure longer periods we need to use an inducible system. The system we chose to use is the Tet-Off system. Measure miRNA and control gene expression levels using Luminex NextGen and RT-PCR

Cell Lines Maintained 26 different cancer cell lines. Due to time constraints only able to collect data from 9 different cancer cell lines: HD-MY-Z, 697, HL60, K562, Ramos (RA1), RS4;11, SEMK2, U937, and HEL cell lines. Triplicates of every cell line done at 6 time points each for a total of 162 samples.

Control Gene Degradation c-myb These graphs show that using treatment with Actinomycin-D does work. These 2 genes, c-myb and c-myc both have known half lives of less than 2 hours. In all 6 cancer cell lines, both genes are shown to have 50% degradation before 2 hours. Therefore this validates our sample quality

Control Gene Degradation c-myb This is the log 2 graph of the data. We were able to calculate half life by taking the inverse of the slope. For the rest of my presentation this is how I will be presenting the information compared to the previous slide.

Control Gene Degradation c-myc These graphs show that using treatment with Actinomycin-D does work. These 2 genes, c-myb and c-myc both have known half lives of less than 2 hours. In all 6 cancer cell lines, both genes are shown to have 50% degradation before 2 hours. Therefore this validates our methodology.

MicroRNA Profiling Used Luminex NextGen Lu, J Nature 2005

RT-PCR Validation Use additional methodology to validate results Use RT-PCR for validation Measure miR-125a in K562 cells Measure u6 small RNA as a control

RT-PCR Validation To confirm this data we ran the RNA samples from some cell lines using RT-PCR to see if the data matched using a different method. Here is an example of HD-MY-Z using RT-PCR. The reason why the data is going up, as if there is more miRNA being produced is because the control gene, u6, is being degraded and miR-125a is not being degraded, therefore causing an upward curve. If you look at the raw data, the amount of miR-125a remains constant throughout all of the timepoints.

RT-PCR Validation

RT-PCR Validation

What Does This All Mean? Most miRNAs have long t1/2 (over 24 hours) We have not observed any fast degrading miRNAs (t1/2 less than 8 hours)

Why would nature create a class of stable cellular regulators? Question pose to the audience. Most regulators share very short half-lives to be easily manipulated to. What could be possible reasons as to why miRNAs degrade very slow. Put forth the possibility that miRNAs are so small that there is not a mechanism that can degrade the microRNA fast.

Acknowledgements Our Team: Jun Lu Hao Zhang Judy Wang Golub Lab: Todd Golub Jinyan Du Broad Institute: Shawna Young Bruce Birren Lucia Vielma Maura Silverstein