We broadly investigate the mechanisms used in cells to regulate gene expression at the translational level. Current research is focused on the question of how ribosomes and mRNAs are dissociated (recycled) following the completion of translation at stop codons (or irreversible stalling events). Without this recycling process, ribosomes would accumulate on mRNAs, limiting the cell's ability to make new protein. Ribosome recycling begins when the stop codon is decoded by the canonical release factors, eRF1 and eRF3. Following GTP hydrolysis by eRF3, the ATPase Rli1 (ABCE1 in higher eukaryotes) separates the 60S subunit from the mRNA-bound 40S ribosome. It is unknown what then removes the 40S subunit from the mRNA. In prior work, we established that lack of Rli1 in the cell leads to a surprising accumulation of ribosomes in the 3'UTR and the translation of short open reading frames. The recycling factor ABCE1 is known to be upregulated in many types of cancer, suggesting that ribosome recycling is critical in cancer cells, potentially as a way to ensure an adequate supply of recycled ribosomes for new rounds of translation during rapid proliferation. We believe this process to also be critical during the innate immune response because the activity of ABCE1 is thought to modulated by genes that are upregulated by interferon. A better understanding of the mechanism of ribosome recycling is therefore important for addressing challenges to human health. The lab primarily employs high-throughput sequencing methods, such as mRNA-Seq and ribosome footprint profiling with computational analysis. We also use an array of biochemical approaches, such as western blots and reporter assays, to complement this work. Finally, we are developing tools for imaging single polysomes in living yeast and mammalian cells, using fluorescence microscopy and reporters consisting of arrayed GFP molecules (SunTag). We recently investigated the role of the protein Hcr1 (eIF3j) in yeast for its role in ribosome recycling. We used ribosome profiling and reporter assays to ask the question of whether Hcr1 facilitates removal of the 60S ribosomal subunit from the mRNA-bound 40S subunit or the 40S ribosome from the mRNA -- two potential roles suggested by previous work. Our data clearly reveal that Hcr1 promotes 60S subunit recycling because the yeast strain missing this factor is a close phenocopy of the Rli1-depleted strain and likely works to enhance Rli1 ATPase activity. Intriguingly, we also found that loss of Hcr1 triggers increased expression of the RLI1 mRNA, suggesting the cell actively senses ribosome recycling fidelity and tunes Rli1 levels to ensure sufficient levels of recycling. We have also investigated the yeast factors Tma64, Tma20, and Tma22 (eIF2D, MCT-1, and DENR in mammals), which have been proposed to recycle 40S ribosomes in lysate-based assays. Our work has now strongly suggested that these factors are required for ribosome recycling in vivo. Without them, we have found that ribosomes enter 3'UTRs and reinitiate new translation, likely by at least two mechanisms. First, we found evidence (ribosome profiling peaks on 3'UTR AUG codons and via western blot of reporter proteins) that that these factors promote recycling of 40S ribosomes. Second, we observed evidence of AUG-independent reinitiation (80S scanning). Our research has therefore shown that these factors play a critical role in preventing translation of 3'UTRs. Mutation of DENR has been shown to be associated with autism and overexpression of MCT-1 is a lymphoma driver in humans, suggesting that the peptides produced in the absence of these factors may be important for human health. Most recently, we have employed a new form of ribosome profiling by footprinting the 40S (as opposed to 80S) ribosome. Data from this experiment now directly demonstrate that loss of Tma64, Tma20, and Tma22 leads to widespread accumulation of 40S ribosomes on stop codons. We therefore conclude that these factors are 40S recycling factors and that the 40S ribosomes that accumulate in their absence are competent to reinitiate translation in 3'UTRs by multiple mechanisms. In addition, we have found that the observation of accumulation of 40S ribosomes on stop codons in this mutant strain by 40S ribosome profiling can serve as a signal of non-canonical termination events across the transcriptome. Surprisingly, we have found widespread evidence of such events internal to coding sequences. We believe such events are indicative of leaky scanning, where the 40S ribosome fails to find the main AUG start codon and instead initiates translation downstream, and in some cases, in a different reading frame. Initial analysis suggests such events occur on about 11% of genes, a number more substantial than previously thought, and can trigger degradation of the mRNA via the nonsense mediated decay pathway (since most out of frame ORFs end with termination codons near the 5' end of the gene). We anticipate that this technique will be useful for detection of other cases of non-canonical translation events. We are also investigating the functional roles for 3'UTR ribosomes and the possibility that loss of efficient recycling under stressful environments can alter fitness. We are examining the effects of nutrient deprivation (yeast) and simulated viral infection (human cell lines), for example, in reducing the efficiency of termination and recycling. Our ribosome profiling results now suggest many stresses induce translation of 3'UTRs but that the mechanism involved under glucose loss in yeast is highly specific for 40S reinitiation. We have also shown these peptides can be detected by western blot and we are now cataloging the repertoire of peptides that are synthesized from 3'UTR sequences for future investigation of functional activities.
|Guydosh, Nicholas R; Kimmig, Philipp; Walter, Peter et al. (2017) Regulated Ire1-dependent mRNA decay requires no-go mRNA degradation to maintain endoplasmic reticulum homeostasis in S. pombe. Elife 6:|
|Guydosh, Nicholas R; Green, Rachel (2017) Translation of poly(A) tails leads to precise mRNA cleavage. RNA 23:749-761|
|de Vasconcellos, Jaira F; Tumburu, Laxminath; Byrnes, Colleen et al. (2017) IGF2BP1 overexpression causes fetal-like hemoglobin expression patterns in cultured human adult erythroblasts. Proc Natl Acad Sci U S A 114:E5664-E5672|
|Young, David J; Guydosh, Nicholas R (2016) Silence without stress. Elife 5:|
|Woolstenhulme, Christopher J; Guydosh, Nicholas R; Green, Rachel et al. (2015) High-precision analysis of translational pausing by ribosome profiling in bacteria lacking EFP. Cell Rep 11:13-21|
|Guydosh, Nicholas R; Green, Rachel (2014) Dom34 rescues ribosomes in 3' untranslated regions. Cell 156:950-62|