Molecular mechanism of the ribosome and functions of translational regulation

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

The termination step of translation is critical for releasing the completed protein from the ribosome so that it can carry out functions in the cell. The termination process begins when the ribosome reaches the stop codon and a heterodimer of release factors (eRF1/3) is recruited to catalyze this process. Termination is generally efficient compared to the rare process of readthrough, where an amino acid is misincorporated instead. In such cases, the ribosome continues translating downstream of the stop codon, creating a C-terminal extension on the protein. However, many genes permit readthrough to occur at much higher frequency. Our goal is to uncover the mechanism for how these programmed readthrough events occur, how they are regulated, and how C-terminally extended proteins affect function. We have developed assays to directly observe readthrough events in the cell using fluorescence microscopy and reporter genes that encode protein arrays that become fluorescent during translation (suntag or moontag). Using these, we are now characterizing differences in readthrough kinetics (stochastic vs burst-like) between programmed readthrough events. Following termination, the process of ribosome recycling (removal of ribosomes from mRNAs) is critical for maintaining a sufficient supply of ribosomes to initiate new translation events in the cell. Loss of recycling efficiency is known to occur under conditions of neuronal aging, iron deficiency, and blood cell differentiation, highlighting the importance of this process. Since ribosomes are composed of 40S and 60S subunits, it is thought that the process occurs in two steps. Our previous work showed the importance of Rli1 and Hcr1 in removing 60S ribosomal subunits from mRNAs. We hypothesized that the proteins Tma64 and Tma20/22 could play a role in removing the remaining 40S subunits since their loss results in apparently unrecycled ribosomes reinitiating translation downstream of stop codons in yeast. However, we lacked direct evidence of unrecycled 40S ribosomes accumulating at stop codons since our assay used conventional ribosome profiling to find unrecycled 80S (not 40S) ribosomes. To obtain direct evidence of the recycling defect, we enhanced a protocol for ribosome profiling of 40S subunits to determine whether they were not recycled in the absence of these factors. We found clear increases in 40S footprint peaks on stop codons, directly establishing a role for Tma64 and Tma20/22 in 40S recycling. We found that the identity of the final sense codon of the gene could influence this activity, suggesting that recycling of the 40S also likely involves unbinding of the tRNA corresponding to this codon. We also found Tma64 performs a much smaller share of recycling. We also showed that mutants in the human ortholog of Tma22 (DENR) that are associated with autism led to recycling defects, thereby linking neurological disease to ribosome recycling. We are also interested in understanding how the cell monitors translating ribosomes for indications that additional regulation is required to tune the process or correct for an aberrant event. One such signal is collisions between translating ribosomes that result in formation of disomes. In our ongoing work, we are examining mechanisms by which cells detect disomes, particularly under stress conditions, by using a modified ribosome profiling approach to detect disome footprints. Another example is premature translation termination, where the ribsosome ends translation before reaching the 3' end of the gene. Such events trigger a pathway called nonsense-mediated decay (NMD) that leads to degradation of the mRNA and is important for eliminating aberrant mRNAs that do not encode full-length proteins. 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 the 40S ribosome profiling technique we developed to identify stop codons that were being actively used for premature translation termination. One class of stop codons we found includes those internal to coding sequences, indicative of a process called leaky scanning. Leaky scanning occurs when the 40S ribosome binds to an mRNA but fails to find the main AUG start codon, and instead initiates translation downstream to translate a short (out of frame) ORF that results in a premature termination event. We also found evidence for premature translation termination on long undecoded transcript isoforms (LUTIs), a key class of RNA that are thought to be generated as a consequence of transcription events that are primarily regulatory in nature. In particular, far upstream promoters are used 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 ensuring these transcripts are silenced and do not produce a protein product. Our work has also examined how translation changes during viral infection and promotes the innate immune response. In particular, we have examined the role of RNase L, an endonuclease that is activated when viral RNA is detected in the cell. RNase L activation is known to cause widespread mRNA decay that is thought to lead to eventual apoptosis and elimination of the infected cell. However, it is unclear whether the degraded transcriptome could still be translated and potentially facilitate this process, or under low-level activation, trigger mechanisms to clear the virus. To address this, we used ribosome profiling on RNase L activated cells to examine how the residual transcriptome was translated. Strikingly, we found a strong increase in ribosome occupancy in the 3' untranslated region downstream of the stop codon. While it was proposed that RNase L could modulate translation termination or ribosome recycling to cause this effect, the data are inconsistent with this hypothesis and do not mimic known mutants of the translation termination or recycling machinery. Instead, we observed increased translation in non-coding regions generally, including 5' untranslated regions and out-of-frame ORFs in coding sequences. Moreover, the effects depended on the cleavage activity of RNase L, suggesting a model where ribosomes initiate translation on mRNA decay fragments and translate any ORF that is encountered. Consistent with this, we found mRNA decay fragments were detectable in the cell. We now plan to ask whether these translation events affect the stability of the fragments, perhaps allowing them to carry out other roles, such as activation of RNA binding proteins. Finally, we also collaborated with lab of Alexander Mankin to reveal that yeast ribosomes could be genetically modified to respond to bacterial antibiotics in predictable ways, suggesting that antibiotic drugs may be repurposed for use in eukaryotic systems.