Talk abstracts
Talk on Friday 04:15-04:30pm submitted by Katherine Querry
Ribosomal release factors Hbs1 and Pelota regulate translation of coding regions following upstream open reading frames
Katherine Querry (Department of Cell Biology, University of Pittsburgh School of Medicine ), Narayanan Nampoothiri, Chris Garbark, Nina Gralewski-Goel, Abigail Carney (Department of Cell Biology, University of Pittsburgh School of Medicine ), Mykola Roiuk, Aurelio Teleman (Faculty of Biosciences, Heidelberg University, Heidelberg, Germany. ), Deepika Vasudevan (Department of Cell Biology, University of Pittsburgh School of Medicine )
Abstract:
Over 40% of mammalian mRNAs contain upstream open reading frames (uORFs), which function to inhibit translation of downstream coding regions1. The best studied of these mRNAs encodes for the transcription factor ATF4, a master regulator of the Integrated Stress Response (ISR) pathway. Across phyla, the 5’ leader of ATF4 mRNA contains two uORFs that regulate translation of ATF4, depending on initiator methionyl tRNA (Met-tRNAiMet) levels. Under homeostasis, after termination at the first uORF, the ribosome can continue scanning and reinitiate at the second uORF, which overlaps with the start codon of ATF4, thus blocking ATF4 synthesis. ISR activation results in reduced Met-tRNAiMet availability, which results in a delay in reinitiation, thus permitting synthesis of ATF4 rather than the inhibitory second uORF. We performed a Drosophila RNAi screen using an ATF4 reporter as bait to identify ribosomal factors that may be involved in the regulation of reinitiation. This screen identified Hbs1, a GTPase which aids in post-termination ribosomal recycling when in complex with Pelota. We hypothesize Hbs1 and Pelota act on the stop codon of the first uORF to release the 60s subunit and the polypeptide chain so that the 40s subunit can reinitiate at the ATF4 start codon. Consistently, we see that depletion of Hbs1 and Pelota results in decreased expression of an ATF4 5’ leader reporter. To test the physiological relevance of this regulatory mechanism, we used a Drosophila retinitis pigmentosa disease model where we have previously shown ATF4 to be required in the eye to delay degeneration. Using electroretinograms to measure transduction in the visual system, we observe that loss of Hbs1 and Pelota show retinal degradation similar to ATF4 mutants. Intriguingly, human patients with deletions in Hbs1 have also been shown to have retinitis pigmentosa2, lending further credibility to our results. We are currently pursuing genome-wide ribosome profiling approaches to identify other uORF-containing mRNAs that are regulated by Hbs1 and Pelota and extending our analyses to other known ribosome release factors as well.
References:
1. Vattem KM, Wek RC. Reinitiation involving upstream ORFs regulates ATF4 mRNA translation in mammalian cells. Proc Natl Acad Sci U S A. 2004 Aug 3;101(31):11269-74. doi: 10.1073/pnas.0400541101.
2. O'Connell AE, Gerashchenko MV, O'Donohue MF, Rosen SM, Huntzinger E, Gleeson D, Galli A, Ryder E, Cao S, Murphy Q, Kazerounian S, Morton SU, Schmitz-Abe K, Gladyshev VN, Gleizes PE, Séraphin B, Agrawal PB. Mammalian Hbs1L deficiency causes congenital anomalies and developmental delay associated with Pelota depletion and 80S monosome accumulation. PLoS Genet. 2019 Feb 1;15(2):e1007917. doi: 10.1371/journal.pgen.1007917.
Keywords: ribosome recycling factors, uORFs, ATF4