Molecular mechanism of the ribosome and functions of translational regulation

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To investigate how translation is regulated to control gene expression, the lab has primarily employed high-throughput sequencing methods, such as mRNA-Seq and ribosome footprint profiling with computational analysis. We are also developing tools for imaging single polysomes in living yeast and mammalian cells by using fluorescence microscopy and reporters consisting of arrayed GFP molecules (SunTag). We also have implemented specialized forms of ribosome profiling by footprinting the 40S, as opposed to 80S, ribosome or collisions between two 80S ribosomes (disomes). We have shown that 40S footprints are typically found at start codons, where the 40S subunit waits to join with the 60S subunit, and at stop codons, after translation is complete and the 60S subunit has been removed. We can now enhance these signals with mutations in yeast that slow down 60S joining at start codons or dissociation of 40S subunits at stop codons. We then use this approach this to detect cryptic translation on non-canonical open reading frames. In contrast, disome footprinting detects cases where two 80S ribosomes collide during translation, leading to a footprint that is twice as long as that protected by a single ribosome. Our work has revealed that disome formation is common and occurs both stochastically and at programmed ribosome stalling events. Intriguingly, we also found that disomes form at stop codons and can move into the 3'UTR when we inhibit the recycling process that removes ribosomes after translation is complete. Such moving disomes may be more broadly critical for clearing stalled ribosomes. Collisions between ribosomes (disomes) are generally thought to be a signal of stress and are known to recruit ribosome quality control (RQC) factors, such as the E3 ubiquitin ligase Hel2/ZNF598 (yeast/mammals), that promote their resolution, trigger mRNA decay, and more broadly activate stress response pathways. Loss of this pathway leads to the production of toxic peptides and neurodegeneration. However, ribosomes naturally collide during the course of translation, raising the question of whether the cell can distinguish disomes that are harmful from those that perform a function. To address this, we globally identified sites where ribosomes collide using disome profiling. Intriguingly, we found that all disomes are recognized by the RQC pathway and showed that loss of Hel2 reduces disome formation. This pathway therefore is widely used to detect many classes of disomes. Our work also revealed that Hel2 is not the only sensor of collided ribosomes. We found that the eIF2alpha kinase Gcn2 is activated when Hel2 is lost. Moreover, we found that the traditional trigger of Gcn2, ribosome stalling induced by starvation of amino acids, was also detected by Hel2. We therefore have established that ribosome collisions are detected by multiple stress-sensing pathways and the integration of these signals may help the cell to differentially respond to harmful vs functional ribosome collisions. Much as disome formation events trigger the RQC pathway, premature translation termination events also trigger a stress pathway called nonsense-mediated decay (NMD) that leads to degradation of the mRNA. This pathway is triggered by aberrant transcripts that encode mutations that encode a premature stop codon within a coding sequence. However, the NMD pathway is known to target many apparently normal transcripts and is therefore thought to play additional roles in gene regulation. To search for cryptic translation events that would cause premature translation termination in normal cells, we employed 40S ribosome profiling, RNA-Seq, and other approaches. We found such events internal to coding sequences, indicative of leaky scanning, where the 40S ribosome fails to find the main AUG start codon and instead initiates translation downstream. We also found evidence for translation of long undecoded transcript isoforms (LUTIs). In these cases, far upstream promoters are used under normal conditions to make long transcripts that encode upstream open reading frames (uORFs). Premature termination after translation of these uORFs results in NMD, suggesting that NMD is important for fully silencing these transcripts. We are now exploring how changes in environmental conditions change promoter usage to favor of isoforms where the uORFs are not transcribed, allowing the main ORF be translated and preventing NMD. Our work has also examined how translation is influenced by activation of RNase L, a branch of the innate immune response. RNase L activation is known to cause widespread mRNA decay via endonucleolytic cleavage of transcripts. How the resultant transcriptome is translated is unknown. We used ribosome profiling on RNase L activated cells and found a strong increase in ribosome occupancy in 3'UTR regions. While it was thought that RNase L can modulate the translation termination and recycling processes and therefore give rise to these effects, our data are inconsistent with this hypothesis. Instead, we observe increased translation in non-coding regions generally, including 5'UTRs and out-of-frame ORFs in coding sequences. Moreover, the effects are dependent on the cleavage activity of RNase L. One model to explain these results is that ribosomes can initiate translation on mRNA decay fragments and translate the encoded ORFs. We plan to further investigate how peptides produced from this process are relevant to the innate immune response and whether these translation events affect the stability or proposed functions of these fragments, such as activation of dsRNA sensors in the cell. Finally, we also collaborated with lab of Yihong Ye to reveal that ufmylation is an important ribosome modification that is used for a ER-specific RQC pathway.